miR-10a rejuvenates aged human mesenchymal stem cells and improves heart function after myocardial infarction through KLF4

miR-10a rejuvenates aged human mesenchymal stem cells and improves heart function after... Background: Aging is one of the key factors that regulate the function of human bone marrow mesenchymal stem cells (hBM-MSCs) and related changes in microRNA (miRNA) expression. However, data reported on aging- related miRNA changes in hBM-MSCs are limited. Methods: We demonstrated previously that miR-10a is significantly decreased in aged hBM-MSCs and restoration of the miR-10a level attenuated cell senescence and increased the differentiation capacity of aged hBM-MSCs by repressing Krüpple-like factor 4 (KLF4). In the present study, miR-10a was overexpressed or KLF4 was downregulated in old hBM-MSCs by lentiviral transduction. The hypoxia-induced apoptosis, cell survival, and cell paracrine function of aged hBM-MSCs were investigated in vitro. In vivo, miR-10a-overexpressed or KLF4-downregulated old hBM- MSCs were implanted into infarcted mouse hearts after myocardial infarction (MI). The mouse cardiac function of cardiac angiogenesis was measured and cell survival of aged hBM-MSCs was investigated. Results: Through lentivirus-mediated upregulation of miR-10a and downregulation of KLF4 in aged hBM-MSCs in vitro, we revealed that miR-10a decreased hypoxia-induced cell apoptosis and increased cell survival of aged hBM- MSCs by repressing the KLF4–BAX/BCL2 pathway. In vivo, transplantation of miR-10a-overexpressed aged hBM- MSCs promoted implanted stem cell survival and improved cardiac function after MI. Mechanistic studies revealed that overexpression of miR-10a in aged hBM-MSCs activated Akt and stimulated the expression of angiogenic factors, thus increasing angiogenesis in ischemic mouse hearts. Conclusions: miR-10a rejuvenated aged hBM-MSCs which improved angiogenesis and cardiac function in injured mouse hearts. Keywords: Aging, MicroRNA, Rejuvenation, Myocardial infarction * Correspondence: gzlijiao@163.com Jun Dong and Zhenhui Zhang contributed equally to this work. Guangzhou Institute of Cardiovascular Disease, Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China Toronto General Research Institute, University Health Network, Toronto, Canada Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 2 of 16 Background cell apoptosis. In human malignant neuroblastoma Previous studies proved that bone marrow (BM) stem SK-N-DZ and IMR-32 cell lines, upregulation of KLF4 cells play an important role in improving heart function inhibited cell growth and increased cell apoptosis by ac- and delaying cardiac remodeling [1, 2] after ischemic tivating caspase-3 [15]. In leukemia cells, KLF4 upregu- damage through reducing fibrosis and increasing angio- lated Bax and downregulated Bcl-2 to induce cell genesis. However, preclinical animal research does not apoptosis, potentially through binding to the sites which necessarily translate into clinical results. Discrepancy in correspond to the promoters of Bcl-2 and Bax [16]. the effects of stem cell therapy [3, 4] may be due to the Furthermore, upregulation of KLF4 in the B-lymphoma age of the donor [5]. The age of stem cell donors might cell line induced cell apoptosis through activation of significantly impact the recipient’s endogenous responses BAK1 [17]. All of this evidence led us to postulate that [6]. Our group found that the proliferation and differen- miR-10a may mitigate cellular apoptosis of aged tiation ability of aged human bone marrow mesenchymal hBM-MSCs through its target gene KLF4. stem cells (hBM-MSCs) was decreased whereas cell sen- The efficacy of stem cell therapy for ischemic diseases escence was increased [7]. Effective methods to rejuven- was compromised by the poor survival of MSCs [18]. In ate aged hBM-MSCs to improve their regenerative the current study, we investigated whether upregulation capability may be required to maximize the beneficial ef- of miR-10a or downregulation of KLF4 can rejuvenate fects of stem cell therapy. aged hBM-MSCs and improve aged hBM-MSC survival Previous evidence has suggested that miRNAs regulate when transplanted into ischemic mouse hearts. In this cell growth, differentiation, replication, survival, and sen- regard, hBM-MSCs were harvested from young and old escence [8, 9]. We have reported that miR-26 attenuated patients undergoing cardiac surgery. Overexpression or myocardial hypertrophy by repressing expression of the inhibition of miR-10a or KLF4 in hBM-MSCs was GSK3b signaling pathway [10]. miR-30 decreased achieved through lentiviral transduction. The effects of autophagy-mediated angiotensin II-related myocardial miR-10a on hBM-MSC apoptosis, survival, and para- hypertrophy [11]. Furthermore, studies have shown that crine function were investigated in vitro. In vivo, the aging process may change the expression pattern of miR-10a-overexpressed hBM-MSCs were implanted into miRNAs. This aging-related change in miRNA expres- the border region of mouse hearts following myocardial sion may play an important role in the regulation of cel- infarction (MI), and cardiac function and related bio- lular function [12]. Our previous study found that logical changes were investigated. The molecular mecha- miR-196a, miR-486-5p, miR-664-star, and miR-378-star nisms of miR-10a on hBM-MSC function were further were significantly increased whereas miR-10a, miR-708, investigated. and miR-3197 were decreased in old hBM-MSCs. We also found that miR-10a increased differentiation and Methods decrease senescence in old hBM-MSCs through the tar- Human bone marrow mesenchymal stem cell isolation, get gene Krüpple-like factor 4 (KLF4) [7]. However, culture, and identification other studies have suggested that miR-10a may have This study was approved by the Research Ethics Com- additional effects other than regulating cell senescence mittee of The Second Affiliated Hospital of Guangzhou and differentiation. Indeed, some studies have shown Medical University. Human bone marrow (BM) was col- that miR-10a and KLF4 regulate cell apoptosis in other lected during cardiac valve surgery at The Second cell types [13, 14]. We speculated that miR10a may also Affiliated Hospital of Guangzhou Medical University. have additional effects related to cellular apoptosis and After anesthesia induction but before completion of the survival to regulate aged hBM-MSC function. sternotomy, 10 ml of sternal marrow was aspirated from The effect of miR-10a on cellular apoptosis has been the patients. Young human bone marrow mesenchymal reported recently. Li et al. [13] found that miR-10a and stem cells (hBM-MSCs) were obtained from patients miR-10b were significantly increased in CD34 marrow 18–30 years old, and old hBM-MSCs from patients 65– cells from 28 patients with myelodysplastic syndrome 80 years old. All patients were free of other pathological compared with healthy donors. They also found that conditions. downregulation of miR10a/10b in clonal cells interfered The hBM-MSCs were cultured as described previously with cell proliferation and enhanced cell apoptosis by [7]. Briefly, after centrifugation through a Ficoll gradient activating the NF-κB-dependent p53 pathway [13]. (1.077 g/ml density; GE Healthcare, Kretztechnik, Zipf, Another study has also shown that miR-10a inhibited Austria), cells were separated and the mononuclear cell KLF4 and RB1-inducible coiled-coil 1(RB1CC1) regu- fraction was plated. After 48 h, nonadherent cells were lated cell apoptosis in acute myeloid leukemia (AML) removed by changing the culture medium. The adherent [14]. On the other hand, KLF4, one of the miR-10a tar- cells were passaged when confluency reached 80%. get genes, has been reported to play a role in regulating hBM-MSCs were characterized by flow cytometry after Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 3 of 16 staining with antibodies against CD29, CD31, CD34, Briefly, C57BL/6 mice were intubated and ventilated CD44, CD45, and CD166 (MultiSciences Biotech Co., with 2% isoflurane. Through a left thoracotomy, the left Shanghai, China) as described previously. anterior descending coronary artery was ligated. Cardiac function was measured with echocardiography at differ- Viral vector construction and transduction ent time points before and after MI as indicated in Re- Lentiviral constructs for overexpression of miR-10a sults. At the study end, the scar area and thickness were (O-10a), KLF4 (O-KLF4), or inhibition of miR-10a and measured by planimetry. The infarct area was defined as KLF4 (O-anti10a and O-antiKLF4) in hBM-MSCs were the entire area of the left ventricle that contained a scar purchased from GenePharma as reported previously [7]. in myocardial sections stained with Masson’s trichrome. The Lenti-miR-10a sequence was TACCCTGTAGATCC Three days prior to MI and cell transplantation, mice GAATTTGTG. The Lenti-anti-10a sequence was CACA were given daily doses of cyclosporine A (5 mg/kg) to AATTCGGATCTACAGGGTA. The Lenti-anti-KLF4 se- induce the immunosuppression duration of the quence was GCCACCCACACTTGTGATTAC. The experiments. Lenti-control sequence was TTCTCCGAACGTGT CACGT. To construct LV-KLF4, the entire CDS region of Immunofluorescent staining KLF4 (NCBI reference sequence: NM_004235.4) was sub- Hearts were fixed in 2% paraformaldehyde (PFA) for cloned into the lentiviral vector. All of the lentiviral con- 24 h after being well perfused with PFA and were then structs expressed GFP. Viral solution was diluted with stored in 0.5 M sucrose at 4 °C overnight. Hearts were serum-free medium and polybrene (5 mg/ml) was added then embedded with OCT, and 5-μm-thick frozen sec- to the culture medium to transduce hBM-MSCs for 72 h. tions were prepared. Slides were incubated with one of the following primary antibodies: Alexa488-conjugated Cell survival and apoptosis evaluation anti-GFP (catalog number A21311, 1:400; Invitrogen) or Young (Y) and old hBM-MSCs (O) were cultured for 72 h anti-α-SMA (catalog number A-2547, 1:400; Sigma), at under hypoxia conditions (0.1% O ). Cell survival was de- room temperature for 2 h. Incubation with the respect- termined using the Cell Counting Kit-8 (CCK8; Dojindo, ive Alexa488 or Alexa568-conjugated secondary anti- Kumamoto, Japan) according to the manufacturer’sin- bodies (all 1:400; Invitrogen) or Isolectin B4 (catalog structions. In brief, cells were seeded into 96-well plates at number I21412, 1:100; ThermoFisher) was carried out at a density of 1 × 10 cells/well. After incubation, 5 μlof room temperature with light protection for 1 h. The nu- CCK-8 reagent was added to each well. The absorbance clei were identified with DAPI. was measured at 450 nm after 2 h of incubation at 37 °C. Terminal deoxynucleotidyl transferase dUTP nick end la- beling (TUNEL; Roche, Laval, QC, Canada) was carried out according to the manufacturer’s instructions. The number Western blotting of TUNEL cells in five randomly selected high-power For western blot analysis, 50 μg of lysate was fraction- fields per dish was determined and averaged with a Nikon ated through a 4% stacking and 10% running SDS-PAGE Eclipase Ti fluorescent microscope. Cell apoptosis was gel, and the fractionated proteins were transferred to a expressed as the percentage of total cells (DAPI ). PVDF membrane. Blots were blocked for 1 h at room temperature with blocking buffer. The antibodies (VEGF, Real-time reverse transcription-polymerase chain reaction SDF, phosphor (ser473)-AKT, and total AKT, all 1:1000) Total RNA was isolated with TRIzol reagent (Invitrogen, reacted with the blots overnight at 4 °C. After washing Grand Island, NY, USA) and cDNA was synthesized using (3 × 5 min in 1 × TBS–0.1% Tween 20), the blots were Moloney murine leukemia virus reverse transcriptase and incubated with horseradish peroxidase-conjugated sec- random primer. Real-time polymerase chain reaction was ondary antibody at 1:2000 dilution for 1 h at room conducted using SensiFAST SYBR Green PCR Master Mix temperature. Visualization was performed with en- (Bioline USA Inc., Taunton, MA, USA) with the following hanced chemiluminescence. For quantification, densi- parameters: 95 °C for 2 min, 95°C for 5 s, and 60 °C for tometry of the target bands was divided by the 30 s for 40 cycles. The oligonucleotide primer sequences corresponding densitometry of the GAPDH (catalog are presented in Additional file 1: Table S1. number mab374, 1:5000; Millipore) band using ImageJ software. To inhibit Akt phosphorylation in the Myocardial infarction and cardiac function measurement miR-10a-overexpressed old hBM-MSCs, 50 μM Akt In- The animal protocol was approved by Research Ethics hibitor VI (catalog number 124013; Merck) was used to Committee of The Second Affiliated Hospital of treat the cells for 72 h under hypoxia conditions follow- Guangzhou Medical University (2014013). Coronary oc- ing the manufacturer’s instructions. The inhibited ex- clusion was performed as described previously [19]. pression of P-AKT was conferred by western blotting. Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 4 of 16 Enzyme-linked immunosorbent assay file 2: Figure S1). The proapoptotic protein expression of hBM-MSCs in different groups were cultured in PUMA was also significantly higher whereas the antia- serum-free DMEM medium under hypoxia conditions poptotic protein expression of MCL1 was significantly for 72 h. The supernatant and cell lysate were collected lower in O hBM-MSCs compared with Y hBM-MSCs re- and the protein concentration was determined using a spectively (Fig. 1c). The ratio of BAX/BCL2 protein was Bio-Rad DC protein assay kit. The level of VEGF (cata- increased in O hBM-MSCs compared with Y log number DVE00; R&D Systems) and SDF (catalog hBM-MSCs (Fig. 1d). The protein expression of cleaved number DSA00; R&D Systems) were determined using caspase-3 and inhibitor of caspase-activated DNase an enzyme-linked immunosorbent assay (ELISA) follow- (ICAD) was also increased in O hBM-MSCs compared ing the manufacturer’s instructions. with Y hBM-MSCs (Fig. 1e). Furthermore, caspase-3 ac- tivity was significantly higher in O hBM-MSCs than in Y Caspase activity assay hBM-MSCs (Fig. 1f). The expression of miR-10a was A caspase fluorescent assay kit specific for caspase-3 significantly decreased in O hBM-MSCs compared with (Biovision, Mountain View, CA, USA) was used to de- Y hBM-MSCs (Fig. 1g). To the contrary, the expression tect caspase activation by measuring the cleavage of a of KLF4, which was one of the targets of miR-10a, was synthetic fluorescent substrate. In brief, cell lysates were significantly increased in O hBM-MSCs compared with prepared with the lysis buffer provided by the assay kit Y hBM-MSCs (Fig. 1h). All of these data implied the and centrifuged at 10,000 × g for 1 min, and the super- possible link between the downregulation of miR-10a natants were collected. With bovine serum albumin as and the increased O hBM-MSC apoptosis. the standard for protein content, equal amounts of pro- tein were reacted with the synthetic fluorescent sub- Upregulation of miR-10a in old hBM-MSCs decreased strates at 37 °C for 1.5 h, and absorbance at 405 nm was hypoxia-induced apoptosis and increased cell survival read on a microplate reader. The fold-increase in Next, to further test whether miR-10a was related to O caspase-3 activity versus control was determined. hBM-MSC apoptosis, miR-10a was overexpressed in O hBM-MSCs (Additional file 3: Figure S2A) and cellular Statistical analysis apoptosis was evaluated. The percentage of apoptotic All values are expressed as mean ± SD. Analyses were cells (TUNEL ) was decreased in miR-10a-upregulated performed using GraphPad InStat software (version 6). O hBM-MSCs (O-10a) compared with the control Student’s t test was used for two-group comparisons. vector-transduced O hBM-MSCs (O-c) that were cul- Comparisons of parameters among three or more groups tured for 72 h under hypoxia conditions (Fig. 2a). In were analyzed using one-way analysis of variance agreement, cell survival was increased in the O-10a (ANOVA) followed by Tukey for single-factor variables group compared with the O-c group (Fig. 2b). The proa- or two-way ANOVA for two-factor variables with re- poptotic mRNA expression of BAX and PUMA was de- peated measures over time, followed by Bonferroni crease in the O-10a group compared with the O-c group post-hoc tests. Differences were considered statistically (Additional file 4: Figure S3). On the contrary, the antia- significant at p < 0.05. poptotic mRNA expression of BCL2 and MCL1 was in- creased in the O-10a group compared with the O-c Results group (Additional file 4: Figure S3). The proapoptotic Cell apoptosis was increased in old hBM-MSCs under protein expression of PUMA was decreased whereas the hypoxia conditions antiapoptotic protein expression of MCL1 was increased Young (Y) and old (O) hBM-MSCs were cultured for in the O-10a group compared with the O-c group re- 72 h under hypoxia conditions, followed by comparison spectively (Fig. 2c). The ratio of BAX/BCL2 protein in of cell survival and apoptosis. The percentage of apop- the O-10a group was decreased compared with the O-c totic cells (TUNEL ) was significantly higher in the O group (Fig. 2d). The protein expression of cleaved group compared with the Y group of hBM-MSCs caspase-3 and ICAD was decreased in the O-10a group (Fig. 1a). In agreement, cell survival was decreased in O compared with the O-c group (Fig. 2e). Furthermore, hBM-MSCs compared with Y hBM-MSCs by CCK-8 caspase-3 activity was significantly lower in the O-10a assay (Fig. 1b). The proapoptotic mRNA expression of group compared with the O-c group (Fig. 2f). These BAX and PUMA was significantly higher in O findings suggested that restoring the miR-10a level re- hBM-MSCs compared with Y hBM-MSCs duced hypoxia-induced apoptosis in O hBM-MSCs. We (Additional file 2: Figure S1). On the contrary, the antia- also examined the effects of miR-10a on Y hBM-MSCs poptotic mRNA expression of BCL2 and MCL1 (BCL2 and found that cell apoptosis was decreased family apoptosis regulator) was significantly lower in O (Additional file 5: Figure S4A) and cell survival hBM-MSCs compared with Y hBM-MSCs (Additional (Additional file 5: Figure S4B) was increased in Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 5 of 16 a b cd fg h Fig. 1 Cell apoptosis increased in old hBM-MSCs under hypoxia conditions. Young (Y) and old (O) hBM-MSCs cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in Y and O hBM-MSCs. b Cell survival evaluated in Y and O hBM-MSCs c Protein expression of MCL1 and PUMA evaluated by western blot analysis in Y and O hBM-MSCs. d Ratio of Bax/BCL2 quantified in Y and O hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in Y and O hBM-MSCs. f Caspase-3 activity measured in Y and O hBM-MSCs. Expression of (g) miR-10a and (h) KLF4 compared in Y and O hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RQ relative quantity, RFU relative fluorescence units miR-10a-upregulated Y hBM-MSCs (Y-10a) compared conditions (Fig. 3a). In agreement, cell survival was in- with the control vector-transduced Y hBM-MSCs (Y-c). creased in the O-antiKLF4 group compared with the The effects of miR-10a on Y hBM-MSC apoptosis and O-c group (Fig. 3b). The proapoptotic mRNA expression survival followed the same trend as in O hBM-MSCs of BAX and PUMA was decreased in the O-antiKLF4 but at a much lower magnitude. group compared with the O-c group (Additional file 6: Figure S5). On the contrary, the antiapoptotic mRNA Downregulation of KLF4 expression in old hBM-MSCs expression of BCL2 and MCL1 was increased in the decreased hypoxia-induced apoptosis and increased cell O-antiKLF4 group compared with the O-c group survival (Additional file 6: Figure S5). The proapoptotic protein KLF4 is one of the targets of miR-10a and was upregu- expression of PUMA was decreased whereas the antia- lated in O hBM-MSCs. To determine whether KLF4 was poptotic protein expression of MCL1 was increased in involved in miR-10a-mediated cellular apoptosis, we ef- the O-antiKLF4 group compared with the O-c group re- fectively inhibited KLF4 in O hBM-MSCs (Additional spectively (Fig. 3c). The ratio of BAX/BCL2 protein in file 3: Figure S2B) and evaluated cell apoptosis. The per- the O-antiKLF4 group was decreased when compared centage of apoptotic cells (TUNEL ) was decreased in with the O-c group (Fig. 3d). Moreover, the protein ex- the KLF4-inhibited O hBM-MSCs (O-antiKLF4) com- pression of cleaved caspase-3 and ICAD was decreased pared with the control vector-transduced O hBM-MSCs in the O-antiKLF4 group compared with the O-c group (O-c) that were cultured for 72 h under hypoxia (Fig. 3e). Caspase-3 activity was significantly lower in the Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 6 of 16 a b cd Fig. 2 Overexpression of miR-10a in old hBM-MSCs decreased hypoxia-induced apoptosis and increased cell survival. miR-10a transduced into old hBM-MSCs by lentiviral vector (O-10a). Control vector-transduced old hBM-MSCs (O-c) served as control. Cells cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c and O-10a hBM-MSCs. b Cell survival evaluated in O-c and O-10a hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-c and O-10a hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-c and O-10a hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase- activated DNase (ICAD) assayed in O-c and O-10a hBM-MSCs. f Caspase-3 activity measured in O-c and O-10a hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units O-antiKLF4 group compared with the O-c group O-KLF4 groups compared with the O-c group (Fig. 4b). (Fig. 3f). The proapoptotic mRNA expression of BAX and PUMA was significantly higher in the O-anti10a and O-KLF4 Downregulation of miR-10a or overexpression of KLF4 in groups compared with the O-c group (Additional file 7: old hBM-MSCs increased hypoxia-induced apoptosis and Figure S6). On the contrary, the antiapoptotic mRNA ex- decreased cell survival pression of BCL2 and MCL1was significantly lower in the All of this evidence suggested that miR-10a, through sup- O-anti10a and O-KLF4 groups compared with the O-c pression of KLF4, may rescue O hBM-MSCs from group (Additional file 7: Figure S6). The proapoptotic pro- hypoxia-induced apoptosis. To test whether the reverse tein expression of PUMA was increased whereas the anti- would post a detrimental effect, miR-10a was effectively apoptotic protein expression of MCL1 was decreased in inhibited in O hBM-MSCs (Additional file 3: Figure S2C) the O-anti10a and O-KLF4 groups compared with the or KLF4 was overexpressed in O hBM-MSCs (Additional O-c group respectively (Fig. 4c). The ratio of BAX/ BCL2 file 3: Figure S2D). The percentage of apoptotic cells protein in the O-anti10a and O-KLF4 groups was in- (TUNEL ) was significantly higher in miR-10a-inhibited creased when compared with the O-c group (Fig. 4d). The old hBM-MSCs (O-anti10a) and KLF4-overexpressed old protein expression of cleaved caspase-3 and ICAD was in- hBM-MSCs (O-KLF4) compared with the control creased in the O-anti10a and O-KLF4 groups compared vector-transduced old hBM-MSCs (O-c) that were cul- with the O-c group (Fig. 4e). Caspase-3 activity was sig- tured for 72 h under hypoxia conditions (Fig. 4a). In agree- nificantly higher in the O-anti10a and O-KLF4 groups ment, cell survival was decreased in the O-anti10a and compared with the O-c group (Fig. 4f). Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 7 of 16 a b cd Fig. 3 Downregulation of KLF4 in old hBM-MSCs decreased hypoxia-induced apoptosis and increased cell survival. KLF4 was effectively inhibited in old hBM-MSCs by lentiviral vector (O-antiKLF4). Control vector-transduced old hBM-MSCs (O-c) served as control. Cells cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c and O-antiKLF4 hBM-MSCs. b Cell survival evaluated in O-c and O-antiKLF4 hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-c and O-antiKLF4 hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-c and O-antiKLF4 hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in O-c and O-antiKLF4 hBM-MSCs. f Caspase-3 activity measured in O-c and O-antiKLF4 hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units The antiapoptotic effect of miR-10a is attenuated by the significantly higher in the O-10a-KLF4 group com- restoration of KLF4 pared with O-10a hBM-MSCs. On the contrary, the To further confirm that KLF4 is indeed the direct antiapoptotic mRNA expression of BCL2 and MCL1 target of miR-10a in mediating hypoxia-induced O was significantly lower in the O-10a-KLF4 group hBM-MSC apoptosis, KLF4 expression was restored compared with O-10a hBM-MSCs (Additional file 8: by a rescue experiment (Additional file 3:Figure Figure S7). The proapoptotic protein expression of S2E). For this purpose, miR-10a-overexpressed O PUMA was increased whereas the antiapoptotic pro- hBM-MSCs were transduced with a lentivirus which tein expression of MCL1 was decreased in the carried the KLF4 vector and the restoration of KLF4 O-10a-KLF4 group compared with the O-10a group expression was confirmed (O-10a-KLF4). After restor- respectively (Fig. 5c). The ratio of BAX/ BCL2 protein ing the expression of KLF4, miR10a lost its antiapop- was increased in the O-10a-KLF4 group compared totic effect on O hBM-MSCs. Cell apoptosis was with the O-10a group (Fig. 5d). The protein expres- increased in the O-10a-KLF4 group compared with sion of active caspase-3 and ICAD was increased in the O-10a group (Fig. 5a). In agreement, cell survival the O-10a-KLF4 group compared with O-10a was decreased in the O-10a-KLF4 group compared hBM-MSCs (Fig. 5e). Caspase-3 activity was also in- with the O-10a group (Fig. 5b). The proapoptotic creased in the O-10a-KLF4 group compared O-10a mRNA expression of BAX and PUMA was hBM-MSCs (Fig. 5f). Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 8 of 16 a b Fig. 4 Downregulation of miR-10a or overexpression of KLF4 in old hBM-MSCs increased hypoxia-induced apoptosis and decreased cell survival. Lentiviral vectors used to transduce old hBM-MSCs to downregulate miR-10a expression (O-anti10a) or overexpress KLF4 (O-KLF4). Control vector-transduced old hBM-MSCs (O-c) served as control. Cells cultured for 72 h under hypoxia condition. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c, O-anti10a, and O-KLF4 hBM-MSCs. b Cell survival evaluated in O-c, O-anti10a, and O-KLF4 hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-c, O-anti10a, and O-KLF4 hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-c, O-anti10a, and O-KLF4 hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in O-c, O-anti10a, and O-KLF4 hBM-MSCs. f Caspase-3 activity measured in O, O-anti10a, and O-KLF4 hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units Implantation of miR-10a-overexpressed or KLF4- MI in the four experimental groups. After MI, there downregulated old hBM-MSCs into infarcted mouse was a significant decrease in fractional shortening (FS; hearts improved cardiac function after MI Fig. 6b) and ejection fraction (EF; Fig. 6c) and an in- To evaluate whether modifying miR-10a or KLF4 crease in left ventricular internal end-systolic dimen- levels in O hBM-MSCs can maximize the beneficial sion (LVIDs; Fig. 6d) and left ventricular internal effects of stem cell therapy, miR-10a-overexpressed or end-diastolic dimension (LVIDd; Fig. 6e)inthe O-c KLF4-downregulated old hBM-MSCs were implanted group compared with the Y-c group. However, there into infarcted mouse hearts. Cardiac function was de- was an improvement in all of these parameters in the termined by echocardiography in mice that received O-10a and O-antiKLF4 groups when compared with implantation of control medium (Media), control the O-c group (Fig. 6a–e). vector-transduced young hBM-MSCs (Y-c), control Similarly, the infarct size at 28 days post MI was sig- vector-transduced old hBM-MSCs (O-c), nificantly larger in the O-c group than the Y-c group miR-10a-overexpressed old hBM-MSCs (O-10a), or (Fig. 6f, g). On the other hand, scar thickness was signifi- KLF4-inhibited old hBM-MSCs (O-antiKLF4) into the cantly lower in the O-c group than the Y-c group border region immediately following MI. Cardiac (Fig. 6h). However, the infarct size was smaller and the function was evaluated at baseline (before MI) and at scar thickness was greater in the O-10a and O-antiKLF4 14 and 28 days after MI. Fig. 6a shows representative groups when compared with the O-c group (Fig. 6f–h). M-mode echocardiographic images taken 28 days post More viable myocardium was found by Trichrome’s Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 9 of 16 a b cd ef Fig. 5 Antiapoptotic effect of miR-10a attenuated by restoration of KLF4. Lentivirus which carries KLF4 vector used to infect miR-10a-upregulated old hBM-MSCs (O-10a) to restore KLF4 expression (O-10a-KLF4). miR-10a-upregulated old hBM-MSCs (O-10a) also infected by the control lentivirus (O-10a-c). Cells cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-10a and O-10a-KLF4 hBM-MSCs. b Cell survival evaluated in O-10a and O-10a-KLF4 hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-10a and O-10a-KLF4 hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-10a and O-10a-KLF4 hBM- MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in O-10a and O-10a-KLF4 hBM-MSCs. f Caspase-3 activity evaluated in O-10a and O-10a-KLF4 hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units staining in the Y-c group than in the O-c group. How- least apoptosis in the group receiving Y hBM-MSCs ever, there was more viable myocardium in the O-10a (Y-c). However, overexpression of miR-10a (O-10a) or and O-antiKLF4 groups when compared with the O-c inhibition of KLF4 expression (O-antiKLF4) increased group (Fig. 6i, j). All of these data clearly demonstrated cell survival but decreased cell apoptosis when com- that modifying the miR-10a or KLF4 level in O pared with the group receiving O hBM-MSCs (O-c). To hBM-MSCs enhanced the beneficial effects of stem cell seek possible downstream mediators, western blot ana- therapy and further improved cardiac function. lyses were performed to examine the activation of AKT. As shown in Fig. 7e, although total AKT expression was miR-10a upregulation or KLF4 downregulation increased similar for all the groups, phospho-Akt expression was old hBM-MSC survival and decreased apoptosis by highest in the Y-c group and was increased in the O-10a activating AKT and O-antiKLF4 groups compared with the O-c group. To evaluate the antiapoptotic effect of miR-10a in vivo, The same pattern was found in the in-vitro cultured the survival of implanted cells was detected by hBM-MSCs. This showed that total AKT expression was lentiviral-mediated GFP expression in the border region similar for hBM-MSCs from all of the conditions while of the mouse hearts at 3 days (Fig. 7a, b) and 7 days phospho-AKT was highest in Y hBM-MSCs and was in- (Fig. 7c, d) post MI. Cell apoptosis was confirmed by creased in the O-10a and O-antiKLF4 groups compared TUNEL staining. There was highest cell survival and with O hBM-MSCs (Fig. 7f). To build up the direct link Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 10 of 16 b c f g i Fig. 6 Implantation of miR-10a-overexpressed or KLF4-downregulated old hBM-MSCs into ischemic area of mouse hearts improved cardiac function after MI. miR-10a-overexpressed or KLF4-downregulated old hBM-MSCs (3 × 10 cells/mouse) implanted into infarcted mouse hearts. Cardiac function determined by echocardiography in mice that received implantation of control medium (Media), control vector-transduced young hBM-MSCs (Y-c), control vector-transduced old hBM-MSCs (O-c), miR-10a-overexpressed old hBM-MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) into border region immediately following MI. Cardiac function measured by echocardiography at baseline (before MI, 0 day), 14 and 28 days after MI in all groups. a Representative M-mode echocardiographic images. b Fractional shortening. c Ejection fraction. d Left ventricular internal end-systolic dimension (LVIDs). e Left ventricular internal end-diastolic dimension (LVIDd). f Each panel shows one representative whole sectioned heart (from base to apex) at 28 days after MI from five individual groups (Media, Y-c, O-c, O-10a, or O-antiKLF4) to show scar areas (arrows) (g) and scar size thickness (h). i Representative images of Masson Trichrome’s staining at 28 days after MI. j Viable myocardium (identified as red with Trichrome’s staining) in ischemia zone quantified and expressed as percentage of total infarct area. n = 6/group. Mean ± SD. *P < 0.05, Y-c vs other groups; #P < 0.05, O-c vs other groups. LV left ventricle, MI myocardial infarction between the activation of Akt and increased cell survival, whereas inactivation of Akt reversed this effect. Collect- we used an inhibitor (Akt Inhibitor VI) to inhibit the ively, these data revealed that miR-10a, through activation phosphorylation (activation) of Akt in the of AKT, increased hBM-MSC survival, thus improving miR-10a-overexpressed old hBM-MSCs (O-10a-P-AKT cardiac function. Inh; Additional file 9: Figure S8A). After culture for 72 h under hypoxia conditions, apoptosis was detected with miR-10a-overexpressed or KLF4-downregulated hBM- the TUNEL assay (Additional file 9: Figure S8B) and cell MSCs increased angiogenesis in infarcted mouse hearts survival was detected by CCK-8 assay in control To determine whether angiogenesis is also affected by vector-transduced (O-c), miR-10a-overexpressed (O-10a), regulating the miR-10a or KLF4 level in O hBM-MSCs, and O-10a-P-AKT Inh old hBM-MSCs. We found that capillary and arteriole densities were quantified by iso- cell apoptosis was increased and cell survival was de- lectin stain (Fig. 8a, b) and α-smooth muscle actin creased in the O-10a-P-AKT Inh group compared to the (α-SMA) stain (Fig. 8c, d) respectively in all four experi- O-10a group. We believe these results showed that mental groups at 3 and 7 days post MI. More capillaries miR-10a, through activating Akt, increased cell survival and arteriole were formed in the group receiving Y Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 11 of 16 a b cd ef Fig. 7 miR-10a overexpression or KLF4 downregulation increased old hBM-MSC survival and decreased apoptosis by activating AKT. miR-10a- overexpressed or KLF4-downregulated old hBM-MSCs (3 × 10 cells/mouse) implanted into infarcted mouse hearts. Cell survival and biochemical changes determined in mice that received implantation of control vector-transduced young hBM-MSCs (Y-c), control vector-transduced old hBM- MSCs (O-c), miR-10a-overexpressed old hBM-MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) into border region immediately following MI. Survival of implanted cells detected by green fluorescent protein (GFP) expression carried by lentiviral vector in border region of infarcted mouse hearts and cell apoptosis assayed by TUNEL staining at 3 days (a, b)and 7days (c, d)post MI. e Expression of total AKT and phospho-(ser473)-AKT detected in border region of infarcted mouse hearts tissue. f Expression of phosphor-(ser473)-AKT also detected in hBM-MSCs. n = 6/group. Mean ± SD. *P <0.05, Y-c vs other groups; #P < 0.05, O-c vs other groups. DAPI 4′,6-diamidino-2-phenylindole, hBM-MSC human mesenchymal stem cell, KLF4 Krüpple-like factor 4, MI myocardial infarction, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling hBM-MSCs (Y-c) compared to the group receiving O and SDF mRNA (Additional file 10: Figure S9A), and hBM-MSCs (O-c). However, overexpression of miR-10a protein (Fig. 8e, f) was decreased in the heart tissue (O-10a) or inhibition of KLF4 (O-antiKLF4) increased from the border region of mice in the O hBM-MSC capillary and arteriole formation when compared with group (O-c) compared to the Y hBM-MSC group (Y-c). the group receiving O hBM-MSC implantation only. However, overexpression of miR-10a (O-10a) or inhib- Furthermore, the expression of angiogenic factors, VEGF ition of KLF4 e (O-antiKLF4) increased VEGF and SDF Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 12 of 16 a b ef gh Fig. 8 miR-10a-upregulated or KLF4-downregulated hBM-MSCs increased mice heart angiogenesis after MI. miR-10a-overexpressed or KLF4- downregulated old hBM-MSCs (3 × 10 cells/mouse) implanted into infarcted mouse hearts. Angiogenesis and expression of angiogenic factors determined in mice that received implantation of control vector-transduced young hBM-MSCs (Y-c), control vector-transduced old hBM-MSCs (O-c), miR-10a-overexpressed old hBM-MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) into border region immediately following MI. Capillary and arteriole densities quantified by isolectin stain (a, b)and α-smooth muscle actin (α-SMA) stain respectively at 3 and 7 days (c, d)postMI. e, f VEGF and SDF protein expression evaluated by western blot analysis from border region of infarcted mouse hearts implanted with Y-c, O-c, O-10a, and O-antiKLF4 hBM-MSCs respectively. g, h VEGF and SDF protein expression evaluated by western blot analysis in Y-c, O-c, O-10a, and O-antiKLF4 hBM-MSCs. i VEGF and SDF protein in cell culture supernatant of Y-c, O-c, O-10a, and O-antiKLF4 hBM-MSCs assayed by ELISA. n = 6/group. Mean ± SD. *P <0.05, Y-c vs other groups; #P < 0.05, O-c vs other groups. DAPI 4′,6-diamidino-2-phenylindole, hBM-MSC human mesenchymal stem cell, KLF4 Krüpple-like factor 4, MI myocardial infarction, SDF stromal cell-derived factor, VEGF vascular endothelial growth factor mRNA and protein expression when compared with KLF4 (O-antiKLF4) increased VEGF and SDF mRNA the O-c group (Fig. 8e, f). Thesametrend wasfound (Additional file 10: Figure S9B) and protein expres- in the in-vitro cultured hBM-MSCs. The VEGF and sion compared with that in O hBM-MSCs (Fig. 8g, SDF mRNA (Additional file 10: Figure S9B) and protein h). The secretion of VEGF and SDF in the cell cul- (Fig. 8g, h) expression was decreased in O hBM-MSCs ture supernatant was also increased in the O-10a and (O-c) compared with Y hBM-MSCs (Y-c). However, O-antiKLF4 groups compared with O hBM-MSCs overexpression of miR-10a (O-10a) or inhibition of (Fig. 8i). Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 13 of 16 Discussion [14]. miR-10a is also reported to regulate cell apop- The present study demonstrated that restoring the tosis in the human cumulus–oocytes complex [26]. In miR-10a level in O hBM-MSCs increased cell survival addition, Bim, the proapoptotic factor, is directly tar- and decreased apoptosis under in-vitro hypoxia and geted by miR-10a, resulting in repressing Casp9 which in-vivo ischemic conditions by repressing the expression is a crucial factor in the apoptotic pathway [27]. miR-10a of KLF4. Accordingly, restoration of the miR-10a level in delivered by exosomes sustains the number of O hBM-MSCs increased the antiapoptotic-related pro- chemotherapy-damaged granulosa cells and reduces the tein expression and decreased the proapoptosis-related number of chemotherapy-damaged apoptotic granulosa protein expression. Furthermore, inhibition of miR-10a cells following nitrogen mustard treatment for 24–48 h target gene KLF4 also increased both cell survival and [28]. All of these findings support the notion that miR-10a the antiapoptotic-related protein expression while it de- is a prosurvival factor, which counteracts the apoptotic creased cellular apoptosis and the proapoptosis-related signals. Consistent with previous data, we also found that protein expression. The molecular mechanism study re- upregulation of miR-10a in aged hBM-MSCs decreased vealed that transplantation of miR-10a-overexpressed or hypoxia-induced apoptosis and increased cell survival. To KLF4-downregulated O hBM-MSCs activated AKT in the contrary, downregulation of miR-10a in aged infarcted mouse hearts, which led to increased survival hBM-MSCs increased hypoxia-induced apoptosis and of implanted cells. Furthermore, transplantation of decreased cell survival. Implantation of miR-10a or anti-KLF4-pretreated O hBM-MSCs in- miR-10a-overexpressed old hBM-MSCs into the ischemic creased the expression and secretion of angiogenic fac- area of mouse hearts improved cell survival and cardiac tors VEGF and SDF, which increased angiogenesis in function after MI. infarcted mouse hearts. All of these effects of miR-10a In our previous paper, using a luciferase reporter assay, ultimately led to enhanced efficacy of stem cell therapy we reported that KLF4 is a direct target of miR-10a. and the improvement of cardiac function after MI. Also, miR-10a decreased cell senescence in aged BM-MSCs are an optimal cell type for therapeutic ap- hBM-MSCs by repressing KLF4 [7]. KLF4 can function proaches for various pathological conditions including as both a repressor and an activator of transcription fac- cardiovascular disease since the cells can be easily iso- tors related to cell cycle regulation, apoptosis, and differ- lated and expanded in vitro [20]. However, the beneficial entiation. The expression of KLF4 can be increased by effects of BM-MSC therapy are restricted by the low sur- DNA damage, serum deprivation, and contact inhibition vival rate and decreased proliferative capacity of the aged [29]. Recent research suggests KLF4 as a proapoptotic cells after cell transplantation [21]. The ischemic envir- factor. A combination of KLF4 plasmid and apigenin onment of the infarcted heart triggers a strong inflam- treatment increases apoptosis in the human malignant matory and oxidative stress reaction and overproduction neuroblastoma SK-N-DZ and IMR-32 cell lines com- of factors related to apoptosis, which all lead to in- pared with control vector or single treatment [15]. After creased BM-MSC apoptosis and decreased cell survival a full-length complementary DNA or an antisense oligo- [22]. Various attempts have been made to improve nucleotide of KLF4 was transfected into a human im- BM-MSC survival under hypoxic and ischemic conditions mortalized myelogenous leukemia line (K562 cells), cell [23]. Here, we have shown that restoring miR-10a in aged growth was decreased and cell apoptosis was increased hBM-MSCs increased cell survival and decreased apop- [16]. Epigenetic inhibition of KLF4 in B-cell lymphomas tosis when transplanted into the ischemic mouse hearts. and particularly in classic Hodgkin lymphoma cases in- MicroRNAs play an important role in cell prolifera- creases lymphoma survival and decreases apoptosis [17]. tion,differentiation,and apoptosis[8]. Upregulation Research in resveratrol has found that KLF4 and early of specific miRNAs increased BM-MSC survival, mak- growth response-1 expression was increased by resvera- ing them more effective to repair the infarct damage trol, resulting in transcription factor 3 activation and cel- [24, 25]. miR-10a has also been reported as a prosur- lular apoptosis [30]. KLF4 expression is increased in vival factor. Previous studies have found that expres- murine astrocytes when exposed to X-ray radiation, sion of miR10a/10b was controlled by TWIST-1 and resulting in more double-strand DNA breaking, and at via the NF-κB and P53 axis, to control last cell apoptosis [31]. The apoptosis rate of SK-BR-3 TNF-α-induced (and stroma-dependent) apoptosis in breast cancer cells is increased and cell tumorigenicity is clonal myeloid cells. Therefore, the TWIST-1/miR10/ decreased by upregulation of KLF4 [32]. However, the p53 axis can serve as a potential new target for thera- role of KLF4 on hBM-MSC apoptosis still remains un- peutic interventions in advanced myelodysplastic syn- clear. In this study, we found that downregulation of dromes [13]. On the other hand, knockdown of KLF4 in aged hBM-MSCs decreased hypoxia-induced miR-10a in the NPM1 mutated cell line OCI-AML3 apoptosis and increased cell survival. To the contrary, decreased cellular survival and clonogenic growth upregulation of KLF4 in aged hBM-MSCs increased Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 14 of 16 hypoxia-induced apoptosis and decreased cell survival. was significantly lower in O-antiKLF4 than in control vector-transduced O Accordingly, upregulation of KLF4 resulted in decreased hBM-MSCs (B). Lentiviral vector carrying anti-miR-10a sequence used to transduce old hBM-MSCs (O-anti10a). miR-10a expression was significantly expression of the antiapoptotic proteins BCL2 and lower in O-anti10a than in control vector-transduced O hBM-MSCs (C). MCL1 but increased the expression of proapoptotic pro- Lentiviral vector carrying KLF4 sequence used to transduce old hBM- teins BAX and PUMA, and caused activation of caspase-3, MSCs (O-KLF4). KLF4 expression was significantly higher in O-KLF4 than in control vector-transduced O hBM-MSCs (D). Lentivirus which carries KLF4 leading to completion of the apoptotic machinery. The im- vector used to infect miR-10a-upregulated old hBM-MSCs (O-10a) to plantation of KLF4-downregulated old hBM-MSCs into restore KLF4 expression (O-10a-KLF4). miR-10a-upregulated old the ischemic area of mouse hearts improved cell survival hBM-MSCs (O-10a) also infected by the control lentivirus (O-10a-c). KLF4 expression restored in O-10a-KLF4 compared to O-10a-c hBM-MSCs (E). and cardiac function after MI. We believed that KLF4 n = 5/group. Mean ± SD. P < 0.05 O-c vs O-10a, O-anti-KLF4, O-anti-10a, plays a proapoptotic role in aged hBM-MSCs. # and O-KLF4; P < 0.05 O-10a-KLF4 vs O-10a-c and O-10a (PDF 41 kb) AKT is a key factor involved in cell survival, prolifera- Additional file 4: Figure S3. Overexpression of miR-10a in old tion, and metabolism [33]. Our data show that miR-10a hBM-MSCs decreased apoptotic gene expression. Quantification of mRNA expression of BAX and PUMA (proapoptotic), BCL2 and MCL1 overexpression or KLF4 downregulation activated AKT (antiapoptotic) in O and O-10a hBM-MSCs after culture for 72 h under both in aged hBM-MSCs and in ischemic mouse hearts. hypoxia conditions. n = 6/group. Mean ± SD. *P < 0.05 (PDF 37 kb) We believe the antiapoptotic effect of miR-10a may be Additional file 5: Figure S4. Overexpression of miR-10a in both young mediated through activation of AKT. In addition, we and old hBM-MSCs decreased hypoxia-induced apoptosis and increased cell survival. miR-10a transduced into young (Y-10a) and old (O-10a) found that miR-10a overexpression or KLF4 downregu- hBM-MSCs by lentiviral vector. Control vector-transduced young lation increased VEGF and SDF expression and secre- hBM-MSCs (Y-c) and old hBM-MSCs (O-c) served as controls. Cells tion. VEGF is a key factor for angiogenesis [34]. It has cultured for 72 h under hypoxia conditions. (A) Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in Y-c, been reported that VEGF inhibits posthypoxic MSC Y-10a, O-c, and O-10ahBM-MSCs. (B) Cell survival evaluated in Y-c, Y-10a, death and increases MSC survival and regeneration in O-c, and O-10a hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05, Y-c vs ischemic conditions [35, 36]. SDF also confers increased Y-10a; #P < 0.05, O-c vs O-10a (PDF 94 kb) vasculogenesis and angiogenesis [37]. Our data sug- Additional file 6: Figure S5. Downregulation of KLF4 in old hBM-MSCs decreased apoptotic gene expression. Quantification of mRNA expression gested that miR-10a increased the survival of aged of BAX and PUMA (proapoptotic), BCL2 and MCL1 (antiapoptotic) in O hBM-MSCs which in turn secreted more VEGF and SDF and O-antiKLF4 hBM-MSCs after culture for 72 h under hypoxia to stimulate angiogenesis in the infarcted hearts. Im- conditions. n = 6/group. Mean ± SD. *P < 0.05 (PDF 37 kb) proved cell survival and enhanced angiogenesis acted Additional file 7: Figure S6. Downregulation of miR-10a or overexpression of KLF4 in old hBM-MSCs increased apoptotic gene synergistically to preserve heart function after MI. expression. Quantification of mRNA expression of BAX and PUMA (pr-apoptotic), BCL2 and MCL1 (antiapoptotic) in O, O-anti10a and O-KLF4 hBM-MSCs after culture for 72 h under hypoxia conditions. Conclusions n = 6/group. Mean ± SD. *P < 0.05 (PDF 38 kb) Our study demonstrated that miR-10a decreased aged Additional file 8: Figure S7. Antiapoptotic effect of miR-10a attenuated hBM-MSC apoptosis and increased cell survival by restoration of KLF4 quantified by RT-qPCR. Quantification of mRNA through suppression of KLF4. Transplantation of expression of BAX and PUMA (proapoptotic), BCL2 and MCL1 (antiapoptotic) in O-10a-c, O-10a-KLF4, and O-c hBM-MSCs after culture for 72 h under miR-10a-overexpressed or KLF4-downregulated old hypoxia conditions. n =6/group. Mean ±SD. *P <0.05 (PDF 57 kb) hBM-MSCs activated AKT and stimulated angiogen- Additional file 9: Figure S8. miR-10a overexpression increased old hBM- esis in the ischemic hearts, thereby improving cardiac MSC survival and decreased apoptosis by activating AKT. Expression of Akt function. miR-10a and KLF4-modified stem cells phosphorylation in miR-10a-overexpressed old hBM-MSCs (O-10a) inhibited by Akt Inhibitor VI during 72-h culture under hypoxia conditions. (A) could be a potent vehicle to combine cell and gene Expression of phosphor-(ser473)-AKT detected in O-c, O-10a, and Akt therapies to improve heart function after injury. Inhibitor VI added O-10a hBM-MSCs (O-10a-P-AKT Inh). (B) Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c, O-10a, and O-10a-P-AKT Inh hBM-MSCs. (C) Cell survival Additional files evaluated in O-c, O-10a, and O-10a-P-AKT Inh hBM-MSCs. n =5/group. Mean ± SD. P < 0.05, O-10a-P-AKT Inh vs O-10a (PDF 184 kb) Additional file 1: Table S1. qRT-PCR primer and miRNA RT primer Additional file 10: Figure S9. Expression of VEGF and SDF mRNA in sequences (DOCX 19 kb) mouse hearts after MI and hBM-MSCs. (A) Expression of VEGF and SDF Additional file 2: Figure S1. Proapoptotic and antiapoptotic gene mRNA determined in border region of infarcted mouse hearts that received implantation of control vector-transduced young hBM-MSCs (Y-c), control expression in old hBM-MSCs under hypoxia conditions. Quantification of mRNA expression of BAX and PUMA (proapoptotic), BCL2 and MCL1 vector-transduced old hBM-MSCs (O-c), miR-10a-overexpressed old hBM- (antiapoptotic) in Y and O hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05 MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) following MI. (PDF 36 kb) (B) Expression of VEGF and SDF mRNA determined by RT-qPCR in Y-c, O-c, O-10a, or O-antiKLF4 hBM-MSCs after hypoxia for 72 h. n =6/group. Mean ± Additional file 3: Figure S2. Expression of miR-10a and KLF4 in old SD. *P < 0.05, Y-c vs other groups; P < 0.05, O-c vs other groups (PDF 39 kb) hBM-MSCs regulated by lentiviral vector. Lentiviral vector carrying miR- 10a sequence used to transduce old hBM-MSCs (O-10a) and control vector-transduced old hBM-MSCs (O-c) served as control. miR-10a Abbreviations expression was significantly higher in O-10a than in control vector- BM: Bone marrow; CCK8: Cell Counting Kit-8; EF: Ejection fraction; transduced (O) hBM-MSCs (A). Lentiviral vector carrying KLF4 siRNA FS: Fractional shortening; hBM-MSC: Human mesenchymal stem cell; sequence used to transduce old hBM-MSCs (O-anti-KLF4). KLF4 expression KLF4: Krüpple-like factor 4; LVIDd: Left ventricular internal end-diastolic Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 15 of 16 dimension; LVIDs: Left ventricular internal end-systolic dimension; 6. Zhuo Y, Li SH, Chen MS, et al. Aging impairs the angiogenic response to MI: Myocardial infarction; SMA: Smooth muscle actin; TUNEL: Terminal ischemic injury and the activity of implanted cells: combined consequences deoxynucleotidyl transferase dUTP nick end labeling for cell therapy in older recipients. J Thorac Cardiovasc Surg. 2010;139:1286–94. 7. Li J, Dong J, Zhang ZH, et al. miR-10a restores human mesenchymal stem cell differentiation by repressing KLF4. J Cell Physiol. 2013;228:2324–36. Acknowledgements The authors thank Dr Ren-Ke Li and Shu-Hong Li (Toronto General Research 8. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33. Institute, Canada) for their valuable advice and editorial assistance with the manuscript. 9. Noren HN, Abdelmohsen K, Gorospe M, et al. microRNA expression patterns reveal differential expression of target genes with age. PLoS One. 2010;5: e10724. Funding 10. Zhang ZH, Li J, Liu BR, et al. MicroRNA-26 was decreased in rat cardiac This work was supported by grants from the National Natural Science hypertrophy model and may be a promising therapeutic target. J Foundation of China (81602334, 81401156), the Science and Technology Cardiovasc Pharmacol. 2013;62:312–9. Planning Project of Guangdong Province, China (2016A020215166, 11. Pan W, Zhong Y, Cheng C, et al. MiR-30-regulated autophagy mediates 2017A020215084), the Natural Science Foundation of Guangdong Province, angiotensin II-induced myocardial hypertrophy. PLoS One. 2013;8:e53950. China (2016A030310285), the Educational Commission of Guangdong Province, China (2015KQNCX125, 2016KTSCX113, 2016KQNCX130), and the 12. Dellago H, Preschitz-Kammerhofer B, Terlecki-Zaniewicz L, et al. High levels Bureau of Education of Guangzhou Municipality, China (1201581618). of oncomiR-21 contribute to the senescence-induced growth arrest in normal human cells and its knock-down increases the replicative lifespan. Aging Cell. 2013;12:446–58. Availability of data and materials 13. Li X, Xu F, Chang C, et al. Transcriptional regulation of miR-10a/b by TWIST- All data generated and/or analyzed during this study are included in this 1 in myelodysplastic syndromes. Haematologica. 2013;98:414–9. published article and its supplementary information files. 14. Bryant A, Palma CA, Jayaswal V, et al. miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression Authors’ contributions induces cell death. Mol Cancer. 2012;11:8. JD and ZZ contributed to the design of the study, analysis and interpretation of 15. Mohan N, Ai W, Chakrabarti M, Banik NL, Ray SK. KLF4 overexpression and the data, and wrote the manuscript. HH contributed to the analysis and apigenin treatment down regulated anti-apoptotic Bcl-2 proteins and interpretation of the data. PM and CC contributed to the design of the study matrix metalloproteinases to control growth of human malignant and the analysis and interpretation of the data. JL, WH, and CT collected the neuroblastoma SK-N-DZ and IMR-32 cells. Mol Oncol. 2013;7:464–74. in-vivo data and contributed to the analysis and interpretation of the data. CZ 16. Li Z, Zhao J, Li Q, et al. KLF4 promotes hydrogen-peroxide-induced contributed to the analysis and interpretation of the data. JL designed the study apoptosis of chronic myeloid leukemia cells involving the bcl-2/bax and wrote the manuscript. All authors read and approved the final manuscript. pathway. Cell Stress Chaperones. 2010;15:905–12. 17. Guan H, Xie L, Leithauser F, et al. KLF4 is a tumor suppressor in B-cell non- Ethics approval and consent to participate Hodgkin lymphoma and in classic Hodgkin lymphoma. Blood. 2010;116: This study was conducted in accordance with the Declaration of Helsinki 1469–78. and was approved by the Research Ethics Committee of The Second 18. Zhang L, Dong XW, Wang JN, et al. PEP-1-CAT-transduced mesenchymal Affiliated Hospital of Guangzhou Medical University (File no. 2014013). stem cells acquire an enhanced viability and promote ischemia-induced angiogenesis. PLoS One. 2012;7:e52537. Competing interests 19. Li SH, Sun Z, Brunt KR, et al. Reconstitution of aged bone marrow with The authors declare that they have no competing interests. young cells repopulates cardiac-resident bone marrow-derived progenitor cells and prevents cardiac dysfunction after a myocardial infarction. Eur Heart J. 2013;34:1157–67. Publisher’sNote 20. Wang S, Qu X, Zhao RC. Clinical applications of mesenchymal stem cells. J Springer Nature remains neutral with regard to jurisdictional claims in Hematol Oncol. 2012;5:19. published maps and institutional affiliations. 21. McGinley LM, McMahon J, Stocca A, et al. Mesenchymal stem cell survival in the infarcted heart is enhanced by lentivirus vector-mediated heat shock Author details protein 27 expression. Hum Gene Ther. 2013;24:840–51. Guangzhou Institute of Cardiovascular Disease, Department of Cardiology, 22. Wang J, Li Z, Zhang Y, et al. CX43 change in LPS preconditioning against The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou apoptosis of mesenchymal stem cells induced by hypoxia and serum 510260, China. Department of Oncology, The Second Affiliated Hospital of deprivation is associated with ERK signaling pathway. Mol Cell Biochem. Guangzhou Medical University, Guangzhou, China. Department of Intensive 2013;380:267–75. Care Unit, The Second Affiliated Hospital of Guangzhou Medical University, 23. Hou M, Liu J, Liu F, Liu K, Yu B. C1q tumor necrosis factor-related protein-3 Guangzhou, China. 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Autologous mesenchymal stem cells regulation of the human cumulus-oocyte complex. Hum Reprod. 2013;28: mobilize cKit+ and CD133+ bone marrow progenitor cells and improve 3038–49. regional function in hibernating myocardium. Circ Res. 2011;109:1044–54. 27. Chesler L, Goldenberg DD, Collins R, et al. Chemotherapy-induced apoptosis 3. Dimmeler S, Leri A. Aging and disease as modifiers of efficacy of cell in a transgenic model of neuroblastoma proceeds through p53 induction. therapy. Circ Res. 2008;102:1319–30. Neoplasia. 2008;10:1268–74. 4. Fan M, Chen W, Liu W, et al. The effect of age on the efficacy of human mesenchymal stem cell transplantation after a myocardial infarction. 28. Xiao GY, Cheng CC, Chiang YS, et al. Exosomal miR-10a derived from Rejuvenation Res. 2010;13:429–38. amniotic fluid stem cells preserves ovarian follicles after chemotherapy. Sci 5. Stolzing A, Jones E, McGonagle D, Scutt A. 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miR-10a rejuvenates aged human mesenchymal stem cells and improves heart function after myocardial infarction through KLF4

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Abstract

Background: Aging is one of the key factors that regulate the function of human bone marrow mesenchymal stem cells (hBM-MSCs) and related changes in microRNA (miRNA) expression. However, data reported on aging- related miRNA changes in hBM-MSCs are limited. Methods: We demonstrated previously that miR-10a is significantly decreased in aged hBM-MSCs and restoration of the miR-10a level attenuated cell senescence and increased the differentiation capacity of aged hBM-MSCs by repressing Krüpple-like factor 4 (KLF4). In the present study, miR-10a was overexpressed or KLF4 was downregulated in old hBM-MSCs by lentiviral transduction. The hypoxia-induced apoptosis, cell survival, and cell paracrine function of aged hBM-MSCs were investigated in vitro. In vivo, miR-10a-overexpressed or KLF4-downregulated old hBM- MSCs were implanted into infarcted mouse hearts after myocardial infarction (MI). The mouse cardiac function of cardiac angiogenesis was measured and cell survival of aged hBM-MSCs was investigated. Results: Through lentivirus-mediated upregulation of miR-10a and downregulation of KLF4 in aged hBM-MSCs in vitro, we revealed that miR-10a decreased hypoxia-induced cell apoptosis and increased cell survival of aged hBM- MSCs by repressing the KLF4–BAX/BCL2 pathway. In vivo, transplantation of miR-10a-overexpressed aged hBM- MSCs promoted implanted stem cell survival and improved cardiac function after MI. Mechanistic studies revealed that overexpression of miR-10a in aged hBM-MSCs activated Akt and stimulated the expression of angiogenic factors, thus increasing angiogenesis in ischemic mouse hearts. Conclusions: miR-10a rejuvenated aged hBM-MSCs which improved angiogenesis and cardiac function in injured mouse hearts. Keywords: Aging, MicroRNA, Rejuvenation, Myocardial infarction * Correspondence: gzlijiao@163.com Jun Dong and Zhenhui Zhang contributed equally to this work. Guangzhou Institute of Cardiovascular Disease, Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China Toronto General Research Institute, University Health Network, Toronto, Canada Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 2 of 16 Background cell apoptosis. In human malignant neuroblastoma Previous studies proved that bone marrow (BM) stem SK-N-DZ and IMR-32 cell lines, upregulation of KLF4 cells play an important role in improving heart function inhibited cell growth and increased cell apoptosis by ac- and delaying cardiac remodeling [1, 2] after ischemic tivating caspase-3 [15]. In leukemia cells, KLF4 upregu- damage through reducing fibrosis and increasing angio- lated Bax and downregulated Bcl-2 to induce cell genesis. However, preclinical animal research does not apoptosis, potentially through binding to the sites which necessarily translate into clinical results. Discrepancy in correspond to the promoters of Bcl-2 and Bax [16]. the effects of stem cell therapy [3, 4] may be due to the Furthermore, upregulation of KLF4 in the B-lymphoma age of the donor [5]. The age of stem cell donors might cell line induced cell apoptosis through activation of significantly impact the recipient’s endogenous responses BAK1 [17]. All of this evidence led us to postulate that [6]. Our group found that the proliferation and differen- miR-10a may mitigate cellular apoptosis of aged tiation ability of aged human bone marrow mesenchymal hBM-MSCs through its target gene KLF4. stem cells (hBM-MSCs) was decreased whereas cell sen- The efficacy of stem cell therapy for ischemic diseases escence was increased [7]. Effective methods to rejuven- was compromised by the poor survival of MSCs [18]. In ate aged hBM-MSCs to improve their regenerative the current study, we investigated whether upregulation capability may be required to maximize the beneficial ef- of miR-10a or downregulation of KLF4 can rejuvenate fects of stem cell therapy. aged hBM-MSCs and improve aged hBM-MSC survival Previous evidence has suggested that miRNAs regulate when transplanted into ischemic mouse hearts. In this cell growth, differentiation, replication, survival, and sen- regard, hBM-MSCs were harvested from young and old escence [8, 9]. We have reported that miR-26 attenuated patients undergoing cardiac surgery. Overexpression or myocardial hypertrophy by repressing expression of the inhibition of miR-10a or KLF4 in hBM-MSCs was GSK3b signaling pathway [10]. miR-30 decreased achieved through lentiviral transduction. The effects of autophagy-mediated angiotensin II-related myocardial miR-10a on hBM-MSC apoptosis, survival, and para- hypertrophy [11]. Furthermore, studies have shown that crine function were investigated in vitro. In vivo, the aging process may change the expression pattern of miR-10a-overexpressed hBM-MSCs were implanted into miRNAs. This aging-related change in miRNA expres- the border region of mouse hearts following myocardial sion may play an important role in the regulation of cel- infarction (MI), and cardiac function and related bio- lular function [12]. Our previous study found that logical changes were investigated. The molecular mecha- miR-196a, miR-486-5p, miR-664-star, and miR-378-star nisms of miR-10a on hBM-MSC function were further were significantly increased whereas miR-10a, miR-708, investigated. and miR-3197 were decreased in old hBM-MSCs. We also found that miR-10a increased differentiation and Methods decrease senescence in old hBM-MSCs through the tar- Human bone marrow mesenchymal stem cell isolation, get gene Krüpple-like factor 4 (KLF4) [7]. However, culture, and identification other studies have suggested that miR-10a may have This study was approved by the Research Ethics Com- additional effects other than regulating cell senescence mittee of The Second Affiliated Hospital of Guangzhou and differentiation. Indeed, some studies have shown Medical University. Human bone marrow (BM) was col- that miR-10a and KLF4 regulate cell apoptosis in other lected during cardiac valve surgery at The Second cell types [13, 14]. We speculated that miR10a may also Affiliated Hospital of Guangzhou Medical University. have additional effects related to cellular apoptosis and After anesthesia induction but before completion of the survival to regulate aged hBM-MSC function. sternotomy, 10 ml of sternal marrow was aspirated from The effect of miR-10a on cellular apoptosis has been the patients. Young human bone marrow mesenchymal reported recently. Li et al. [13] found that miR-10a and stem cells (hBM-MSCs) were obtained from patients miR-10b were significantly increased in CD34 marrow 18–30 years old, and old hBM-MSCs from patients 65– cells from 28 patients with myelodysplastic syndrome 80 years old. All patients were free of other pathological compared with healthy donors. They also found that conditions. downregulation of miR10a/10b in clonal cells interfered The hBM-MSCs were cultured as described previously with cell proliferation and enhanced cell apoptosis by [7]. Briefly, after centrifugation through a Ficoll gradient activating the NF-κB-dependent p53 pathway [13]. (1.077 g/ml density; GE Healthcare, Kretztechnik, Zipf, Another study has also shown that miR-10a inhibited Austria), cells were separated and the mononuclear cell KLF4 and RB1-inducible coiled-coil 1(RB1CC1) regu- fraction was plated. After 48 h, nonadherent cells were lated cell apoptosis in acute myeloid leukemia (AML) removed by changing the culture medium. The adherent [14]. On the other hand, KLF4, one of the miR-10a tar- cells were passaged when confluency reached 80%. get genes, has been reported to play a role in regulating hBM-MSCs were characterized by flow cytometry after Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 3 of 16 staining with antibodies against CD29, CD31, CD34, Briefly, C57BL/6 mice were intubated and ventilated CD44, CD45, and CD166 (MultiSciences Biotech Co., with 2% isoflurane. Through a left thoracotomy, the left Shanghai, China) as described previously. anterior descending coronary artery was ligated. Cardiac function was measured with echocardiography at differ- Viral vector construction and transduction ent time points before and after MI as indicated in Re- Lentiviral constructs for overexpression of miR-10a sults. At the study end, the scar area and thickness were (O-10a), KLF4 (O-KLF4), or inhibition of miR-10a and measured by planimetry. The infarct area was defined as KLF4 (O-anti10a and O-antiKLF4) in hBM-MSCs were the entire area of the left ventricle that contained a scar purchased from GenePharma as reported previously [7]. in myocardial sections stained with Masson’s trichrome. The Lenti-miR-10a sequence was TACCCTGTAGATCC Three days prior to MI and cell transplantation, mice GAATTTGTG. The Lenti-anti-10a sequence was CACA were given daily doses of cyclosporine A (5 mg/kg) to AATTCGGATCTACAGGGTA. The Lenti-anti-KLF4 se- induce the immunosuppression duration of the quence was GCCACCCACACTTGTGATTAC. The experiments. Lenti-control sequence was TTCTCCGAACGTGT CACGT. To construct LV-KLF4, the entire CDS region of Immunofluorescent staining KLF4 (NCBI reference sequence: NM_004235.4) was sub- Hearts were fixed in 2% paraformaldehyde (PFA) for cloned into the lentiviral vector. All of the lentiviral con- 24 h after being well perfused with PFA and were then structs expressed GFP. Viral solution was diluted with stored in 0.5 M sucrose at 4 °C overnight. Hearts were serum-free medium and polybrene (5 mg/ml) was added then embedded with OCT, and 5-μm-thick frozen sec- to the culture medium to transduce hBM-MSCs for 72 h. tions were prepared. Slides were incubated with one of the following primary antibodies: Alexa488-conjugated Cell survival and apoptosis evaluation anti-GFP (catalog number A21311, 1:400; Invitrogen) or Young (Y) and old hBM-MSCs (O) were cultured for 72 h anti-α-SMA (catalog number A-2547, 1:400; Sigma), at under hypoxia conditions (0.1% O ). Cell survival was de- room temperature for 2 h. Incubation with the respect- termined using the Cell Counting Kit-8 (CCK8; Dojindo, ive Alexa488 or Alexa568-conjugated secondary anti- Kumamoto, Japan) according to the manufacturer’sin- bodies (all 1:400; Invitrogen) or Isolectin B4 (catalog structions. In brief, cells were seeded into 96-well plates at number I21412, 1:100; ThermoFisher) was carried out at a density of 1 × 10 cells/well. After incubation, 5 μlof room temperature with light protection for 1 h. The nu- CCK-8 reagent was added to each well. The absorbance clei were identified with DAPI. was measured at 450 nm after 2 h of incubation at 37 °C. Terminal deoxynucleotidyl transferase dUTP nick end la- beling (TUNEL; Roche, Laval, QC, Canada) was carried out according to the manufacturer’s instructions. The number Western blotting of TUNEL cells in five randomly selected high-power For western blot analysis, 50 μg of lysate was fraction- fields per dish was determined and averaged with a Nikon ated through a 4% stacking and 10% running SDS-PAGE Eclipase Ti fluorescent microscope. Cell apoptosis was gel, and the fractionated proteins were transferred to a expressed as the percentage of total cells (DAPI ). PVDF membrane. Blots were blocked for 1 h at room temperature with blocking buffer. The antibodies (VEGF, Real-time reverse transcription-polymerase chain reaction SDF, phosphor (ser473)-AKT, and total AKT, all 1:1000) Total RNA was isolated with TRIzol reagent (Invitrogen, reacted with the blots overnight at 4 °C. After washing Grand Island, NY, USA) and cDNA was synthesized using (3 × 5 min in 1 × TBS–0.1% Tween 20), the blots were Moloney murine leukemia virus reverse transcriptase and incubated with horseradish peroxidase-conjugated sec- random primer. Real-time polymerase chain reaction was ondary antibody at 1:2000 dilution for 1 h at room conducted using SensiFAST SYBR Green PCR Master Mix temperature. Visualization was performed with en- (Bioline USA Inc., Taunton, MA, USA) with the following hanced chemiluminescence. For quantification, densi- parameters: 95 °C for 2 min, 95°C for 5 s, and 60 °C for tometry of the target bands was divided by the 30 s for 40 cycles. The oligonucleotide primer sequences corresponding densitometry of the GAPDH (catalog are presented in Additional file 1: Table S1. number mab374, 1:5000; Millipore) band using ImageJ software. To inhibit Akt phosphorylation in the Myocardial infarction and cardiac function measurement miR-10a-overexpressed old hBM-MSCs, 50 μM Akt In- The animal protocol was approved by Research Ethics hibitor VI (catalog number 124013; Merck) was used to Committee of The Second Affiliated Hospital of treat the cells for 72 h under hypoxia conditions follow- Guangzhou Medical University (2014013). Coronary oc- ing the manufacturer’s instructions. The inhibited ex- clusion was performed as described previously [19]. pression of P-AKT was conferred by western blotting. Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 4 of 16 Enzyme-linked immunosorbent assay file 2: Figure S1). The proapoptotic protein expression of hBM-MSCs in different groups were cultured in PUMA was also significantly higher whereas the antia- serum-free DMEM medium under hypoxia conditions poptotic protein expression of MCL1 was significantly for 72 h. The supernatant and cell lysate were collected lower in O hBM-MSCs compared with Y hBM-MSCs re- and the protein concentration was determined using a spectively (Fig. 1c). The ratio of BAX/BCL2 protein was Bio-Rad DC protein assay kit. The level of VEGF (cata- increased in O hBM-MSCs compared with Y log number DVE00; R&D Systems) and SDF (catalog hBM-MSCs (Fig. 1d). The protein expression of cleaved number DSA00; R&D Systems) were determined using caspase-3 and inhibitor of caspase-activated DNase an enzyme-linked immunosorbent assay (ELISA) follow- (ICAD) was also increased in O hBM-MSCs compared ing the manufacturer’s instructions. with Y hBM-MSCs (Fig. 1e). Furthermore, caspase-3 ac- tivity was significantly higher in O hBM-MSCs than in Y Caspase activity assay hBM-MSCs (Fig. 1f). The expression of miR-10a was A caspase fluorescent assay kit specific for caspase-3 significantly decreased in O hBM-MSCs compared with (Biovision, Mountain View, CA, USA) was used to de- Y hBM-MSCs (Fig. 1g). To the contrary, the expression tect caspase activation by measuring the cleavage of a of KLF4, which was one of the targets of miR-10a, was synthetic fluorescent substrate. In brief, cell lysates were significantly increased in O hBM-MSCs compared with prepared with the lysis buffer provided by the assay kit Y hBM-MSCs (Fig. 1h). All of these data implied the and centrifuged at 10,000 × g for 1 min, and the super- possible link between the downregulation of miR-10a natants were collected. With bovine serum albumin as and the increased O hBM-MSC apoptosis. the standard for protein content, equal amounts of pro- tein were reacted with the synthetic fluorescent sub- Upregulation of miR-10a in old hBM-MSCs decreased strates at 37 °C for 1.5 h, and absorbance at 405 nm was hypoxia-induced apoptosis and increased cell survival read on a microplate reader. The fold-increase in Next, to further test whether miR-10a was related to O caspase-3 activity versus control was determined. hBM-MSC apoptosis, miR-10a was overexpressed in O hBM-MSCs (Additional file 3: Figure S2A) and cellular Statistical analysis apoptosis was evaluated. The percentage of apoptotic All values are expressed as mean ± SD. Analyses were cells (TUNEL ) was decreased in miR-10a-upregulated performed using GraphPad InStat software (version 6). O hBM-MSCs (O-10a) compared with the control Student’s t test was used for two-group comparisons. vector-transduced O hBM-MSCs (O-c) that were cul- Comparisons of parameters among three or more groups tured for 72 h under hypoxia conditions (Fig. 2a). In were analyzed using one-way analysis of variance agreement, cell survival was increased in the O-10a (ANOVA) followed by Tukey for single-factor variables group compared with the O-c group (Fig. 2b). The proa- or two-way ANOVA for two-factor variables with re- poptotic mRNA expression of BAX and PUMA was de- peated measures over time, followed by Bonferroni crease in the O-10a group compared with the O-c group post-hoc tests. Differences were considered statistically (Additional file 4: Figure S3). On the contrary, the antia- significant at p < 0.05. poptotic mRNA expression of BCL2 and MCL1 was in- creased in the O-10a group compared with the O-c Results group (Additional file 4: Figure S3). The proapoptotic Cell apoptosis was increased in old hBM-MSCs under protein expression of PUMA was decreased whereas the hypoxia conditions antiapoptotic protein expression of MCL1 was increased Young (Y) and old (O) hBM-MSCs were cultured for in the O-10a group compared with the O-c group re- 72 h under hypoxia conditions, followed by comparison spectively (Fig. 2c). The ratio of BAX/BCL2 protein in of cell survival and apoptosis. The percentage of apop- the O-10a group was decreased compared with the O-c totic cells (TUNEL ) was significantly higher in the O group (Fig. 2d). The protein expression of cleaved group compared with the Y group of hBM-MSCs caspase-3 and ICAD was decreased in the O-10a group (Fig. 1a). In agreement, cell survival was decreased in O compared with the O-c group (Fig. 2e). Furthermore, hBM-MSCs compared with Y hBM-MSCs by CCK-8 caspase-3 activity was significantly lower in the O-10a assay (Fig. 1b). The proapoptotic mRNA expression of group compared with the O-c group (Fig. 2f). These BAX and PUMA was significantly higher in O findings suggested that restoring the miR-10a level re- hBM-MSCs compared with Y hBM-MSCs duced hypoxia-induced apoptosis in O hBM-MSCs. We (Additional file 2: Figure S1). On the contrary, the antia- also examined the effects of miR-10a on Y hBM-MSCs poptotic mRNA expression of BCL2 and MCL1 (BCL2 and found that cell apoptosis was decreased family apoptosis regulator) was significantly lower in O (Additional file 5: Figure S4A) and cell survival hBM-MSCs compared with Y hBM-MSCs (Additional (Additional file 5: Figure S4B) was increased in Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 5 of 16 a b cd fg h Fig. 1 Cell apoptosis increased in old hBM-MSCs under hypoxia conditions. Young (Y) and old (O) hBM-MSCs cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in Y and O hBM-MSCs. b Cell survival evaluated in Y and O hBM-MSCs c Protein expression of MCL1 and PUMA evaluated by western blot analysis in Y and O hBM-MSCs. d Ratio of Bax/BCL2 quantified in Y and O hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in Y and O hBM-MSCs. f Caspase-3 activity measured in Y and O hBM-MSCs. Expression of (g) miR-10a and (h) KLF4 compared in Y and O hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RQ relative quantity, RFU relative fluorescence units miR-10a-upregulated Y hBM-MSCs (Y-10a) compared conditions (Fig. 3a). In agreement, cell survival was in- with the control vector-transduced Y hBM-MSCs (Y-c). creased in the O-antiKLF4 group compared with the The effects of miR-10a on Y hBM-MSC apoptosis and O-c group (Fig. 3b). The proapoptotic mRNA expression survival followed the same trend as in O hBM-MSCs of BAX and PUMA was decreased in the O-antiKLF4 but at a much lower magnitude. group compared with the O-c group (Additional file 6: Figure S5). On the contrary, the antiapoptotic mRNA Downregulation of KLF4 expression in old hBM-MSCs expression of BCL2 and MCL1 was increased in the decreased hypoxia-induced apoptosis and increased cell O-antiKLF4 group compared with the O-c group survival (Additional file 6: Figure S5). The proapoptotic protein KLF4 is one of the targets of miR-10a and was upregu- expression of PUMA was decreased whereas the antia- lated in O hBM-MSCs. To determine whether KLF4 was poptotic protein expression of MCL1 was increased in involved in miR-10a-mediated cellular apoptosis, we ef- the O-antiKLF4 group compared with the O-c group re- fectively inhibited KLF4 in O hBM-MSCs (Additional spectively (Fig. 3c). The ratio of BAX/BCL2 protein in file 3: Figure S2B) and evaluated cell apoptosis. The per- the O-antiKLF4 group was decreased when compared centage of apoptotic cells (TUNEL ) was decreased in with the O-c group (Fig. 3d). Moreover, the protein ex- the KLF4-inhibited O hBM-MSCs (O-antiKLF4) com- pression of cleaved caspase-3 and ICAD was decreased pared with the control vector-transduced O hBM-MSCs in the O-antiKLF4 group compared with the O-c group (O-c) that were cultured for 72 h under hypoxia (Fig. 3e). Caspase-3 activity was significantly lower in the Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 6 of 16 a b cd Fig. 2 Overexpression of miR-10a in old hBM-MSCs decreased hypoxia-induced apoptosis and increased cell survival. miR-10a transduced into old hBM-MSCs by lentiviral vector (O-10a). Control vector-transduced old hBM-MSCs (O-c) served as control. Cells cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c and O-10a hBM-MSCs. b Cell survival evaluated in O-c and O-10a hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-c and O-10a hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-c and O-10a hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase- activated DNase (ICAD) assayed in O-c and O-10a hBM-MSCs. f Caspase-3 activity measured in O-c and O-10a hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units O-antiKLF4 group compared with the O-c group O-KLF4 groups compared with the O-c group (Fig. 4b). (Fig. 3f). The proapoptotic mRNA expression of BAX and PUMA was significantly higher in the O-anti10a and O-KLF4 Downregulation of miR-10a or overexpression of KLF4 in groups compared with the O-c group (Additional file 7: old hBM-MSCs increased hypoxia-induced apoptosis and Figure S6). On the contrary, the antiapoptotic mRNA ex- decreased cell survival pression of BCL2 and MCL1was significantly lower in the All of this evidence suggested that miR-10a, through sup- O-anti10a and O-KLF4 groups compared with the O-c pression of KLF4, may rescue O hBM-MSCs from group (Additional file 7: Figure S6). The proapoptotic pro- hypoxia-induced apoptosis. To test whether the reverse tein expression of PUMA was increased whereas the anti- would post a detrimental effect, miR-10a was effectively apoptotic protein expression of MCL1 was decreased in inhibited in O hBM-MSCs (Additional file 3: Figure S2C) the O-anti10a and O-KLF4 groups compared with the or KLF4 was overexpressed in O hBM-MSCs (Additional O-c group respectively (Fig. 4c). The ratio of BAX/ BCL2 file 3: Figure S2D). The percentage of apoptotic cells protein in the O-anti10a and O-KLF4 groups was in- (TUNEL ) was significantly higher in miR-10a-inhibited creased when compared with the O-c group (Fig. 4d). The old hBM-MSCs (O-anti10a) and KLF4-overexpressed old protein expression of cleaved caspase-3 and ICAD was in- hBM-MSCs (O-KLF4) compared with the control creased in the O-anti10a and O-KLF4 groups compared vector-transduced old hBM-MSCs (O-c) that were cul- with the O-c group (Fig. 4e). Caspase-3 activity was sig- tured for 72 h under hypoxia conditions (Fig. 4a). In agree- nificantly higher in the O-anti10a and O-KLF4 groups ment, cell survival was decreased in the O-anti10a and compared with the O-c group (Fig. 4f). Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 7 of 16 a b cd Fig. 3 Downregulation of KLF4 in old hBM-MSCs decreased hypoxia-induced apoptosis and increased cell survival. KLF4 was effectively inhibited in old hBM-MSCs by lentiviral vector (O-antiKLF4). Control vector-transduced old hBM-MSCs (O-c) served as control. Cells cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c and O-antiKLF4 hBM-MSCs. b Cell survival evaluated in O-c and O-antiKLF4 hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-c and O-antiKLF4 hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-c and O-antiKLF4 hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in O-c and O-antiKLF4 hBM-MSCs. f Caspase-3 activity measured in O-c and O-antiKLF4 hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units The antiapoptotic effect of miR-10a is attenuated by the significantly higher in the O-10a-KLF4 group com- restoration of KLF4 pared with O-10a hBM-MSCs. On the contrary, the To further confirm that KLF4 is indeed the direct antiapoptotic mRNA expression of BCL2 and MCL1 target of miR-10a in mediating hypoxia-induced O was significantly lower in the O-10a-KLF4 group hBM-MSC apoptosis, KLF4 expression was restored compared with O-10a hBM-MSCs (Additional file 8: by a rescue experiment (Additional file 3:Figure Figure S7). The proapoptotic protein expression of S2E). For this purpose, miR-10a-overexpressed O PUMA was increased whereas the antiapoptotic pro- hBM-MSCs were transduced with a lentivirus which tein expression of MCL1 was decreased in the carried the KLF4 vector and the restoration of KLF4 O-10a-KLF4 group compared with the O-10a group expression was confirmed (O-10a-KLF4). After restor- respectively (Fig. 5c). The ratio of BAX/ BCL2 protein ing the expression of KLF4, miR10a lost its antiapop- was increased in the O-10a-KLF4 group compared totic effect on O hBM-MSCs. Cell apoptosis was with the O-10a group (Fig. 5d). The protein expres- increased in the O-10a-KLF4 group compared with sion of active caspase-3 and ICAD was increased in the O-10a group (Fig. 5a). In agreement, cell survival the O-10a-KLF4 group compared with O-10a was decreased in the O-10a-KLF4 group compared hBM-MSCs (Fig. 5e). Caspase-3 activity was also in- with the O-10a group (Fig. 5b). The proapoptotic creased in the O-10a-KLF4 group compared O-10a mRNA expression of BAX and PUMA was hBM-MSCs (Fig. 5f). Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 8 of 16 a b Fig. 4 Downregulation of miR-10a or overexpression of KLF4 in old hBM-MSCs increased hypoxia-induced apoptosis and decreased cell survival. Lentiviral vectors used to transduce old hBM-MSCs to downregulate miR-10a expression (O-anti10a) or overexpress KLF4 (O-KLF4). Control vector-transduced old hBM-MSCs (O-c) served as control. Cells cultured for 72 h under hypoxia condition. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c, O-anti10a, and O-KLF4 hBM-MSCs. b Cell survival evaluated in O-c, O-anti10a, and O-KLF4 hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-c, O-anti10a, and O-KLF4 hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-c, O-anti10a, and O-KLF4 hBM-MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in O-c, O-anti10a, and O-KLF4 hBM-MSCs. f Caspase-3 activity measured in O, O-anti10a, and O-KLF4 hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units Implantation of miR-10a-overexpressed or KLF4- MI in the four experimental groups. After MI, there downregulated old hBM-MSCs into infarcted mouse was a significant decrease in fractional shortening (FS; hearts improved cardiac function after MI Fig. 6b) and ejection fraction (EF; Fig. 6c) and an in- To evaluate whether modifying miR-10a or KLF4 crease in left ventricular internal end-systolic dimen- levels in O hBM-MSCs can maximize the beneficial sion (LVIDs; Fig. 6d) and left ventricular internal effects of stem cell therapy, miR-10a-overexpressed or end-diastolic dimension (LVIDd; Fig. 6e)inthe O-c KLF4-downregulated old hBM-MSCs were implanted group compared with the Y-c group. However, there into infarcted mouse hearts. Cardiac function was de- was an improvement in all of these parameters in the termined by echocardiography in mice that received O-10a and O-antiKLF4 groups when compared with implantation of control medium (Media), control the O-c group (Fig. 6a–e). vector-transduced young hBM-MSCs (Y-c), control Similarly, the infarct size at 28 days post MI was sig- vector-transduced old hBM-MSCs (O-c), nificantly larger in the O-c group than the Y-c group miR-10a-overexpressed old hBM-MSCs (O-10a), or (Fig. 6f, g). On the other hand, scar thickness was signifi- KLF4-inhibited old hBM-MSCs (O-antiKLF4) into the cantly lower in the O-c group than the Y-c group border region immediately following MI. Cardiac (Fig. 6h). However, the infarct size was smaller and the function was evaluated at baseline (before MI) and at scar thickness was greater in the O-10a and O-antiKLF4 14 and 28 days after MI. Fig. 6a shows representative groups when compared with the O-c group (Fig. 6f–h). M-mode echocardiographic images taken 28 days post More viable myocardium was found by Trichrome’s Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 9 of 16 a b cd ef Fig. 5 Antiapoptotic effect of miR-10a attenuated by restoration of KLF4. Lentivirus which carries KLF4 vector used to infect miR-10a-upregulated old hBM-MSCs (O-10a) to restore KLF4 expression (O-10a-KLF4). miR-10a-upregulated old hBM-MSCs (O-10a) also infected by the control lentivirus (O-10a-c). Cells cultured for 72 h under hypoxia conditions. a Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-10a and O-10a-KLF4 hBM-MSCs. b Cell survival evaluated in O-10a and O-10a-KLF4 hBM-MSCs. c Protein expression of MCL1 and PUMA evaluated by western blot analysis in O-10a and O-10a-KLF4 hBM-MSCs. d Ratio of Bax/BCL2 quantified in O-10a and O-10a-KLF4 hBM- MSCs. e Protein expression of cleaved caspase-3 and inhibitor of caspase-activated DNase (ICAD) assayed in O-10a and O-10a-KLF4 hBM-MSCs. f Caspase-3 activity evaluated in O-10a and O-10a-KLF4 hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05. DAPI 4′,6-diamidino-2-phenylindole, KLF4 Krüpple-like factor 4, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, RFU relative fluorescence units staining in the Y-c group than in the O-c group. How- least apoptosis in the group receiving Y hBM-MSCs ever, there was more viable myocardium in the O-10a (Y-c). However, overexpression of miR-10a (O-10a) or and O-antiKLF4 groups when compared with the O-c inhibition of KLF4 expression (O-antiKLF4) increased group (Fig. 6i, j). All of these data clearly demonstrated cell survival but decreased cell apoptosis when com- that modifying the miR-10a or KLF4 level in O pared with the group receiving O hBM-MSCs (O-c). To hBM-MSCs enhanced the beneficial effects of stem cell seek possible downstream mediators, western blot ana- therapy and further improved cardiac function. lyses were performed to examine the activation of AKT. As shown in Fig. 7e, although total AKT expression was miR-10a upregulation or KLF4 downregulation increased similar for all the groups, phospho-Akt expression was old hBM-MSC survival and decreased apoptosis by highest in the Y-c group and was increased in the O-10a activating AKT and O-antiKLF4 groups compared with the O-c group. To evaluate the antiapoptotic effect of miR-10a in vivo, The same pattern was found in the in-vitro cultured the survival of implanted cells was detected by hBM-MSCs. This showed that total AKT expression was lentiviral-mediated GFP expression in the border region similar for hBM-MSCs from all of the conditions while of the mouse hearts at 3 days (Fig. 7a, b) and 7 days phospho-AKT was highest in Y hBM-MSCs and was in- (Fig. 7c, d) post MI. Cell apoptosis was confirmed by creased in the O-10a and O-antiKLF4 groups compared TUNEL staining. There was highest cell survival and with O hBM-MSCs (Fig. 7f). To build up the direct link Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 10 of 16 b c f g i Fig. 6 Implantation of miR-10a-overexpressed or KLF4-downregulated old hBM-MSCs into ischemic area of mouse hearts improved cardiac function after MI. miR-10a-overexpressed or KLF4-downregulated old hBM-MSCs (3 × 10 cells/mouse) implanted into infarcted mouse hearts. Cardiac function determined by echocardiography in mice that received implantation of control medium (Media), control vector-transduced young hBM-MSCs (Y-c), control vector-transduced old hBM-MSCs (O-c), miR-10a-overexpressed old hBM-MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) into border region immediately following MI. Cardiac function measured by echocardiography at baseline (before MI, 0 day), 14 and 28 days after MI in all groups. a Representative M-mode echocardiographic images. b Fractional shortening. c Ejection fraction. d Left ventricular internal end-systolic dimension (LVIDs). e Left ventricular internal end-diastolic dimension (LVIDd). f Each panel shows one representative whole sectioned heart (from base to apex) at 28 days after MI from five individual groups (Media, Y-c, O-c, O-10a, or O-antiKLF4) to show scar areas (arrows) (g) and scar size thickness (h). i Representative images of Masson Trichrome’s staining at 28 days after MI. j Viable myocardium (identified as red with Trichrome’s staining) in ischemia zone quantified and expressed as percentage of total infarct area. n = 6/group. Mean ± SD. *P < 0.05, Y-c vs other groups; #P < 0.05, O-c vs other groups. LV left ventricle, MI myocardial infarction between the activation of Akt and increased cell survival, whereas inactivation of Akt reversed this effect. Collect- we used an inhibitor (Akt Inhibitor VI) to inhibit the ively, these data revealed that miR-10a, through activation phosphorylation (activation) of Akt in the of AKT, increased hBM-MSC survival, thus improving miR-10a-overexpressed old hBM-MSCs (O-10a-P-AKT cardiac function. Inh; Additional file 9: Figure S8A). After culture for 72 h under hypoxia conditions, apoptosis was detected with miR-10a-overexpressed or KLF4-downregulated hBM- the TUNEL assay (Additional file 9: Figure S8B) and cell MSCs increased angiogenesis in infarcted mouse hearts survival was detected by CCK-8 assay in control To determine whether angiogenesis is also affected by vector-transduced (O-c), miR-10a-overexpressed (O-10a), regulating the miR-10a or KLF4 level in O hBM-MSCs, and O-10a-P-AKT Inh old hBM-MSCs. We found that capillary and arteriole densities were quantified by iso- cell apoptosis was increased and cell survival was de- lectin stain (Fig. 8a, b) and α-smooth muscle actin creased in the O-10a-P-AKT Inh group compared to the (α-SMA) stain (Fig. 8c, d) respectively in all four experi- O-10a group. We believe these results showed that mental groups at 3 and 7 days post MI. More capillaries miR-10a, through activating Akt, increased cell survival and arteriole were formed in the group receiving Y Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 11 of 16 a b cd ef Fig. 7 miR-10a overexpression or KLF4 downregulation increased old hBM-MSC survival and decreased apoptosis by activating AKT. miR-10a- overexpressed or KLF4-downregulated old hBM-MSCs (3 × 10 cells/mouse) implanted into infarcted mouse hearts. Cell survival and biochemical changes determined in mice that received implantation of control vector-transduced young hBM-MSCs (Y-c), control vector-transduced old hBM- MSCs (O-c), miR-10a-overexpressed old hBM-MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) into border region immediately following MI. Survival of implanted cells detected by green fluorescent protein (GFP) expression carried by lentiviral vector in border region of infarcted mouse hearts and cell apoptosis assayed by TUNEL staining at 3 days (a, b)and 7days (c, d)post MI. e Expression of total AKT and phospho-(ser473)-AKT detected in border region of infarcted mouse hearts tissue. f Expression of phosphor-(ser473)-AKT also detected in hBM-MSCs. n = 6/group. Mean ± SD. *P <0.05, Y-c vs other groups; #P < 0.05, O-c vs other groups. DAPI 4′,6-diamidino-2-phenylindole, hBM-MSC human mesenchymal stem cell, KLF4 Krüpple-like factor 4, MI myocardial infarction, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling hBM-MSCs (Y-c) compared to the group receiving O and SDF mRNA (Additional file 10: Figure S9A), and hBM-MSCs (O-c). However, overexpression of miR-10a protein (Fig. 8e, f) was decreased in the heart tissue (O-10a) or inhibition of KLF4 (O-antiKLF4) increased from the border region of mice in the O hBM-MSC capillary and arteriole formation when compared with group (O-c) compared to the Y hBM-MSC group (Y-c). the group receiving O hBM-MSC implantation only. However, overexpression of miR-10a (O-10a) or inhib- Furthermore, the expression of angiogenic factors, VEGF ition of KLF4 e (O-antiKLF4) increased VEGF and SDF Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 12 of 16 a b ef gh Fig. 8 miR-10a-upregulated or KLF4-downregulated hBM-MSCs increased mice heart angiogenesis after MI. miR-10a-overexpressed or KLF4- downregulated old hBM-MSCs (3 × 10 cells/mouse) implanted into infarcted mouse hearts. Angiogenesis and expression of angiogenic factors determined in mice that received implantation of control vector-transduced young hBM-MSCs (Y-c), control vector-transduced old hBM-MSCs (O-c), miR-10a-overexpressed old hBM-MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) into border region immediately following MI. Capillary and arteriole densities quantified by isolectin stain (a, b)and α-smooth muscle actin (α-SMA) stain respectively at 3 and 7 days (c, d)postMI. e, f VEGF and SDF protein expression evaluated by western blot analysis from border region of infarcted mouse hearts implanted with Y-c, O-c, O-10a, and O-antiKLF4 hBM-MSCs respectively. g, h VEGF and SDF protein expression evaluated by western blot analysis in Y-c, O-c, O-10a, and O-antiKLF4 hBM-MSCs. i VEGF and SDF protein in cell culture supernatant of Y-c, O-c, O-10a, and O-antiKLF4 hBM-MSCs assayed by ELISA. n = 6/group. Mean ± SD. *P <0.05, Y-c vs other groups; #P < 0.05, O-c vs other groups. DAPI 4′,6-diamidino-2-phenylindole, hBM-MSC human mesenchymal stem cell, KLF4 Krüpple-like factor 4, MI myocardial infarction, SDF stromal cell-derived factor, VEGF vascular endothelial growth factor mRNA and protein expression when compared with KLF4 (O-antiKLF4) increased VEGF and SDF mRNA the O-c group (Fig. 8e, f). Thesametrend wasfound (Additional file 10: Figure S9B) and protein expres- in the in-vitro cultured hBM-MSCs. The VEGF and sion compared with that in O hBM-MSCs (Fig. 8g, SDF mRNA (Additional file 10: Figure S9B) and protein h). The secretion of VEGF and SDF in the cell cul- (Fig. 8g, h) expression was decreased in O hBM-MSCs ture supernatant was also increased in the O-10a and (O-c) compared with Y hBM-MSCs (Y-c). However, O-antiKLF4 groups compared with O hBM-MSCs overexpression of miR-10a (O-10a) or inhibition of (Fig. 8i). Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 13 of 16 Discussion [14]. miR-10a is also reported to regulate cell apop- The present study demonstrated that restoring the tosis in the human cumulus–oocytes complex [26]. In miR-10a level in O hBM-MSCs increased cell survival addition, Bim, the proapoptotic factor, is directly tar- and decreased apoptosis under in-vitro hypoxia and geted by miR-10a, resulting in repressing Casp9 which in-vivo ischemic conditions by repressing the expression is a crucial factor in the apoptotic pathway [27]. miR-10a of KLF4. Accordingly, restoration of the miR-10a level in delivered by exosomes sustains the number of O hBM-MSCs increased the antiapoptotic-related pro- chemotherapy-damaged granulosa cells and reduces the tein expression and decreased the proapoptosis-related number of chemotherapy-damaged apoptotic granulosa protein expression. Furthermore, inhibition of miR-10a cells following nitrogen mustard treatment for 24–48 h target gene KLF4 also increased both cell survival and [28]. All of these findings support the notion that miR-10a the antiapoptotic-related protein expression while it de- is a prosurvival factor, which counteracts the apoptotic creased cellular apoptosis and the proapoptosis-related signals. Consistent with previous data, we also found that protein expression. The molecular mechanism study re- upregulation of miR-10a in aged hBM-MSCs decreased vealed that transplantation of miR-10a-overexpressed or hypoxia-induced apoptosis and increased cell survival. To KLF4-downregulated O hBM-MSCs activated AKT in the contrary, downregulation of miR-10a in aged infarcted mouse hearts, which led to increased survival hBM-MSCs increased hypoxia-induced apoptosis and of implanted cells. Furthermore, transplantation of decreased cell survival. Implantation of miR-10a or anti-KLF4-pretreated O hBM-MSCs in- miR-10a-overexpressed old hBM-MSCs into the ischemic creased the expression and secretion of angiogenic fac- area of mouse hearts improved cell survival and cardiac tors VEGF and SDF, which increased angiogenesis in function after MI. infarcted mouse hearts. All of these effects of miR-10a In our previous paper, using a luciferase reporter assay, ultimately led to enhanced efficacy of stem cell therapy we reported that KLF4 is a direct target of miR-10a. and the improvement of cardiac function after MI. Also, miR-10a decreased cell senescence in aged BM-MSCs are an optimal cell type for therapeutic ap- hBM-MSCs by repressing KLF4 [7]. KLF4 can function proaches for various pathological conditions including as both a repressor and an activator of transcription fac- cardiovascular disease since the cells can be easily iso- tors related to cell cycle regulation, apoptosis, and differ- lated and expanded in vitro [20]. However, the beneficial entiation. The expression of KLF4 can be increased by effects of BM-MSC therapy are restricted by the low sur- DNA damage, serum deprivation, and contact inhibition vival rate and decreased proliferative capacity of the aged [29]. Recent research suggests KLF4 as a proapoptotic cells after cell transplantation [21]. The ischemic envir- factor. A combination of KLF4 plasmid and apigenin onment of the infarcted heart triggers a strong inflam- treatment increases apoptosis in the human malignant matory and oxidative stress reaction and overproduction neuroblastoma SK-N-DZ and IMR-32 cell lines com- of factors related to apoptosis, which all lead to in- pared with control vector or single treatment [15]. After creased BM-MSC apoptosis and decreased cell survival a full-length complementary DNA or an antisense oligo- [22]. Various attempts have been made to improve nucleotide of KLF4 was transfected into a human im- BM-MSC survival under hypoxic and ischemic conditions mortalized myelogenous leukemia line (K562 cells), cell [23]. Here, we have shown that restoring miR-10a in aged growth was decreased and cell apoptosis was increased hBM-MSCs increased cell survival and decreased apop- [16]. Epigenetic inhibition of KLF4 in B-cell lymphomas tosis when transplanted into the ischemic mouse hearts. and particularly in classic Hodgkin lymphoma cases in- MicroRNAs play an important role in cell prolifera- creases lymphoma survival and decreases apoptosis [17]. tion,differentiation,and apoptosis[8]. Upregulation Research in resveratrol has found that KLF4 and early of specific miRNAs increased BM-MSC survival, mak- growth response-1 expression was increased by resvera- ing them more effective to repair the infarct damage trol, resulting in transcription factor 3 activation and cel- [24, 25]. miR-10a has also been reported as a prosur- lular apoptosis [30]. KLF4 expression is increased in vival factor. Previous studies have found that expres- murine astrocytes when exposed to X-ray radiation, sion of miR10a/10b was controlled by TWIST-1 and resulting in more double-strand DNA breaking, and at via the NF-κB and P53 axis, to control last cell apoptosis [31]. The apoptosis rate of SK-BR-3 TNF-α-induced (and stroma-dependent) apoptosis in breast cancer cells is increased and cell tumorigenicity is clonal myeloid cells. Therefore, the TWIST-1/miR10/ decreased by upregulation of KLF4 [32]. However, the p53 axis can serve as a potential new target for thera- role of KLF4 on hBM-MSC apoptosis still remains un- peutic interventions in advanced myelodysplastic syn- clear. In this study, we found that downregulation of dromes [13]. On the other hand, knockdown of KLF4 in aged hBM-MSCs decreased hypoxia-induced miR-10a in the NPM1 mutated cell line OCI-AML3 apoptosis and increased cell survival. To the contrary, decreased cellular survival and clonogenic growth upregulation of KLF4 in aged hBM-MSCs increased Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 14 of 16 hypoxia-induced apoptosis and decreased cell survival. was significantly lower in O-antiKLF4 than in control vector-transduced O Accordingly, upregulation of KLF4 resulted in decreased hBM-MSCs (B). Lentiviral vector carrying anti-miR-10a sequence used to transduce old hBM-MSCs (O-anti10a). miR-10a expression was significantly expression of the antiapoptotic proteins BCL2 and lower in O-anti10a than in control vector-transduced O hBM-MSCs (C). MCL1 but increased the expression of proapoptotic pro- Lentiviral vector carrying KLF4 sequence used to transduce old hBM- teins BAX and PUMA, and caused activation of caspase-3, MSCs (O-KLF4). KLF4 expression was significantly higher in O-KLF4 than in control vector-transduced O hBM-MSCs (D). Lentivirus which carries KLF4 leading to completion of the apoptotic machinery. The im- vector used to infect miR-10a-upregulated old hBM-MSCs (O-10a) to plantation of KLF4-downregulated old hBM-MSCs into restore KLF4 expression (O-10a-KLF4). miR-10a-upregulated old the ischemic area of mouse hearts improved cell survival hBM-MSCs (O-10a) also infected by the control lentivirus (O-10a-c). KLF4 expression restored in O-10a-KLF4 compared to O-10a-c hBM-MSCs (E). and cardiac function after MI. We believed that KLF4 n = 5/group. Mean ± SD. P < 0.05 O-c vs O-10a, O-anti-KLF4, O-anti-10a, plays a proapoptotic role in aged hBM-MSCs. # and O-KLF4; P < 0.05 O-10a-KLF4 vs O-10a-c and O-10a (PDF 41 kb) AKT is a key factor involved in cell survival, prolifera- Additional file 4: Figure S3. Overexpression of miR-10a in old tion, and metabolism [33]. Our data show that miR-10a hBM-MSCs decreased apoptotic gene expression. Quantification of mRNA expression of BAX and PUMA (proapoptotic), BCL2 and MCL1 overexpression or KLF4 downregulation activated AKT (antiapoptotic) in O and O-10a hBM-MSCs after culture for 72 h under both in aged hBM-MSCs and in ischemic mouse hearts. hypoxia conditions. n = 6/group. Mean ± SD. *P < 0.05 (PDF 37 kb) We believe the antiapoptotic effect of miR-10a may be Additional file 5: Figure S4. Overexpression of miR-10a in both young mediated through activation of AKT. In addition, we and old hBM-MSCs decreased hypoxia-induced apoptosis and increased cell survival. miR-10a transduced into young (Y-10a) and old (O-10a) found that miR-10a overexpression or KLF4 downregu- hBM-MSCs by lentiviral vector. Control vector-transduced young lation increased VEGF and SDF expression and secre- hBM-MSCs (Y-c) and old hBM-MSCs (O-c) served as controls. Cells tion. VEGF is a key factor for angiogenesis [34]. It has cultured for 72 h under hypoxia conditions. (A) Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in Y-c, been reported that VEGF inhibits posthypoxic MSC Y-10a, O-c, and O-10ahBM-MSCs. (B) Cell survival evaluated in Y-c, Y-10a, death and increases MSC survival and regeneration in O-c, and O-10a hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05, Y-c vs ischemic conditions [35, 36]. SDF also confers increased Y-10a; #P < 0.05, O-c vs O-10a (PDF 94 kb) vasculogenesis and angiogenesis [37]. Our data sug- Additional file 6: Figure S5. Downregulation of KLF4 in old hBM-MSCs decreased apoptotic gene expression. Quantification of mRNA expression gested that miR-10a increased the survival of aged of BAX and PUMA (proapoptotic), BCL2 and MCL1 (antiapoptotic) in O hBM-MSCs which in turn secreted more VEGF and SDF and O-antiKLF4 hBM-MSCs after culture for 72 h under hypoxia to stimulate angiogenesis in the infarcted hearts. Im- conditions. n = 6/group. Mean ± SD. *P < 0.05 (PDF 37 kb) proved cell survival and enhanced angiogenesis acted Additional file 7: Figure S6. Downregulation of miR-10a or overexpression of KLF4 in old hBM-MSCs increased apoptotic gene synergistically to preserve heart function after MI. expression. Quantification of mRNA expression of BAX and PUMA (pr-apoptotic), BCL2 and MCL1 (antiapoptotic) in O, O-anti10a and O-KLF4 hBM-MSCs after culture for 72 h under hypoxia conditions. Conclusions n = 6/group. Mean ± SD. *P < 0.05 (PDF 38 kb) Our study demonstrated that miR-10a decreased aged Additional file 8: Figure S7. Antiapoptotic effect of miR-10a attenuated hBM-MSC apoptosis and increased cell survival by restoration of KLF4 quantified by RT-qPCR. Quantification of mRNA through suppression of KLF4. Transplantation of expression of BAX and PUMA (proapoptotic), BCL2 and MCL1 (antiapoptotic) in O-10a-c, O-10a-KLF4, and O-c hBM-MSCs after culture for 72 h under miR-10a-overexpressed or KLF4-downregulated old hypoxia conditions. n =6/group. Mean ±SD. *P <0.05 (PDF 57 kb) hBM-MSCs activated AKT and stimulated angiogen- Additional file 9: Figure S8. miR-10a overexpression increased old hBM- esis in the ischemic hearts, thereby improving cardiac MSC survival and decreased apoptosis by activating AKT. Expression of Akt function. miR-10a and KLF4-modified stem cells phosphorylation in miR-10a-overexpressed old hBM-MSCs (O-10a) inhibited by Akt Inhibitor VI during 72-h culture under hypoxia conditions. (A) could be a potent vehicle to combine cell and gene Expression of phosphor-(ser473)-AKT detected in O-c, O-10a, and Akt therapies to improve heart function after injury. Inhibitor VI added O-10a hBM-MSCs (O-10a-P-AKT Inh). (B) Cell apoptosis assayed by TUNEL staining. Percentage of apoptotic cells (TUNEL ) quantified in O-c, O-10a, and O-10a-P-AKT Inh hBM-MSCs. (C) Cell survival Additional files evaluated in O-c, O-10a, and O-10a-P-AKT Inh hBM-MSCs. n =5/group. Mean ± SD. P < 0.05, O-10a-P-AKT Inh vs O-10a (PDF 184 kb) Additional file 1: Table S1. qRT-PCR primer and miRNA RT primer Additional file 10: Figure S9. Expression of VEGF and SDF mRNA in sequences (DOCX 19 kb) mouse hearts after MI and hBM-MSCs. (A) Expression of VEGF and SDF Additional file 2: Figure S1. Proapoptotic and antiapoptotic gene mRNA determined in border region of infarcted mouse hearts that received implantation of control vector-transduced young hBM-MSCs (Y-c), control expression in old hBM-MSCs under hypoxia conditions. Quantification of mRNA expression of BAX and PUMA (proapoptotic), BCL2 and MCL1 vector-transduced old hBM-MSCs (O-c), miR-10a-overexpressed old hBM- (antiapoptotic) in Y and O hBM-MSCs. n = 6/group. Mean ± SD. *P < 0.05 MSCs (O-10a), or KLF4-inhibited old hBM-MSCs (O-antiKLF4) following MI. (PDF 36 kb) (B) Expression of VEGF and SDF mRNA determined by RT-qPCR in Y-c, O-c, O-10a, or O-antiKLF4 hBM-MSCs after hypoxia for 72 h. n =6/group. Mean ± Additional file 3: Figure S2. Expression of miR-10a and KLF4 in old SD. *P < 0.05, Y-c vs other groups; P < 0.05, O-c vs other groups (PDF 39 kb) hBM-MSCs regulated by lentiviral vector. Lentiviral vector carrying miR- 10a sequence used to transduce old hBM-MSCs (O-10a) and control vector-transduced old hBM-MSCs (O-c) served as control. miR-10a Abbreviations expression was significantly higher in O-10a than in control vector- BM: Bone marrow; CCK8: Cell Counting Kit-8; EF: Ejection fraction; transduced (O) hBM-MSCs (A). Lentiviral vector carrying KLF4 siRNA FS: Fractional shortening; hBM-MSC: Human mesenchymal stem cell; sequence used to transduce old hBM-MSCs (O-anti-KLF4). KLF4 expression KLF4: Krüpple-like factor 4; LVIDd: Left ventricular internal end-diastolic Dong et al. Stem Cell Research & Therapy (2018) 9:151 Page 15 of 16 dimension; LVIDs: Left ventricular internal end-systolic dimension; 6. Zhuo Y, Li SH, Chen MS, et al. Aging impairs the angiogenic response to MI: Myocardial infarction; SMA: Smooth muscle actin; TUNEL: Terminal ischemic injury and the activity of implanted cells: combined consequences deoxynucleotidyl transferase dUTP nick end labeling for cell therapy in older recipients. J Thorac Cardiovasc Surg. 2010;139:1286–94. 7. Li J, Dong J, Zhang ZH, et al. miR-10a restores human mesenchymal stem cell differentiation by repressing KLF4. J Cell Physiol. 2013;228:2324–36. Acknowledgements The authors thank Dr Ren-Ke Li and Shu-Hong Li (Toronto General Research 8. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33. Institute, Canada) for their valuable advice and editorial assistance with the manuscript. 9. Noren HN, Abdelmohsen K, Gorospe M, et al. microRNA expression patterns reveal differential expression of target genes with age. PLoS One. 2010;5: e10724. Funding 10. Zhang ZH, Li J, Liu BR, et al. MicroRNA-26 was decreased in rat cardiac This work was supported by grants from the National Natural Science hypertrophy model and may be a promising therapeutic target. J Foundation of China (81602334, 81401156), the Science and Technology Cardiovasc Pharmacol. 2013;62:312–9. Planning Project of Guangdong Province, China (2016A020215166, 11. Pan W, Zhong Y, Cheng C, et al. MiR-30-regulated autophagy mediates 2017A020215084), the Natural Science Foundation of Guangdong Province, angiotensin II-induced myocardial hypertrophy. PLoS One. 2013;8:e53950. China (2016A030310285), the Educational Commission of Guangdong Province, China (2015KQNCX125, 2016KTSCX113, 2016KQNCX130), and the 12. Dellago H, Preschitz-Kammerhofer B, Terlecki-Zaniewicz L, et al. High levels Bureau of Education of Guangzhou Municipality, China (1201581618). of oncomiR-21 contribute to the senescence-induced growth arrest in normal human cells and its knock-down increases the replicative lifespan. Aging Cell. 2013;12:446–58. Availability of data and materials 13. Li X, Xu F, Chang C, et al. Transcriptional regulation of miR-10a/b by TWIST- All data generated and/or analyzed during this study are included in this 1 in myelodysplastic syndromes. Haematologica. 2013;98:414–9. published article and its supplementary information files. 14. Bryant A, Palma CA, Jayaswal V, et al. miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression Authors’ contributions induces cell death. Mol Cancer. 2012;11:8. JD and ZZ contributed to the design of the study, analysis and interpretation of 15. Mohan N, Ai W, Chakrabarti M, Banik NL, Ray SK. KLF4 overexpression and the data, and wrote the manuscript. HH contributed to the analysis and apigenin treatment down regulated anti-apoptotic Bcl-2 proteins and interpretation of the data. PM and CC contributed to the design of the study matrix metalloproteinases to control growth of human malignant and the analysis and interpretation of the data. JL, WH, and CT collected the neuroblastoma SK-N-DZ and IMR-32 cells. Mol Oncol. 2013;7:464–74. in-vivo data and contributed to the analysis and interpretation of the data. CZ 16. Li Z, Zhao J, Li Q, et al. KLF4 promotes hydrogen-peroxide-induced contributed to the analysis and interpretation of the data. JL designed the study apoptosis of chronic myeloid leukemia cells involving the bcl-2/bax and wrote the manuscript. All authors read and approved the final manuscript. pathway. Cell Stress Chaperones. 2010;15:905–12. 17. Guan H, Xie L, Leithauser F, et al. KLF4 is a tumor suppressor in B-cell non- Ethics approval and consent to participate Hodgkin lymphoma and in classic Hodgkin lymphoma. Blood. 2010;116: This study was conducted in accordance with the Declaration of Helsinki 1469–78. and was approved by the Research Ethics Committee of The Second 18. Zhang L, Dong XW, Wang JN, et al. PEP-1-CAT-transduced mesenchymal Affiliated Hospital of Guangzhou Medical University (File no. 2014013). stem cells acquire an enhanced viability and promote ischemia-induced angiogenesis. PLoS One. 2012;7:e52537. Competing interests 19. Li SH, Sun Z, Brunt KR, et al. Reconstitution of aged bone marrow with The authors declare that they have no competing interests. young cells repopulates cardiac-resident bone marrow-derived progenitor cells and prevents cardiac dysfunction after a myocardial infarction. Eur Heart J. 2013;34:1157–67. Publisher’sNote 20. Wang S, Qu X, Zhao RC. Clinical applications of mesenchymal stem cells. J Springer Nature remains neutral with regard to jurisdictional claims in Hematol Oncol. 2012;5:19. published maps and institutional affiliations. 21. 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Stem Cell Research & TherapySpringer Journals

Published: May 30, 2018

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