Background: Chemoresistance to temozolomide (TMZ) is a major challenge in the treatment of glioblastoma (GBM). We previously found that miR-519a functions as a tumor suppressor in glioma by targeting the signal transducer and activator of transcription 3 (STAT3)-mediated autophagy oncogenic pathway. Here, we investigated the effects of miR-519a on TMZ chemosensitivity and autophagy in GBM cells. Furthermore, the underlying molecular mechanisms and signaling pathways were explored. Methods: In the present study, two stable TMZ-resistant GBM cell lines were successfully generated by exposure of parental cells to a gradually increasing TMZ concentration. After transfecting U87-MG/TMZ and U87-MG cells with miR-519a mimic or inhibitor, a series of biochemical assays such as MTT, apoptosis, and colony formation were performed to determine the chemosensitive response to TMZ. The autophagy levels in GBM cells were detected by transmission electron microscopy, LC3B protein immunofluorescence, and Western blotting analysis. Stable knockdown and overexpression of miR-519a in GBM cells were established using lentivirus. A xenograft nude mouse model and in situ brain model were used to examine the in vivo effects of miR-519a. Tumor tissue samples were collected from 48 patients with GBM and were used to assess the relationship between miR-519a and STAT3 expression. Results: TMZ treatment significantly upregulated miR-519a in U87-MG cells but not in U87-MG/TMZ cells. Moreover, the expression of miR-519a and baseline autophagy levels was lower in U87-MG/TMZ cells as compared to U87-MG cells. miR-519a dramatically enhanced TMZ-induced autophagy and apoptotic cell death in U87-MG/TMZ cells, while inhibition of miR-519a promoted TMZ resistance and reduced TMZ-induced autophagy in U87-MG cells. Furthermore, miR-519a induced autophagy through modification of STAT3 expression. The in vivo results showed that miR-519a can enhance apoptosis and sensitized GBM to TMZ treatment by promoting autophagy and targeting the STAT3/Bcl-2/ Beclin-1 pathway. In human GBM tissues, we found an inverse correlation between miR-519a and STAT3 expression. Conclusions: Our results suggested that miR-519a increased the sensitivity of GBM cells to TMZ therapy. The positive effects of miR-519a may be mediated through autophagy. In addition, miR-519a overexpression can induce autophagy by inhibiting STAT3/Bcl-2 pathway. Therefore, a combination of miR-519a and TMZ may represent an effective therapeutic strategy in GBM. Keywords: miR-519a, Signal transducer and activator of transcription 3, Glioblastoma, Autophagy, Chemoresistance * Correspondence: email@example.com Hong Li and Lei Chen contributed equally to this work. Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, People’sRepublic ofChina Nanfang Neurology Research Institution, Nanfang Hospital, Guangzhou 510515, Guangdong Province, People’s Republic of China 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. Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 2 of 16 Background STAT3 may regulate autophagy in distinct ways . For Glioblastoma (GBM) is the most common primary malig- instance, nuclear STAT3 may upregulate BCL2 expres- nant brain tumor in adults . Multimodality treatment sion and lead to autophagy inhibition . Therefore, a such as cytoreductive surgery followed by radiotherapy better understanding of the role of STAT3 signaling in with concomitant and adjuvant temozolomide (TMZ) regulating autophagy may provide new insights into the chemotherapy has been widely accepted as the new stand- mechanisms of chemoresistance and the potential strat- ard of care for patients with newly diagnosed GBM. egies to overcome TMZ chemoresistance in GBM. However, the prognosis of TMZ-treated patients remains In the present study, we evaluated whether miR-519a dismal, with a median survival of 12.1–14.6 months [2–4]. can affect the chemosensitivity of TMZ in GBM. Fur- Intrinsic or acquired chemoresistance to TMZ is a major thermore, the roles of miR-519a in the modulation of clinical obstacle for the treatment of GBM patients. autophagy via STAT3/Bcl-2/Beclin-1 signaling pathway Therefore, a better understanding of the molecular mech- were investigated. anisms underlying TMZ chemoresistance may lead to im- proved clinical outcomes in GBM patients. Methods Recently, several studies have shown that anticancer Cell lines and reagents therapies can induce autophagy, which constitutes a U87-MG cells were obtained from the Cell Bank of the novel mechanism of chemoresistance in cancer [5–8]. Chinese Academy of Sciences (Shanghai, China) and were Autophagy is a highly evolutionarily conserved process cultured in Dulbecco’s modified Eagle’s medium (DMEM) that occurs in virtually all eukaryotic cells and has been with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, implicated in various physiological and pathological con- USA), 100 U/mL penicillin, and 100 mg/mL streptomycin ditions . In some cases, autophagy induces apoptotic (Gibco) at 37 °C in a humidified incubator with 5% CO . death in GBM cells upon TMZ treatment, and treatment The methods for culturing patient-derived GBM cell line with autophagy inducer rapamycin can further enhance G131212 were described previously . TMZ-resistant chemotherapy-induced apoptosis [10–14]. In other cell lines were generated by iterative pulse exposure of cases, TMZ-induced autophagy may delay cell death U87-MG and G131212 GBM cells to TMZ. The derived [15–17]. Therefore, modulation of autophagy in re- resistant cell lines were designated as U87-MG/TMZ and sponse to TMZ treatment may hold great promise for G131212/TMZ, respectively. Meanwhile, a stock solution circumventing chemotherapeutic resistance and improv- of TMZ (100 mM; cat. no. T2577; Sigma-Aldrich, St. ing anticancer efficacy in GBM patients. However, the Louis, MO, USA) was dissolved in dimethylsulfoxide roles of autophagy in regulating GBM cell death and sur- (DMSO; cat. no. D2650; Sigma-Aldrich) and stored at − vival remain controversial. 20 °C. 3-Methyladenine (3-MA; cat. no. M9281; MicroRNAs (miRs) are small, non-coding RNA mole- Sigma-Aldrich) was prepared freshly in DMEM at 60 °C cules (20–22 nucleotides in length) that negatively regu- and then diluted to 5 mM before use. late gene expression by binding to the 3′-untranslated region (3′UTR) of target mRNAs . miRs have been Oligonucleotides and siRNA transfection shown to be key players in a wide range of biological The miRNA mimic, miRNA inhibitor, STAT3 siRNA, processes, including proliferation, apoptosis, and migra- and scrambled siRNA were synthesized by RiBoBio tion . Recent evidence has indicated that miRs can (China). The oligonucleotide sequences were listed in regulate the chemosensitivity of glioma cells to TMZ by Table S1 (Additional file 1: Table S1). The miRNA over- modulating autophagy signaling . Previously, we expression vector pCMV-MIR519A (MI0003182) and demonstrated that miR-519a is closely related to im- the empty vector control were obtained from OriGene proved prognosis of GBM patients . However, the (Rockville, MD, USA). The miR-519a sponge and empty molecular mechanisms underlying the role of miR-519a vector control were purchased from GeneChem (China). in the chemoresistance of GBM remain unclear. Transfections were performed using Lipofectamine 2000 Signal transducer and activator of transcription 3 reagent (cat. no. 11668-019; Invitrogen, Carlsbad, CA, (STAT3) functions as a signal messenger and transcrip- USA) according to the manufacturer’s instructions. tion factor, which regulates the transcription of down- stream target genes during malignant transformation Cell viability assay and tumor development. Several studies have demon- Cell viability was assessed by using MTT assays. First, strated that STAT3 overexpression in glioma cells can cells were seeded in 96-well plates at a density of 8000 promote tumor progression [22–24]. A growing body of cells per well. After an overnight incubation, the cells evidence has implicated STAT3 in the regulation of au- were treated under the indicated conditions. At the end tophagy, from the assembly of autophagosomes to their of the treatment, 0.5 mg/mL MTT was added to each maturation . In addition, differential localization of well and incubated for 4 h. Then, the supernatants were Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 3 of 16 aspirated carefully, and formazan crystals were dissolved and cells were selected in puromycin (1 μg/mL). The se- in DMSO. Finally, the absorbance was measured at lected cells were sorted by flow cytometry to maintain a 550 nm using Thermo Varioskan Flash reader (Thermo GFP-positive rate for at least 95%. Fisher Scientific, Waltham, MA, USA). Western blotting Colony forming cell assay Western blotting was performed as described previously Cells (200 cells/well) were seeded onto 6-well culture . The antibodies including anti-LC3B (cat. no. 4445), plates and cultured in DMEM supplemented with 10% anti-BECN1/Beclin (cat. no. 3495), anti-STAT3 (cat. no. FBS. The cells were treated with the indicated agents and 12640), anti-phospho-STAT3 (Tyr705; cat. no. 9145), and incubated for 10–14 days at 37 °C and 5% CO . Colonies anti-CASP3/caspase-3 (cat. no. 9915) were purchased were then stained with 0.1% crystal violet (Sigma-Aldrich) from Cell Signaling Technology, while anti-Bax (cat. no. and counted. In some experiments, cells were pre-treated sc-7480) and anti-Bcl-2 (cat. no. sc-509) were obtained for 1 h with 3-MA (Sigma-Aldrich) or rapamycin (Abcam, from Santa Cruz Biotechnology, Santa Cruz, CA, USA. San Francisco, CA, USA), followed by an incubation period of 24 h. For each set of clones, three independent RNA isolation and quantitative reverse transcription assays were carried out. polymerase chain reaction Total miRNA from cultured cells was extracted using Cell apoptosis assay TRIzol reagent (cat. no. 15596-026; Invitrogen), and the GBM cells were transfected with miRNAs (or RNA purity was evaluated by A260/A280 ratio of anti-miRNAs) and/or incubated with 400 μM TMZ for 1.9–2.0. cDNA was synthesized from 1 μg of total RNA 36 h. Subsequently, cells were harvested and stained using PrimeScript RT reagent kit (cat. no. RR047A; with propidium iodide (PI) and annexin V-fluorescein Takara, Shiga, Japan). The primers used for PCR amplifi- isothiocyanate (FITC) for apoptotic analysis. The per- cation were listed in Table S2 (Additional file 2: Table S2). centage of apoptotic cells was calculated as the sum of The expression levels of target genes were quantified on a early and late apoptotic cells located in the lower and Stratagene Mx-3005p instrument (Agilent Technologies upper right quadrants, respectively. Inc., USA) by using Maxima SYBR Green/ROX qPCR Master Mix (cat. no. K0222; Thermo Scientific). Triplicate Green fluorescent protein-LC3 puncta assay samples were examined. GBM green fluorescent protein (GFP)-LC3 stable cells were transfected with miRNAs or anti-miRNAs. Two days Tumor xenograft assays in nude mice after the transfection, cells were fixed with 4% paraformal- BALB/c nude mice (4–5 weeks old) were provided by the dehyde. GFP-LC3 dot formation was observed under a Experimental Animal Center of Southern Medical Univer- confocal laser scanning microscope (FLUOVIEW FV10i; sity. U87-MG cells with stable expression of lentivirus Olympus, Japan). The average number of GFP-LC3 dots/ miR-519a or U87-MG/TMZ cells with stable expression cell was counted in at least 200 cells. of miR-519a shRNA were injected into the left flank of the mice, while control cells were injected into the right Transmission electron microscopy (TEM) flank of the mice. Mice were injected intraperitoneally GBM cells were subjected to different treatments. The (i.p.) with phosphate-buffered saline alone (control) or freshly harvested tumors from mice were fixed overnight TMZ (Merck Co., NJ, USA; 20 mg/kg/mouse) once every with 2.5% glutaraldehyde at 4 °C and post-fixed in 1% other day for 3 weeks, starting on day 3. Tumor volume osmic acid. The fixed samples were then dehydrated and animal weight were assessed every 4 days. Tumor vol- using a graded series of ethanol (70–100%) and embed- ume was calculated using the following formula: volume 3 3 ded in EPON resin. Ultrathin sections were cut with an (mm ) = 4/3 × 3.14 × radius (mm) .Micewerehumanely ultramicrotome and double-stained with uranyl acetate sacrificed on day 24. Tissue blocks were subjected to im- and lead citrate. The stained sections were then exam- munohistochemical staining and TEM analysis. ined using a TEM (H-7650; Hitachi, Tokyo, Japan). For survival analysis in the orthotopic xenograft model, nude mice were randomly divided into four Construction of stable lentiviral clones groups: LV-anti-miR-519a group, LV-anti-NC group, Lentiviral expressing GFP empty vector (NC-LV), GFP LV-anti-NC+TMZ group, and LV-anti-miR-519a+TMZ vector overexpressing miR-519a (LV-miR-519a), or GFP group. In this model, 3 × 10 cells were stereotactically vector inhibiting miR-519a expression (LV-anti-miR-519a) implanted into the right striatum of the mice. Twenty was constructed by Systems Biosciences Inc. (Mountain four days after injection, tumor burden of mice was View, CA, USA). Virus production and cell transduction assessed using magnetic resonance imaging (MRI) scan- in GBM cells were performed as previously described , ner (Bruker Medical Inc., Billerica, MA, USA). The Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 4 of 16 Fig. 1 (See legend on next page.) Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 5 of 16 (See figure on previous page.) Fig. 1 miR-519a sensitized GBM cells to TMZ treatment. a Parental (black ellipses) or resistant (black squares) cells were exposed to increase the concentrations of TMZ in serum-free medium (acute growth inhibition assay) for 72 h. Cell viability was measured by MTT assays. b The expression of miR-519a was detected by qRT-PCR. c Cell viability of DMSO- or TMZ-treated GBM cells transfected with miR-519a or anti-miR-519a. d, e GBM cells transfected with miR-519a or anti-miR-519a were treated with 400 μM TMZ for 36 h. Cells were harvested and stained with PI and annexin V-FITC for apoptotic analysis (**p <0.01 vs. TMZ group). f, g Colony formation assay showing the sensitizing effects of miR-519a on GBM cells after TMZ treatment (**p <0.01 vs. TMZ group). h Western blot analysis of cleaved caspase-3 in GBM cells transfected with miR-519a or anti-miR-519a and then treated with TMZ for 24 h. Data represent the mean (± standard deviations) and are representative of three independent experiments, each performed in triplicate. *p < 0.05, **p <0.01 number of surviving nude mice was recorded, and respective parental sensitive cells. Moreover, TMZ may in- survival analysis was performed by Kaplan-Meier sur- duce the expression of miR-519a in U87-MG cells but not vival curves. in U87-MG/TMZ cells (Additional file 4:FigureS2). To further confirm the role of miR-519a in TMZ che- Patient samples moresistance of GBM cells, we transiently transfected In this study, 24 patients with recurrent GBM treated with U87-MG (or U87-MG/TMZ) cells with miR-519a inhibi- TMZ before second surgery and 24 patients with primary tor (or miR-519a mimic) and evaluated the effects of miR GBM without TMZ treatment were recruited from by using MTT and clonogenic assays. We found that Nanfang Hospital (Guangzhou, China). Tissue samples miR-519a sensitized the U87-MG/TMZ cells to TMZ and were retrieved from the Department of Pathology and confirmed that anti-miR-519a induced TMZ resistance in subjected to sectioning process. Subsequently, the tissue U87-MG cells (Fig. 1c). The results of flow cytometry sections were fixed and immunohistochemically stained analysis revealed that miR-519a overexpression signifi- with anti-LC3B anti-STAT3, and anti-CASP3/caspase-3 cantly increased GBM cell apoptosis caused by TMZ antibodies (cat. no. 4445, 12649, and 9915, respectively; treatment, whereas downregulation of miR-519a inhibited Cell Signaling Technology, Danvers, MA, USA). TMZ-induced apoptosis in U87-MG cells (Fig. 1d, e). Col- ony formation in U87-MG/TMZ cells was markedly lower Statistical analysis than that in the control group, while higher colony forma- All experiments were performed in triplicate and repeated tion was found in U87-MG cells (Fig. 1f). These results at least once. All data were expressed as means ± standard were validated by using the overexpressing vector deviation. If the homogeneity of variance assumption was pCMV-MIR-519a or the inhibition vector miR-519a met, one-way analysis of variance (ANOVA) was per- sponge (Additional file 5: Figure S3). formed to determine the differences between groups, Furthermore, Western blot analysis showed that while least significant difference (LSD) test was used to TMZ-induced cellular apoptosis was greatly enhanced by compare the means of two groups. If the variance was het- miR-519a compared to NC, while knockdown of endogen- erogeneous, Welch test was applied to compare the differ- ous miR-519a decreased TMZ-induced cell apoptosis ences between groups, while Dunnett’s T3 test was used (Fig. 1h). Moreover, miR-519a can effectively sensitize forpairwisecomparisons. p values of less than 0.05 were GBM cells to irradiation treatment (Additional file 6: considered statistically significant. Figure S4). Collectively, these data supported that miR-519a may enhance TMZ chemosensitivity in GBM cells. Results miR-519a sensitized GBM cells to TMZ treatment miR-519a promoted TMZ-induced autophagy in GBM cells A stable TMZ-resistant phenotype of parental U87-MG The results of both immunofluorescence (Fig. 2a) and cells (a GBM cell line) and G131212 cells (patient-derived Western blotting (Fig. 2b) showed that U87-MG/TMZ GBM cells) was established by repetitive pulse exposure to cells had lower autophagic activity than U87-MG cells. increasing concentrations of TMZ for 6 months . Both Meanwhile, the sensitivity of both TMZ-sensitive and TMZ-resistant U87-MG/TMZ and G131212/TMZ cells TMZ-resistant cells was tested, with regard to autophagy exhibited lower sensitivity to TMZ and lower proliferation inducers, including low glucose (LG), chloroquine (CQ), doubling times than the respective parental cells (Fig. 1a and rapamycin (Rapa) at different concentrations. These and Additional file 3: Figure S1). results indicated that U87-MG cells were sensitive to all To determine the effect of miR-519a on the chemosensi- the tested concentrations of rapamycin, whereas the tivity of TMZ, we first examined the expression levels of growth of U87MG/TMZ cells was not affected by rapa- miR-519a in resistant sublines. As shown in Fig. 1b, the ex- mycin (Additional file 7: Figure S5). It is thereby pro- pression of miR-519a was lower in the resistant U87-MG/ posed that the lack of autophagy is a possible TMZ and G131212/TMZ cells compared to their mechanism for TMZ resistance. Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 6 of 16 Fig. 2 (See legend on next page.) Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 7 of 16 (See figure on previous page.) Fig. 2 miR-519a enhanced TMZ-induced autophagy in GBM cells. The expression levels of LC3-II in U87-MG/TMZ and U87-MG cells were evaluated by immunofluorescence assays (a) and Western blotting (b). Red indicates LC3B and blue indicates nuclei. c Both U87-MG/TMZ and U87-MG cells were transfected with GFP-LC3 construct expressing either miR-519a or anti-miR-519a, followed by treatment with 400 μM TMZ. The numbers of GFP-LC3 puncta were quantified using confocal laser scanning microscopy. d Both U87-MG/TMZ and U87-MG cells were transfected with either miR-519a or anti-miR-519a for 24 h, followed by treatment with 400 μM TMZ. Cell samples were prepared for transmission electron microscopy analysis. The arrows indicate autophagic vacuoles. e U87-MG/TMZ cells transfected with miR-519a and parental U87-MG cells transfected with anti-miR-519a were exposed to 400 μM TMZ at different time points (0–24 h). Whole cell lysates were analyzed by Western blotting. f U87-MG/ TMZ cells transfected with or without miR-519a and treated with or without 400 μM TMZ after incubation with 20 nM bafilomycin A1 for 2 h. U87-MG cells transfected with or without anti-miR-519a were treated with 20 nM bafilomycin A1 for 2 h. Cell lysates were analyzed by Western blotting. *p <0.05, **p < 0.01 In order to determine the effects of miR-519a on au- without TMZ treatment. Indeed, pre-treatment of tophagy induction following TMZ treatment, U87-MG/ U87-MG cells with 3-MA (an inhibitor of autophagy) sig- TMZ cells were transfected with miR-519a and nificantly attenuated TMZ-induced cytotoxicity, while U87-MG cells were transfected with anti-miR-519a prior rapamycin enhanced TMZ cytotoxicity in U87-MG/TMZ to TMZ treatment. In addition, GFP-LC3 was stably cells (Additional file 8: Figure S6). These results further expressed in U87-MG and U87-MG/TMZ cells to facili- suggested that autophagy may contribute to the chemo- tate the visualization of autophagy. As compared with sensitivity of TMZ in GBM cells. To confirm whether au- control cells, overexpression of miR-519a enhanced tophagy is responsible for the cellular sensitization during TMZ-induced GFP-LC3 puncta formation in U87-MG/ TMZ chemotherapy enhanced by miR-519a, we assessed TMZ cells, whereas knockdown of miR-519a in U87MG the cell apoptosis rate after the inhibition and induction of cells inhibited the formation of TMZ-induced GFP-LC3 autophagic activity in both U87-MG/TMZ and U87-MG puncta (Fig. 2c). Besides, TEM was used to count the cells, respectively. Treatment with 3-MA significantly at- number of autophagic vacuoles per cell. The results re- tenuated the anti-proliferative effects of miR-519a in vealed that the number of autophagic vacuoles per cell miR-519a-overexpressing U87-MG/TMZ cells, whereas was markedly increased in miR-519a-overexpressing rapamycin treatment reversed the chemoresistance of U87-MG/TMZ cells and decreased in miR-519a-knock- TMZ in U87-MG cells transfected with anti-miR-519a,as down U87-MG cells after TMZ treatment (Fig. 2d). indicated by MTT and colony formation assays, respect- In order to detect the occurrence of autophagy after ively (Fig. 3a, b, e). TMZ treatment in the presence or absence of miR-519a, The results of flow cytometry assays clearly showed we conducted Western blot analysis to examine the that 3-MA significantly attenuated TMZ-induced apop- levels of two autophagy-related proteins: LC3B and tosis in miR-519a-overexpressing U87-MG/TMZ cells, Beclin-1. As shown in Fig. 2e, the expression levels of whereas rapamycin enhanced TMZ-induced apoptosis in LC3-II and Beclin-1 proteins were increased in miR-519- miR-519a-knockdown U87-MG cells (Fig. 3c, dc, d). In a-overexpressing U87-MG/TMZ cells. On the other particular, 3-MA strongly inhibited the transformation hand, knockdown of miR-519a inhibited the expression of LC3-I into LC3-II and decreased the TMZ-induced of LC3-II and Beclin-1 in U87-MG cells. Since the in- activation of caspase-3 in miR-519a-overexpressing creased LC3-II may be due to either autophagy induc- U87-MG/TMZ cells (Fig. 3f). Meanwhile, rapamycin sig- tion or inhibition of autophagic flux , BafA1, an nificantly promoted the LC3-II accumulation and in- inhibitor of fusion between autophagosomes and lyso- creased the TMZ-induced activation of caspase-3 in somes, was used in this study. Twenty-four hours after miR-519a-knockdown U87-MG cells (Fig. 3f). These TMZ treatment, the BafA1-treated negative control cells findings strongly suggested that the enhanced apoptosis displayed markedly increased accumulation of LC3II, of GBM cells induced by the combination of miR-519a and the ectopic expression of miR-519a may enhance and TMZ is dependent on autophagy. these effects (Fig. 2f). Taken together, our data indicated that the inductive effects of miR-519a on autophagy can be resulted from the induction of early stages of autoph- miR-519a induced autophagy through modification of agy, rather than from the suppression of autophagosome STAT3 expression degradation. We have previously demonstrated that miR-519a can target STAT3 in GBM  and speculated that STAT3 miR-519a sensitized GBM cells to TMZ treatment by may be involved in miR-519a-enhanced autophagy after promoting autophagy TMZ treatment. Thus, qRT-PCR and Western blotting Both 3-MA and rapamycin were not able to affect the analysis were performed to determine the expression viability of U87MG and U87MG/TMZ cells, respectively, levels of STAT3 in both U87-MG and U87-MG/TMZ Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 8 of 16 Fig. 3 miR-519a sensitized GBM cells to TMZ treatment partly regulated by autophagy. U87-MG/TMZ cells transfected with miR-519a were treated with or without 400 μM TMZ after incubation with 3-MA (an inhibitor of autophagy) for 2 h. U87-MG cells transfected with anti-miR-519a were treated with or without 200 nM rapamycin for 2 h. The cells were then analyzed for the following: a assessment of proliferation by MTT assay; b, e assessment of colony formation; c, d assessment of apoptosis by FACS analysis of PI-stained cells; and f assessment of caspase-3 activity. *p < 0.05, **p < 0.01 Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 9 of 16 Fig. 4 (See legend on next page.) Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 10 of 16 (See figure on previous page.) Fig. 4 miR-519a induced autophagy through the modification of STAT3 expression. The expression levels of STAT3 in U87-MG/TMZ and U87-MG cells were evaluated by qRT-PCR (a) and Western blotting (b). U87-MG/TMZ cells were transfected with STAT3 siRNA or negative control siRNA and were subjected to immunoblotting with the indicated antibodies (c). STAT3 knockdown affected GFP-LC3 dot aggregation (d, f). Knockdown of STAT3 affected the number of autophagic vacuoles (e, g). AV (autophagic vacuoles) = autophagosomes and lysosomes. Effects of STAT3 on miR-519a-enhanced autophagy and apoptosis were analyzed by immunoblotting in the indicated cells (h). *p < 0.05, **p < 0.01 cells. The results indicated that STAT3 expression was numbers of autophagic vacuoles after TMZ treatment increased in U87-MG/TMZ cells (Fig. 4a, b). (Fig. 5f, g). These in vivo findings suggested that the che- A total of three siRNAs were constructed, and the one mosensitizing effect of miR-519a may be contributed to with the most efficient knockdown of STAT3 was autophagy induction. chosen (Additional file 9: Figure S7). Transfection with Additionally, the antitumor efficacy of miR-519a was STAT3 siRNA inhibited both basal autophagy and examined in an orthotopic G131212/TMZ xenograft TMZ-induced autophagy, thus suggesting that STAT3 model. MRI results on day 24 revealed lower tumor vol- may be involved during the induction of autophagy in umes in miR-519a-overexpressing tumor cells than in GBM cells (Fig. 4c). Furthermore, we constructed control (Fig. 5h). Further results from Kaplan-Meier sur- STAT3-expressing plasmids and siRNA to evaluate vival analysis showed that miR-519a can improve the whether co-transfection with miR-519a mimic or inhibi- survival time after tumor cell implantation (Fig. 5i). tor can counteract the effect of STAT3-expressing plas- These in vivo results supported that miR-519a can mids or STAT3 siRNA in GBM cells. sensitize GBM cells to TMZ. Ectopic expression of STAT3 significantly attenuated the effects of miR-519a on autophagy induction in U87-MG/ miR-519a was associated with chemoresistance of GBM TMZ cells, while suppression of STAT3 can stimulate To further evaluate the clinical role of miR-519a in clin- anti-miR-519a-dependent autophagy in U87-MG cells. ical samples (Additional file 10: Table S3), we performed Moreover, ectopic expression of STAT3 may decrease qRT-PCR assays to detect the expression levels of the number of GFP-LC3 dots (Fig. 4d, f) and autopha- miR-519a and STAT3 in brain tissues from patients with gic vacuoles (Fig. 4e, g) induced by miR-519a.The re- primary and recurrent GBM. We identified downregula- sults of Western blotting analysis also revealed that tion of miR-519a and upregulation of STAT3 in recur- STAT3 significantly attenuated the miR-519a-enhanced rent GBM tissues compared to primary GBM tissues autophagy and apoptosis in GBM cells (Fig. 4h). (Fig. 6a). A significant inverse correlation was found be- Collectively, these findings suggested that STAT3 was tween miR-519a and STAT3 expression levels (Fig. 6b). critical for miR-519a-enhanced autophagy after TMZ Similarly, the results from immunohistochemical stain- treatment in GBM cells. ing showed the increased STAT3 expression and re- duced LC3B and cleaved caspase-3 levels in recurrent miR-519a sensitized GBM cells to TMZ treatment in vivo GBM tissues compared to primary GBM tissues (Fig. 6c). To investigate the effects of miR-519a in vivo, we estab- Therefore, these differences strongly suggested an appar- lished U87-MG/TMZ cells with stable overexpression of ent association between miR-519a, STAT3, and LC3B in miR-519a and U87-MG cells with stable knockdown of GBM patients. miR-519a. Both U87-MG/TMZ and U87-MG cells were infected with LV-miR-519a and LV-anti-miR-519a before Discussion they were applied to a subcutaneous xenograft model. GBM is the most common type of malignant brain After treatment with TMZ, tumors derived from miR-519- tumor . Even after multimodality treatment includ- a-overexpressing U87-MG/TMZ cells grew more slowly ing radical surgery, radiation, and chemotherapy, the and had lower tumor weight than those derived from cells median survival time of GBM is approximately 1 year harboring empty vector. Meanwhile, downregulation of from diagnosis. De novo and acquired resistance to miR-519a expression by LV-anti-miR-519a suppressed the TMZ in GBM cells have emerged as a challenging prob- chemosensitivity of U87-MG cells to TMZ (Fig. 5a–c). lem in clinical practice . Therefore, identifying the Immunohistochemical analysis revealed that miR-519a mechanisms underlying TMZ chemoresistance shed increased the levels of LC3B, Bax, and cleaved caspase-3 light on a novel combination therapy strategy to circum- and decreased the levels of phospho-STAT3 and Bcl-2, vent acquired resistance in GBM patients. Numerous or vice versa in xenograft tumors (Fig. 5d). TEM results studies have reported that miRNA dysfunction may be demonstrated that forced expression of miR-519a in- involved in tumor progression and therapeutic resistance creased the numbers of autophagic vacuoles in GBM tis- [20, 32, 33]. Moreover, the therapeutic potential of miR- sues, whereas knockdown of miR-519a decreased the NAs in cancer, either alone or in combination with Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 11 of 16 Fig. 5 (See legend on next page.) Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 12 of 16 (See figure on previous page.) Fig. 5 miR-519a enhanced the antitumor efficacy of TMZ in vivo. The dissected tumors were collected at the end of drug administration (a), and tumor weight was measured (b). The tumor volume was calculated as 4/3 × 3.14 × radius (mm) (c). Immunohistochemical analysis (d)and Western blot analysis (e) of phospho-STAT3, LC3B, Bcl-2, Bax, and cleaved caspase-3 levels in xenograft tumors. Xenograft tumors were subjected to TEM. AV (autophagic vacuoles) = autophagosomes and lysosomes (f, g). Coronal T2-weighted MRI of tumors acquired from patient-derived GBM cells (G131212/ TMZ) from one animal in each treatment group (red arrow) in the brain samples on day 24 after treatment (h). The survival of mice with orthotopic tumors was measured by Kaplan-Meier survival curves (i). *p < 0.05, **p <0.01 conventional drugs, has been demonstrated in several to transcriptionally activate the apoptosis-inhibitory pro- published studies (reviewed in ). Previously, we re- tein BCL2, which also inhibits the induction of autoph- ported that miR-519a is downregulated in GBM cells, agy by dissociating the Bcl-2/Beclin-1 complex. Upon and overexpression of miR-519a may suppress GBM cell activation, STAT3 upregulates BCL2 expression and proliferation . However, the pivotal role of miR-519a consequently leads to autophagy inhibition [33, 40, 41]. in the modulation of TMZ sensitivity is still not fully In our study, transfection with STAT3 siRNA also inhib- understood. In this study, we demonstrated that the ex- ited both basal autophagy and TMZ-induced autophagy, pression of miR-519a was reduced in chemoresistant suggesting that STAT3 was involved in the induction of GBM tissues and TMZ-resistant cells, thus suggesting autophagy in GBM. Moreover, dysfunction of miRNAs that low levels of miR-519a were associated with TMZ can modulate autophagy through a variety of mecha- resistance. In addition, our results showed that miR-519a nisms in GBM [20, 32, 33]. We previously demonstrated enhanced chemosensitivity in GBM cells, mainly that miR-519a functions as a tumor suppressor in gli- through TMZ-induced autophagy and apoptosis. oma by targeting STAT3 ; therefore, we hypothe- Both prosurvival and prodeath roles of autophagy have sized that miR-519a-mediated prodeath autophagy may been proposed in GBM cells in response to metabolic and occur via targeting of STAT3 to sensitize U87-MG/TMZ therapeutic stress and are dependent on the cellular con- cells to TMZ. Indeed, in this study, we found that text and duration or degree of stress stimuli. Moreover, miR-519a promoted the autophagy of GBM cells by en- accumulating evidence has revealed a correlative relation- hancing dissociation of the Bcl-2/Beclin-1 complex and ship between chemoresistance and reduced autophagic ac- enhanced therapeutic efficacy in vivo and in vitro. We tivity in GBM cells [35–37]. Consistent with previous further improved our understanding of the molecular studies, we found that the basal level of autophagy was basis of miR-519a in GBM. Advances in molecular biol- lower in TMZ-resistant U87-MG/TMZ cells than in ogy have promoted our understanding of the molecular parental GBM cells, suggesting an inverse correlation basis of GBM and provide tools with which to improve between reduced autophagy and chemoresistance therapy. There are many tools, including decoy oligonu- (Additional file 11). Therefore, restoration of autophagic cleotides/antisense oligonucleotide/RNA interference activity in resistant GBM cells may be a promising strategy and guanine-rich oligonucleotides, that have been very to overcome chemoresistance and improve the effective- promising in modulating STAT3 pathway and facilitating ness of chemotherapy. In this study, we demonstrated that the development of new drugs for clinical applications the enhanced autophagy by forced miR-519a expression . miR-519a, as a small molecule, may be a favorable can sensitize GBM cells to TMZ. Additionally, these ef- candidate for analysis in clinical trials. fects were attenuated by co-treatment with autophagic More recently, several studies have suggested that autoph- blockers (3-MA), suggesting the involvement of autopha- agy plays a prodeath role in GBM cells treated with chemo- gic pathway. These results are consistent with previous re- therapeutic agents, by enhancing autophagy-mediated search demonstrating that autophagy induction can lead apoptosis instead of autophagic cell death [41–43]. Mu to the suppression of GBM cell growth [10–14]. Further- et al.  observed an induced autophagy and apop- more, the combination of miR-519a and TMZ induced tosis in GBM cells treated with a combination treat- prodeath autophagy, suggesting that the autophagic re- ment of β-elemene and gefitinib. Bak et al.  sponse of GBM cells to TMZ can be modified when ad- reported that enhanced autophagy contributes to the ministered in combination with other antitumor agents. synergistic effects of vitamin D in TMZ-based GBM Because the autophagy-related signal pathway is com- chemotherapy. Peng-Hsu et al. found that miR-128 plex, additional studies are needed to elucidate the promotes apoptotic death in glioma cells through mechanisms of cell autophagy regulation. Beclin-1 may non-protective autophagy formation. Our results, in bind to and be inhibited by Bcl-2 protein to prevent cell agreement with previous findings [41–43], showed that autophagy [38, 39]. Furthermore, STAT3/Bcl-2/Beclin-1 autophagy inhibition by 3-MA may reduce apoptosis signaling is associated with the induction of autophagy. during combined treatment of miR-519a and TMZ, Previous reports have showed that STAT3 has the ability whereas rapamycin-induced autophagy can enhance Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 13 of 16 Fig. 6 miR-519a was associated with chemoresistance. a miR-519a expression was assessed in primary (n = 24) and recurrent GBM tissue samples (n = 24). b Expression levels of miR-519a were inversely correlated with STAT3 mRNA in tissue samples, as measured by linear regression analysis. c Expression of STAT3, LC3B, and cleaved caspase-3 in primary and recurrent GBM tissue samples were determined by immunochemical staining (magnification × 200). d Schematic illustration of the mechanisms underlying miR-519a induced chemosensitivity to TMZ. Pointed arrows and blunted arrows indicate both activation and repression, respectively. *p < 0.05, **p < 0.01 Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 14 of 16 apoptosis following combined treatment of anti-- of miR-519a was measured by qRT-PCR. a TMZ enhanced the levels of miR-519a and TMZ. Nevertheless, emerging evidence miR-519a in U87-MG cells in a concentration-dependent manner. b TMZ induced miR-519a upregulation in a time-dependent manner. Each bar has suggested a crosstalk between autophagic and represents the mean ± s.d. of three independent experiments. *p < 0.05, apoptotic pathways . **p < 0.01, NS > 0.05 vs. control group. (TIF 1152 kb) Since the autophagy-related pathway network can be Additional file 5: Figure S3. miR-519a sensitized GBM cells to TMZ complex, additional studies are required to elucidate the treatment. a Cell viability of U87-MG/TMZ and U87-MG cells transfected with pCMV-miR-519a or miR-519a sponge and then treated with or without TMZ molecular mechanisms underlying cell autophagy. at various concentrations (or times). b Colony formation in U87-MG/TMZ STAT3/Bcl-2/Beclin-1 signaling has been proposed to be and U87-MG cells transfected with pCMV-miR-519a or miR-519a sponge and associated with autophagy induction. Notably, Beclin-1 then treated with or without TMZ at various concentrations (or times). Each bar represents the mean ± s.d. of three independent experiments. NS > 0.05, may be bound to or inhibited by Bcl-2 protein in order to *p <0.05, **p < 0.01. (TIF 4190 kb) prevent cell autophagy [38, 39]. STAT3 is able to induce Additional file 6: Figure S4. miR-519a enhanced radiosensitivity in GBM BCL2 transcriptional activation, which inhibits the induc- cells. a Cell viability of GBM cells after treatment. Each bar represents the tion of autophagy by dissociating the Bcl-2/Beclin-1 com- mean ± standard deviation of three independent experiments. b Clonogenic survival of GBM cells transfected with miR-519a or anti-miR-519a. Each bar plex [33, 40, 41]. In this study, transfection with STAT3 represents the mean ± s.d. of three independent experiments. *p <0.05, siRNA inhibited both basal autophagy and TMZ-induced **p < 0.01. (TIF 1670 kb) autophagy, suggesting that STAT3 signaling pathway is in- Additional file 7: Figure S5. Cellular viability assay for TMZ-sensitive volved in TMZ-induced autophagy in GBM cells. and -resistant cells. a U87-MG/TMZ and U87-MG cells were cultured in normal medium, with low glucose (LG), or in the presence of chloroquine Additionally, dysfunction of miRNAs can modulate au- (CQ). b The cell viability of U87-MG/TMZ and U87-MG treated with different tophagy in GBM cells through various mechanisms [20, concentrations of rapamycin for 72 h. The cell viability for a and b was 32, 33]. We previously demonstrated that miR-519a func- evaluatedbyMTT assays.Datarepresent the mean(±standarddeviation)of three independent experiments. *p <0.05, **p < 0.01, NS > 0.05 vs. control tions as a tumor suppressor in glioma by targeting STAT3 group. (TIF 816 kb) . In the present study, we confirmed that miR-519a Additional file 8: Figure S6. Effects of 3-MA, rapamycin(Rapa), and/or sensitized U87-MG/TMZ cells to TMZ and triggered their combination with TMZ on the viability of U87-MG/TMZ and U87-MG autophagy-mediated apoptosis via STAT3 pathway. In- cells. a Rapamycin is not able to affect the viability of U87MG/TMZ cells. b 3-MA is not able to affect the viability of U87MG. Each bar represents deed, we found that miR-519a promoted the autophagy of the mean ± s.d. of three independent experiments. NS > 0.05,*p < 0.05. GBM cells via dissociation of Bcl-2/Beclin-1 complex. (TIF 538 kb) These results significantly improved our understanding of Additional file 9: Figure S7. The knockdown efficiency of siSTAT3. Cells the molecular basis of miR-519a in GBM cells with transfected with STAT3 siRNAs (NS, #1, #2, or #3) were treated with or without TMZ (400 μm) for 48 h. qRT-PCR (a) and Western blot analysis (b) TMZ resistance. for the respective target genes were carried out 48 h after transfection. Immunoblots (c) of the extracts for the indicated proteins in U87-MG cells. GAPDH was used as a loading control for Western blots. **p < 0.01 Conclusions vs. Si-NC group. (TIF 599 kb) The results of this study suggested that miR-519a may Additional file 10: Table S3. Clinical information of patients with hold a great potential to overcome TMZ chemoresistance recurrent GBM. (DOCX 105 kb) in GBM. In vivo and in vitro analysis clearly indicated that Additional file 11: Supplemental Material and Methods. (DOCX 104 kb) miR-519a increased TMZ sensitivity by promoting GBM cell apoptosis through autophagy. Additionally, overex- Abbreviations pression of miR-519a can induce autophagy via the inhib- 3′-UTR: 3′-Untranslated region; AV: Autophagic vacuole; GBM: Glioblastoma; ition of STAT3/Bcl-2 signaling pathway. Therefore, GFP: Green fluorescent protein; miR-519a: MicroRNA-519a; miRNA: MicroRNA; miR-519a in combination with TMZ therapy could render PCR: Polymerase chain reaction; TEM: Transmission electron microscopy; TMZ: Temozolomide a more effective therapeutic approach for GBM. Funding Additional files This work was supported by the National Natural Science Foundation of China (grant no. 81772656), Natural Science Foundation of Guangdong Province (grant nos. 2014A030313298 and 2016A030313563), and the Additional file 1: Table S1. Sequences of siRNAs and miRNA used in National Key Clinical Specialist Construction Program of China. this study. (DOCX 58 kb) Additional file 2: Table S2. List of primer sequences used in this study. (DOCX 48 kb) Availability of data and materials Additional file 3: Figure S1. Determination of cell growth rates by All data generated during this study are included in this published article using doubling time assay. U87-MG and U87-MG/TMZ cells displayed [and its supplementary information files]. doubling times of 37.1 and 29.2 h, respectively. Each bar represents the mean ± s.d. of three independent experiments. (TIF 107 kb) Authors’ contributions Additional file 4: Figure S2. TMZ enhanced the expression of miR-519a LYT, QST, and HAN conceived the study. LH, CL, LJJ, LWW, and ZQ carried in U87-MG cells but showed no effect on U87-MG/TMZ cells. U87-MG out the experiments. LYT, QST, GL, and WK analyzed the data. LH and LYT cells and U87-MG/TMZ cells were treated with different concentrations of wrote the manuscript. All authors have read and approved the final version TMZ for 24 h or with 200 μM TMZ for the indicated times. The expression of the manuscript. Li et al. Journal of Hematology & Oncology (2018) 11:70 Page 15 of 16 Ethics approval and consent to participate combination with adjuvant temozolomide and radination (ABTC 0603). 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Journal of Hematology & Oncology
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Published: May 29, 2018