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MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation

MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3... Background: Sarcopenia is a common skeletal disease related to myogenic disorders and muscle atrophy. Current clinical management has limited effectiveness. We sought to investigate the role of miR-1290 in myoblast differentiation and muscle atrophy. Methods: By transfecting miR-1290 into C2C12 cells, we investigated whether miR-1290 regulates myogenesis and myotube atrophy via AKT/P70 signaling pathway. MHC staining was performed to assess myoblast differentiation. Differentiation-relatedMHC,Myod, andMyogprotein levels, and atrophy-related MuRF1 and atrogin-1 were explored by western blot. An LPS-induced muscle atrophy rat model was developed. RT-PCR was conducted to analyze miR-1290 serum levels in muscle atrophy patients and normal controls (NCs). Results: The miR-1290 transfection increased MHC-positive cells and MHC, Myod, and Myog protein levels in the miR- 1290 transfection group, demonstrating that miR-1290 promoted C2C12 myoblast differentiation. Myotube diameter in the miR-1290 transfection group was higher than in the TNF-α-induced model group. Western blot analysis showed decreased MuRF1 and atrogin-1 levels in the miR-1290 transfection group compared with the model group, demonstrating that miR-1290 protected against myoblast cellular atrophy. Luciferase assay and western blot analysis showed that miR-1290 regulation was likely caused by AKT/p70/FOXO3 phosphorylation activation. In the LPS-induced muscle atrophy rat model, miR-1290 mimics ameliorated gastrocnemius muscle loss and increased muscle fiber cross- sectional area. Clinically, miR-1290 serum level was significantly decreased in muscle atrophy patients. Conclusions: We found that miR-1290 enhances myoblast differentiation and inhibits myotube atrophy through Akt/ p70/FoxO3 signaling in vitro and in vivo. In addition, miR-1290 may be a potential therapeutic target for sarcopenia treatment. Keywords: miR-1290, Myoblast differentiation, Atrophy, Akt/P70/FoxO3 * Correspondence: 200100798@163.com; zhengyongjun@fudan.edu.cn Ji Che and Cuidi Xu contributed equally to this work. Department of Pain, Huadong Hospital, Shanghai Key Laboratory of Clinical Geriatric Medicine, Fudan University, No. 221, West YanAn Rd, Shanghai 200040, P.R. China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. Che et al. Skeletal Muscle (2021) 11:6 Page 2 of 12 Background (Thermo Fisher Scientific, CA, USA). TNF-α (AF-315- Skeletal muscle is a crucial component of the human 01A) was obtained from PeproTech. Protein extraction body, occupying about 40–50% of body mass [1]. Skel- Kit, BCA Protein Assay kit, Alexa Fluor 488-labeled goat etal muscle has exercise, metabolism, and endocrine anti-mouse IgG, and Nuclear and Cytoplasmic Protein functions [2] and participates in most activities of the Extraction kit were purchased from Beyotime Institute human body. Abnormal skeletal muscle causes a marked of Biotechnology (Beyotime, Haimeng, China). Trizol decrease in quality of life and presents a great medical was purchased from Sigma-Aldrich (St. Louis, MO, burden on the population, especially elders [3]. Muscle USA). Lipofectamine 2000 (Invitrogen, Waltham, MA, atrophy is a common skeletal disease characterized by USA) was purchased from Invitrogen. The miR-1290 muscle wasting or loss of muscle fibers. The causes of mimic and negative control were synthesized by Gene- muscle atrophy include metabolic diseases or toxins, Pharma (Shanghai, China). Akt inhibitor GDC-0068 such as diabetes, AIDS, toxic drugs, and insufficient (#HY-15186) was purchased from MedChemExpress kidney function [4]. To date, clinical strategies to treat (Suite Q, NJ, USA). Primary antibodies against myosin skeletal muscle atrophy have included surgery, such as heavy chain (MHC) (sc-376157, 1:500), Akt (sc-5298; 1: free muscle transfer; as well as pharmacology interven- 500), P-P70 (sc-8416; 1:500), and P70(sc-8418; 1:500) tion; rehabilitation, such as radiation, muscle stimula- were purchased from Santa Cruz Biotechnology (Dallas, tion, and manual therapy; and myogenic stem cell TX, USA). FOXO3A (10849-1-AP, 1:1000), MyoG transplantation. However, despite numerous advances in (67082, 1:1000), MyoD (18943-1-AP, 1:1000), Lamin the past, current clinical strategies have not achieved full B1(12987-1-AP, 1:5000), and GAPDH (60004-1-Ig, 1: muscle function restoration [5]. Furthermore, the above 5000) were obtained from Proteintech (Wuhan, China). treatments are often accompanied by side effects [6]. MuRF1 (ab183094; 1:5000) and atrogin-1 (ab168372; 1: Commonly, muscle atrophy can be divided into de- 5000) were purchased from Abcam (Cambridge, MA, creased muscle protein synthesis and increased proteoly- USA). Antibodies specific for p-AKT were obtained from sis [6]. The decreased regenerative capacity of the Cell Signaling Technology (Danvers, MA, USA). Second- muscle plays crucial roles in the complex process of ary antibodies were provided by Proteintech (Wuhan, muscle atrophy. Myogenesis initializes the proliferation China). All other chemicals not mentioned were of ana- and differentiation of myoblasts into muscle cells, and lytic grade. consequently the fusion of muscle cells into myotubes, which in turn leads to the maturation of muscle fibers Cell culture [7]. miRNAs are noncoding single-stranded RNAs that The mouse myoblast C2C12 cells were provided by Cya- can block translation or elicit degradation of target genes gen Biosciences, Inc., and cultured in DMEM containing [8]. At present, targeting miRNAs is regarded as a new 1% penicillin-streptomycin and 10% fetal bovine serum therapeutic approach for muscle activities, such as MiR- following the American Type Culture collection instruc- 133 [9], miR-222 [10], and MiR-204-5p [11]. A special tions. Cells were maintained in an incubator at 37 °C miRNA, miR-1290, has been reported to be associated with 5% CO for regular maintenance. For the differenti- with nasopharyngeal development [12], lung cancer bio- ation stage, we changed pre-confluent C2C12 cultures markers [13], and pulmonary fibrosis [14]. However, its from high-serum (10% FBS) to low-serum conditions role in the regulation of C2C12 myogenic differentiation (2% HS) to induce cell cycle exit, commitment to myo- and myotube atrophy, as well as potential-related mech- genic differentiation and fusion between myoblasts to anisms, has not been elucidated. form multinucleated myotubes. Normally, during C2C12 In this study, we demonstrated that miR-1290 en- differentiation, myotube density increased sharply 5 days hanced C2C12 myogenic differentiation and inhibited after confluence. For the differentiation study, C2C12 myotube atrophy in vitro by targeting FOXO3 via the cells with miR-1290 mimic were transfected with Lipo- AKT/P70 pathway. Furthermore, miR-1290 ameliorated fectamine 2000 and set as the transfection group, while the loss of gastrocnemius muscle and increased muscle the C2C12 cells were set as the control group. For the cell area in an LPS-induced muscle atrophy rat model. C2C12 myotube atrophy study, miR-1290 mimic was Additionally, miR-1290 was decreased in muscle atrophy transfected into C2C12 cells by Lipofectamine 2000 fol- patients. lowing the manufacturer’s instructions. After 24 h, TNF- α (20 ng/ml) was added. The C2C12 cells were cultured Materials and methods for 24 h. After transfection of miR-1290 mimic, cell dif- Materials ferentiation was induced by substituting the regular Phosphate-buffered saline (PBS), Dulbecco’s Modified maintenance medium with the differentiation medium Eagle’s Medium (DMEM), penicillin-streptomycin, and (DMEM + 2% horse serum). fetal bovine serum were provided by Thermo Fisher Che et al. Skeletal Muscle (2021) 11:6 Page 3 of 12 C2C12 cell differentiation assay and 10 s at 95 °C by Applied Biosystems® StepOne RT- The MHC immunofluorescence (IF) assay was employed PCR system. The housekeeping gene U6 was run and used to investigate the differentiation role of miR-1290 in as an internal standard for RT-PCR assay with primer: C2C12 cells [15]. C2C12 cells were cultured in six-well (forward 5′-GGAACGATACAGAGAAGATTAGC-3′, plates with prepared coverslips [16]. After adding miR- reverse 5′-TGGAACGCTTCACGAATTTGC-G-3′). 1290 mimic, the C2C12 cells on coverslips were carefully washed using PBS two times, then incubated in 0.25% Western blot Triton X-100 for 15 min. Subsequently, the C2C12 cells Western blotting was performed to explore the related were blocked using blocking reagent, incubated with pri- protein activities in the C2C12 cells. In brief, C2C12 mary anti-MHC overnight, then incubated in Alexa cells after exposure were harvested, and protein of the Fluor 488-labeled goat anti-mouse IgG for 2 h. Finally, cells was isolated. The protein concentration was esti- the cells were counterstained by DAPI for 10 min. The mated by BCA kits and adjusted. Subsequently, protein images were processed using Image Pro Plus 6.0. The ra- was separated and transferred to PVDF membrane. The tio of MHC-positive myotube area versus the total area PVDF membrane was blocked using blocking reagent was observed. and incubated with primary antibodies separately over- night at 4 °C. Then, the PVDF membrane was subjected Giemsa staining and measurement of myotube diameters to incubation with desired secondary anti-rabbit anti- C2C12 myotube diameter was measured as previously body. GAPDH was used as the internal control. The in- reported. Cells were first washed with PBS twice and tensity was analyzed by ChemDoc. fixed in 4% paraformaldehyde for 10 min, after which the myotubes were stained with 10% Giemsa solution RNA interference for 40 min and then observed by an optical microscope The siRNA was transfected into C2C12 cells by Lipofec- (Leica). For each condition, six pictures were randomly tamine 2000 following the manufacturer’s instructions. taken from each well of the six-well plates. The diame- After transfection, the cells were cultured with growth ters of three different sites in each myotube were mea- medium or induction medium for differentiation for 4 sured using ImageJ software, and at least 100 myotubes days. si-FoxO3-1: TGGACGACCTGCTGGATAA, si- in one well were measured. FoxO3-2: CCGAGAACCTCATGGACGA. Dual-luciferase reporter assay Animal experimental design To verify whether FOXO3 was the direct target gene of All animal experiments were approved and followed by miR-1290, luciferase analysis was performed. FOXO3 the Institutional Animal Care and Use Committee of 3’UTR psiCHECK-2 vector containing miR-1290 pre- Fudan University. Sprague Dawley (SD) rats (150–160 g, dicted seed match site or mutant site were purchased 6 weeks old, specific pathogen-free (SPF), healthy, and from HANBIO (Shanghai, China). HEK293T cells were all males) were obtained from Shanghai Sippr BK La- cultured in 12-well plates and co-transfected with lucif- boratory Animals, Ltd., and housed in Fudan University erase reporter vector and miR-1290 using the Lipofecta- at a temperature of 20–25 °C, humidity of 40% ± 5%, mine 2000 following the guidance of a previous report and in a 12-h light/12-h dark cycle environment at SPF [17]. After 48 h of co-culture, the C2C12 cells were har- grade. Rats were provided with free access to adequate vested and washed twice using PBS. The dual-luciferase pellets, and diet and water were SPF grade. A total of 20 assays were employed to explore the fluorescence inten- rats were distributed in two different groups. All rats sity. Luciferase activity was assessed via a Dual- were injected with LPS to establish the atrophy model Luciferase Reporter Assay System (Promega), and the group, and 10 were set as the control group and intra- data were normalized to the control, which was the β- muscularly injected with miR-NC, while 10 were set as gal luciferase activity levels, before data calculation. the treatment group with miR-1290 mimic. LPS (50 μg/ kg) dissolved in saline was injected intraperitoneally into Qualitative real-time polymerase chain reaction (RT-PCR) the rat to induce atrophy following previously described RNA was isolated by Trizol (Takara) and obtained using methods [18]. The miR-NC and mir-1290 mimic were a miRNeasy Mini kit (QIAGEN) in accordance with the administrated 7 days after model establishment. After manufacture manual. Reverse transcription was per- last administration, rats in all groups were euthanized formed using a TaqMan microRNA cDNA Synthesis kit after anesthesia, the gastrocnemius muscles were har- (MT006) in accordance with the manufacturer’s instruc- vested, and the gastrocnemius muscle weight (GW) and tions. Each qPCR reaction solution was prepared using gastrocnemius muscle weight/body weight (GW/BW) ra- 50 ng of cDNA, TB Green® qRT-PCR (1×) in a total vol- tio were recorded. Then, the gastrocnemius was fixed ume of 20 μl with 40 cycles of 5 s at 95 °C, 20 s at 60 °C, using 10% paraformaldehyde, embedded, and cut into Che et al. Skeletal Muscle (2021) 11:6 Page 4 of 12 slices at 5 μm. The slices were stained using hematoxylin Effect of miR-1290 on C2C12 cell proliferation in vitro and eosin (HE), then imaged using a microscope (MF31, As shown in Fig. 2a, miR-1290 transfection led to an in- Mshot, Guangzhou, China). The myofiber cross- crease in the MHC-positive area in the MHC group sectional area was determined following a previous compared with the control group. This result hinted that report. miR-1290 transfection increased differentiation in the C2C12 cells. In addition, the western blot results vali- Clinical serum sample preparation dated that miR-1290 transfection enhanced the protein Serum samples were obtained from 12 knee osteoarth- levels of MHC, MyoD, and MyoG (Fig. 2c). These results ritis patients with muscle atrophy and 6 patients without revealed that differentiation in the miR-1290 transfection muscle atrophy as normal controls. For all patients, group was promoted compared with the group with nor- lower limb muscle strength was measured by a manual mal C2C12 cells. dynamometer, and lean mass was also measured. Protective effect of miR-1290 in myotube atrophy in vitro Analysis of data To further explore the effect of miR1290 on muscle at- Cell experiments were repeated three times. The results rophy, we treated C2C12 myotubes with TNF-α for 24 h were shown as mean ± standard deviation (SD) and de- to establish a muscle atrophy model. Giemsa staining termined using one-way analysis of variance (ANOVA) (Fig. 3a and b) was used to measure cell diameter. Com- to analyze statistical significance. P value < 0.05 was con- pared with the normal control group (21.14 ± 1.07 μm), sidered statistically significant. All statistical analyses myotube diameters in the TNF-α stimulation group were performed using statistical software (SPSS22.0, (15.11 ± 1.06 μm) were significantly decreased. However, Chicago, IL, USA) and are shown by GraphPad Prism myotube diameters increased in the miR-1290 transfec- 6.0 (GraphPad Software Inc., San Diego, CA, USA). tion group (19.55 ± 1.26 μm). Furthermore, western blot analysis showed that the MuRF1 levels were increased in Results the TNF-α stimulation group, but decreased in the miR- MiR-1290 expression is negatively correlated with muscle 1290 mimic group (Fig. 3c). Atrogin-1 showed similar atrophy trends (Fig. 3c). These results demonstrated the protect- Through analyzing the serum expression of miR-1290 ive effect of miR-1290 in myotube atrophy in myoblast via RT-PCR, we concluded that the miR-1290 level of C2C12 cells. muscle atrophy patients was markedly lower compared with normal controls (Fig. 1a). To further assess the as- Protective effect of miR-1290 against myotube atrophy sociation between miR-1290 and the severity of muscle in vivo atrophy, we divided human subjects into three groups To validate the protective effect in vivo, LPS was according to muscle strength (Q1: > 21 kg; Q2: 20–21 employed to create a muscle atrophy model. The miRNA- kg; Q3: < 20 kg) and lean mass (Q1: > 37,000; Q2: 34, 1290 mimic injection attenuated the decrease in gastro- 000–37,000; Q3: < 34,000): Q1, Q2, and Q3 (Fig. 1b and cnemius muscle weight (GW), body weight (BW), and the c). The degree of miR-1290 reduction showed a negative gastrocnemius muscle weight/body weight (GW/BW) ra- correlation with the severity of atrophy. tio (Fig. 4b–d). Moreover, HE staining revealed that LPS Fig. 1 The correlation between miR-1290 and knee osteoarthritis patient with muscle atrophy. a RT-PCR result of miR-1290’s difference in serum. b and c The muscle strength and lean mass of different severity of muscle atrophy. The data was normalized to U6 and statistical difference among different group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 Che et al. Skeletal Muscle (2021) 11:6 Page 5 of 12 Fig. 2 The effect of miR-1290 mimic transfection on C2C12 cells. a and b The MHC staining of C2C12 myoblast with miR-1290 or miR-NC and quantification of MHC area (scale bar, 50 μm). c–f The western blot analysis and quantification of MHC, MyoD, and MyoG after transfection of miRNAs. GAPDH was used as an internal control for western blot analysis. The statistical difference among miR-1290 transfection group and control group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 induced a marked decrease in the muscle fiber cross- 1290-binding site. Results showed that miR-1290 overex- sectional area, but miRNA-1290 mimic injection increased pression notably inhibited luciferase reporter activity from the muscle fiber cross-sectional area (Fig. 4e and f). the vector containing the WT FOXO3 3’UTR, but mutated FOXO3 did not change significantly (Fig. 5b). Moreover, MiR-1290 regulates C2C12 myogenesis and myotube after transfecting miR-1290 mimics in C2C12 cells, we atrophy by directly targeting FOXO3 found gradually increasing FOXO3 expression levels in the To investigate the molecular mechanisms by which miR- cytoplasm and decreasing levels in the nucleus (Fig. 5c). To 1290 mediates C2C12 myogenic regulation and muscle atro- confirm whether FOXO3 affected myoblasts, siFOXO3 was phy, we hypothesized that miR-1290 may promote myo- transfected into C2C12 myoblasts. Western blot analysis genic differentiation and inhibit myotube atrophy by directly showed that siFOXO3 inhibited FOXO3 expression success- targeting FOXO3. To explore whether miR-1290 directly fully (Fig. 5e). The MHC-positive cell areas were markedly targets FOXO3, we purchased wild-type (WT) and mutant increased upon FOXO3 knockdown (Fig. 5g), and protein (MUT) 3’UTR psi-CHECK2, which contained the miR- levels of MyoD and MyoG were also increased (Fig. 5i). Che et al. Skeletal Muscle (2021) 11:6 Page 6 of 12 Fig. 3 The effect of miR-1290 mimic transfection on C2C12 cells under TNF-α. a and b Giemsa staining and myotube diameters among all groups. c–e The western blot analysis and quantification of MuRF1 and atrogin-1 after overexpression of miR-1290 in TNF-α-induced atrophy. GAPDH was used as an internal control for western blot analysis. The statistical difference among miR-1290 transfection group and other groups were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 Taken together, these results suggestthatmiR-1290 pro- factor in an inactive state in the cytoplasm. As shown in motes myoblast differentiation by activating AKT/p70 Fig. 6c, the phosphorylation protein levels of AKT and pathway-mediated FOXO3 phosphorylation, which conse- P70 were increased in the miR-1290 transfection group, quently prevents FOXO3 nuclear translocation. which confirmed that miR-1290 may promote myoblast differentiation via AKT/P70/FOXO3 signaling pathway Role of protein kinase B (AKT)/P70/FOXO3 signaling modulation. Next, we added AKT inhibitor (GDC-0068) pathway in miR-1290’s effect on myogenic differentiation to C2C12 myotubes. Western blot analysis showed de- It is known that activation of AKT induces FOXO3 creased expression of MyoD and MyoG, as well as de- phosphorylation, which maintains the transcription creased MHC measured by IF (Fig. 6a and c). However, Che et al. Skeletal Muscle (2021) 11:6 Page 7 of 12 Fig. 4 Effect of miR-1290 on muscle atrophy rat model. a The typical gastrocnemius muscle after injecting miR-NC and miR-1290 respectively in LPS-induced atrophy mice model. b The gastrocnemius muscle weight (GW) ratio after injecting miR-NC and miR-1290 respectively in LPS- induced atrophy mice model. c and d The body weight and the gastrocnemius muscle weight/body weight (GW/BW) ratio after injecting miR- NC and miR-1290 respectively in LPS-induced atrophy mice model. e HE staining after injecting miR-NC and miR-1290 respectively in LPS-induced atrophy mice model. f The muscle fiber cross-sectional area after injecting miR-NC and miR-1290 respectively in LPS-induced atrophy mice model. The statistical difference among miR-1290 mimic group and NC groups were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 miR-1290 transfection did not increase the expression of Role of protein kinase B (AKT)/P70/FOXO3 signaling MHC, MyoD, and MyoG after GDC-0068 treatment. pathway in miR-1290’s effect on myotube atrophy Furthermore, after adding GDC-0068, miR-1290 did not To explore the effect of miR-1290 on muscle atrophy, we increase the phosphorylation of AKT and P70 (Fig. 6c). measured myotube diameters after treating TNF-α- In C2C12 myoblasts, we observed increased FOXO3 induced C2C12 myotubes with miR-1290 and/or GDC- protein in the cytoplasm and a decreased level in the nu- 0068 (Fig. 7a). As shown in Fig. 7c, miR-1290 significantly cleus after miR-1290 transfection. However, combination increased the phosphorylation of both AKT and P70 and treatment of miR-1290 and GDC-0086 did not increase decreased the expression of MuRF1 and atrogin-1 even FOXO3 in the cytoplasm or decrease FOXO3 in the nu- after TNF-α treatment, while this ability disappeared after cleus of C2C12 myoblasts (Fig. 6c). Taken together, we GDC-0086 treatment. Moreover, miR-1290 transfection conclude that miR-1290 promotes myogenesis by acti- did not activate FOXO3 phosphorylation or promote vating AKT/P70 signaling-mediated FOXO3 nuclear translocation of FOXO3 from the nucleus to the cyto- translocation. plasm after inhibiting the AKT pathway (Fig. 7c). Che et al. Skeletal Muscle (2021) 11:6 Page 8 of 12 Fig. 5 The intrinsic mechanism of miR-1290’s effect. a Bioinformatics analysis was performed to predict the miR-1290-binding seed sequence in the 3’UTR of FoxO3. b The luciferase result of miR-1290 and FOXO3. c and d The western blot analysis and quantification to determine FoxO3 levels in cytoplasm and nucleus in miR-1290-transfected C2C12 myoblast. e and f The knockdown efficiency of FoxO3-specific siRNA was confirmed by western blot. g and h MHC staining was performed after FoxO3 knockdown (scale bar, 50 μm). i and j.The expression of MyoD and MyoG were analyzed by western blot. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and other groups were considered significant at the levels of * P < 0.05, **P <0.01, or ***P <0.001 Discussion The proliferation and differentiation of muscle cells This study was designed to investigate the role of miR- are important biological processes in skeletal myogen- 1290 in C2C12 myoblast cells. In this study, miR-1290 esis, and foremost factors against muscle atrophy [19]. expression accelerated the differentiation of C2C12 cells. When proliferation and differentiation are insufficient, The AKT/P70/ FoxO3 signaling pathway played crucial muscle atrophy occurs when the rate of protein degrad- roles in the myoblast differentiation Importantly, the ation in body tissue exceeds the rate of protein synthesis. promoted differentiation and protective effect against at- The differentiation stage of myoblast cells is a complex rophy were validated using a rat atrophy model. process regulated by specific transcription factors [20]. Che et al. Skeletal Muscle (2021) 11:6 Page 9 of 12 Fig. 6 MiR-1290 activates AKT/P70/FoxO3 signaling pathways during myoblast differentiation. a MHC staining performed after treated miR-1290/ miR-NC with or without GDC-0068 (scale bar, 50 μm). b MHC-positive areas/total areas were quantified using Harmony 4.1 software (n = 6). c–f The western blot analysis and quantification of phosphorylated and all forms of AKT and P70, MyoG, and MyoD, after transfecting miR-1290/miR- NC with or without GDC0068. GDC-0068 inhibited miR-1290-activated phosphorylation of AKT and P70 in C2C12 myoblasts. Western blot to analyze FoxO3 expression levels in cytoplasm and nucleus of C2C12 myoblasts. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and inhibitor group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 First, proteins related to the cell cycle, such as cyclin D1, study, TNF-α downregulated the myoD genes and protein cyclin E, and CDK4, can promote cell proliferation in nor- levels. However, the MyoD and MyoG genes, and protein mal and cancer cells [17]. Meanwhile, transcription factors levels were enhanced after miR-1290 transfection. There- such as MYF5 (Myogenic factor 5), MYF6 (Myogenic fac- fore, miR-1290 promoted the differentiation process. tor 6), MyoD (Myogenic differentiation antigen), MyoG Several proteins and signaling pathways participate in (Myogenin), and MRF4 (Myogenic regulatory factor 4), the regulation of the myoblast differentiation process, also called the myogenic regulatory factor family, can such as AKT, p70, AMP-activated protein kinase regulate differentiation into myotubes [21, 22]. The pro- (AMPK), FoxO3, IGF-1, and TGF-β [24, 25]. AKT and inflammatory cytokine TNF-α elicits muscle proteolysis its downstream p70 can receive upstream stimuli, such and blocks myogenesis by destabilizing MyoD [23]. In this as IGF-1, during myoblast differentiation and muscle Che et al. Skeletal Muscle (2021) 11:6 Page 10 of 12 Fig. 7 Role of Protein kinase B (AKT)/P70/FOXO3 signaling pathway in effect of miR-1290 on myotube atrophy. a Giemsa staining was performed to calculate myotube diameters for TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 treatments. b Cell diameters of five groups were measured. c–f Western blot analysis and quantification of phosphorylated and all forms of AKT and P70 of C1C12 myotubes for TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 treatments. Western blot was performed to analyze the expression of MuRF1 and atrogin-1 in TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 groups. After treatment with miR-1290/miR-NC or with GDC-0068, FoxO3 expression levels in cytoplasm and nucleus of C2C12 myotubes were examined by western blot. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and inhibitor group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 mass maintenance and activation [26]. A previous report YY1. Consequently, the AKT pathway regulated energy indicated that phosphorylation of AKT/mTOR blocked homeostasis processes in myoblasts, including intracellu- FoxO3 translocation to the nucleus, and therefore, lar ATP synthesis, citrate synthase activity, glycolysis, muscle atrophy was inhibited [27]. The mTOR phos- mitochondrial DNA copy maintenance, gene transcrip- phorylation also elicited the downstream targets such as tion, and protein translation, which are linked to energy Che et al. Skeletal Muscle (2021) 11:6 Page 11 of 12 metabolism [28]. In this study, an AKT inhibitor, GDC- alpha; HE: Hematoxylin–eosin; SD: Standard deviation; WT: Wild-type; MYF5: Myogenic factor 5; MYF6: Myogenic factor 6; MyoD: Myogenic 0068, decreased the MHC-positive ratio compared with differentiation antigen; MRF4: Myogenic regulatory factor 4; AMPK: AMP- the miR-1290 transfection group, and downregulated the activated protein kinase; IGF-1: Insulin-like growth factor 1; TGF- MyoD and MyoG genes and proteins levels, thus validat- β: Transforming growth factor-β; mTOR: Mammalian target of rapamycin; ROS: Reactive oxygen species; IKK1: IkB kinase complex; SOCS4: Suppressor of ing the crucial role of AKT in miR-1290’s regulatory ef- cytokine signaling 4; INPP4B: Inositol polyphosphate 4-phosphatase B fect on C2C12 cells. The transcription factor FOXO3 is one member of the human FoxO family [29]. FOXO3 is Acknowledgements widely expressed in skeletal muscle tissue and partici- Not applicable pates in numerous cellular responses such as autophagy, Authors’ contributions apoptosis, stem cell homeostasis, and ROS diminishing (I) Conception and design: JC, CX, XW, and YZ; (II) Administrative support: YZ; [30]. FOXO3 activation in the nucleus accelerates the (III) Provision of study materials or patients: YM, QH, PJ, XW, and YW; (IV) upregulation of Mafx and Murf1 mRNA [31], as well as Collection and assembly of data: JC and CXu; (V) Data analysis and interpretation: JC and CX; (VI) Manuscript writing: all authors; (VII) Final MAFbx and HDAC6 proteins [32]. In this study, FOXO3 approval of manuscript: all authors. was found to be the target of miR-1290. MiR-1290 is a miRNA that often contributes to cancer Funding development. MiR-1290 accelerated cell proliferation This work was supported by grants from Shanghai Key disciplines program of Health and Family Planning (No: 2017ZZ02010) and the National Key R&D and invasion in the pathological processes of gastric can- Program of “Strategic Advanced Electronic Materials” (No: 2017YFB0403803). cer [33], esophageal squamous cell carcinoma [34], lung cancer [13], pancreatic carcinoma [35], and colorectal Availability of data and materials cancer [36]. Therefore, miR-1290 was recognized as a The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. cancer biomarker [37], and thus a potential therapeutic target of anti-cancer compounds [38]. Additionally, miR- Declarations 1290 expression enhanced the production of pulmonary fibrosis markers. Most importantly, miR-1290 acceler- Ethics approval and consent to participate The clinical study was approved by the Ethics Committee of Huadong ated the differentiation of hepatocyte-like cells from Hospital (approval code: 2016K049), this study was carried out in accordance mesenchymal stem cells [39]. This report hinted at the with the World Medical Association’s Declaration of Helsinki. All participants possibility of miR-1290’s effect on myoblast cells. In this were informed about the objectives of the study, and they all signed the Informed Consent Form. study, we found that the miR-1290 mimic successfully All animal care and experimental procedures were approved by and promoted myoblast cell differentiation and exerted a performed following the standards of the Animal Welfare Committees of protective effect against muscle atrophy in vivo. Previous Fudan University in Shanghai, China [No.202006007S]. reports revealed that targets of miRNA contained the BY Consent for publication targeting nuclear factor I/X [34], IkB kinase complex Not applicable (IKK1) [40], suppressor of cytokine signaling 4 (SOCS4) [41], interferon regulatory factor 2 [42], LHX6 [43], and Competing interests inositol polyphosphate 4-phosphatase B (INPP4B) [44]. The authors declare that they have no competing interests. Our study is the first to reveal that FOXO3, a critical Author details skeletal muscle myogenesis regulation protein, is the tar- 1 Department of Pain, Huadong Hospital, Shanghai Key Laboratory of Clinical get of miR-1290. Geriatric Medicine, Fudan University, No. 221, West YanAn Rd, Shanghai 200040, P.R. China. Department of Osteoporosis and Bone Disease, Huadong Hospital, Research Section of Geriatric Metabolic Bone Disease, Shanghai Conclusion Geriatric Institute, Shanghai, China. In summary, the present study revealed the role of miR- Received: 16 November 2020 Accepted: 28 February 2021 1290 in myoblast cell differentiation and muscle atrophy. The miR-1290 mimics accelerated C2C12 cell differenti- ation. The AKT/P70/ FoxO3 signaling pathway plays References 1. Mafakheri S, Flörke RR, Kanngießer S, Hartwig S, Espelage L, De Wendt C, crucial roles in the effect of miR-1290 in myogenesis. Schönberger T, Hamker N, Lehr S, Chadt A, Al-Hasani H. AKT and AMP- The promoted differentiation and protective effects on activated protein kinase regulate TBC1D1 through phosphorylation and its atrophy were validated using a rat atrophy model. interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP. J Biol Chem. 2018;293:17853–62. Abbreviations 2. Romagnoli C, Pampaloni B, Brandi ML. Muscle endocrinology and its AIDS: Acquired immunodeficiency syndrome; miRNAs: MicroRNAs; relation with nutrition. Aging Clin Exp Res. 2019;31:783–92. FOXO3: Forkhead Box O3; AKT: Protein kinase B; PBS: Phosphate-buffered 3. Waltz TB, Fivenson EM, Morevati M, Li C, Becker KG, Bohr VA, Fang EF. saline; DMEM: Dulbecc o’s Modified Eagle’s Medium; MHC: Myosin heavy Sarcopenia, aging and prospective interventional strategies. Curr Med chain; MyoG: Myogenin; MuRF1: Muscle-specific RING Finger 1; Chem. 2018;25:5588–96. IF: Immunofluorescence; qPCR: Real-time polymerase chain reaction; 4. Li J, Chan MC, Yu Y, Bei Y, Chen P, Zhou Q, Cheng L, Chen L, Ziegler O, SD: Sprague Dawley; SPF: Specific pathogen-free; NC: Negative control; Rowe GC, Das S, Xiao J. miR-29b contributes to multiple types of muscle BW: Body weight; GW: Gastrocnemius weight; TNF-α: Tumor necrosis factor atrophy. Nat Commun. 2017;8:15201. Che et al. Skeletal Muscle (2021) 11:6 Page 12 of 12 5. Rao VK, Kapp D, Schroth M. Gene therapy for spinal muscular atrophy: an mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can emerging treatment option for a devastating disease. J Manag Care Spec prevent muscle atrophy in vivo. Nat Cell Biol. 2001;3:1014–9. Pharm. 2018;24:S3–s16. 29. Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, 6. Cao RY, Li J, Dai Q, Li Q, Yang J. Muscle atrophy: present and future. Adv Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL, Schiaffino S, Sandri M. Exp Med Biol. 2018;1088:605–24. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab. 2007;6: 458–71. 7. Sabourin LA, Rudnicki MA. The molecular regulation of myogenesis. Clin Genet. 2000;57:16–25. 30. Morris BJ, Willcox DC, Donlon TA, Willcox BJ. FOXO3: a major gene for human longevity--a mini-review. Gerontology. 2015;61:515–25. 8. Bhaskaran M, Mohan M. MicroRNAs: history, biogenesis, and their evolving 31. Nakao R, Abe T, Yamamoto S, Oishi K. Ketogenic diet induces skeletal role in animal development and disease. Vet Pathol. 2014;51:759–74. muscle atrophy via reducing muscle protein synthesis and possibly 9. Zhang J, Hua C, Zhang Y, Wei P, Tu Y, Wei T. KAP1-associated transcriptional activating proteolysis in mice. Sci Rep. 2019;9:19652. inhibitory complex regulates C2C12 myoblasts differentiation and 32. Wang J, Gao X, Ren D, Zhang M, Zhang P, Lu S, Huan C, Yao Y, Zheng L, mitochondrial biogenesis via miR-133a repression. Cell Death Dis. 2020;11: Bao Z, Zhou J. Triptolide induces atrophy of myotubes by triggering IRS-1 degradation and activating the FoxO3 pathway. Toxicol In Vitro. 2020;65: 10. Gan M, Shen L, Liu L, Guo Z, Wang S, Chen L, Zheng T, Fan Y, Tan Y, Jiang D, Li X, Zhang S, Zhu L. miR-222 is involved in the regulation of genistein 33. Huang J, Shen M, Yan M, Cui Y, Gao Z, Meng X. Exosome-mediated transfer on skeletal muscle fiber type. J Nutr Biochem. 2020;80:108320. of miR-1290 promotes cell proliferation and invasion in gastric cancer via 11. Houzelle A, Dahlmans D, Nascimento EBM, Schaart G, Jörgensen JA, NKD1. Acta Biochim Biophys Sin (Shanghai). 2019;51:900–7. Moonen-Kornips E, Kersten S, Wang X, Hoeks J. MicroRNA-204-5p modulates 34. Mao Y, Liu J, Zhang D, Li B. MiR-1290 promotes cancer progression by mitochondrial biogenesis in C2C12 myotubes and associates with oxidative targeting nuclear factor I/X(NFIX) in esophageal squamous cell carcinoma capacity in humans. J Cell Physiol. 2020;235:9851–63. (ESCC). Biomed Pharmacother. 2015;76:82–93. 12. Chen X, Huang Y, Shi D, Nie C, Luo Y, Guo L, Zou Y, Xie C. LncRNA ZNF667- 35. Tavano F, Gioffreda D, Valvano MR, Palmieri O, Tardio M, Latiano TP, Piepoli AS1 promotes ABLIM1 expression by adsorbing micro RNA-1290 to A, Maiello E, Pirozzi F, Andriulli A. Droplet digital PCR quantification of miR- suppress nasopharyngeal carcinoma cell progression. Onco Targets Ther. 1290 as a circulating biomarker for pancreatic cancer. Sci Rep. 2018;8:16389. 2020;13:4397–409. 36. Li H, Zhang H, Lu G, Li Q, Gu J, Song Y, Gao S, Ding Y. Mechanism analysis 13. Wu Y, Wei J, Zhang W, Xie M, Wang X, Xu J. Serum exosomal miR-1290 is a of colorectal cancer according to the microRNA expression profile. Oncol potential biomarker for lung adenocarcinoma. Onco Targets Ther. 2020;13: Lett. 2016;12:2329–36. 7809–18. 37. Imaoka H, Toiyama Y, Fujikawa H, Hiro J, Saigusa S, Tanaka K, Inoue Y, Mohri 14. Guan S, Wu Y, Zhang Q, Zhou J. TGF-β1 induces CREB1-mediated miR-1290 Y, Mori T, Kato T, Toden S, Goel A, Kusunoki M. Circulating microRNA-1290 upregulation to antagonize lung fibrosis via Napsin A. Int J Mol Med. 2020; as a novel diagnostic and prognostic biomarker in human colorectal cancer. 46:141–8. Ann Oncol. 2016;27:1879–86. 15. D'Andrea P, Civita D, Cok M, Ulloa Severino L, Vita F, Scaini D, Casalis L, 38. Zhou W, Wang S, Ying Y, Zhou R, Mao P. miR-196b/miR-1290 participate in Lorenzon P, Donati I, Bandiera A. Myoblast adhesion, proliferation and the antitumor effect of resveratrol via regulation of IGFBP3 expression in differentiation on human elastin-like polypeptide (HELP) hydrogels. J Appl acute lymphoblastic leukemia. Oncol Rep. 2017;37:1075–83. Biomater Funct Mater. 2017;15:e43–53. 39. Zhou X, Cui L, Zhou X, Yang Q, Wang L, Guo G, Hou Y, Cai W, Han Z, Shi Y, 16. Liang T, Zhou B, Shi L, Wang H, Chu Q, Xu F, Li Y, Chen R, Shen C, Schinckel Han Y. Induction of hepatocyte-like cells from human umbilical cord- AP. lncRNA AK017368 promotes proliferation and suppresses differentiation derived mesenchymal stem cells by defined microRNAs. J Cell Mol Med. of myoblasts in skeletal muscle development by attenuating the function of 2017;21:881–93. miR-30c. FASEB J. 2018;32:377–89. 40. Ta N, Huang X, Zheng K, Zhang Y, Gao Y, Deng L, Zhang B, Jiang H, Zheng 17. Wang H, Shi L, Liang T, Wang B, Wu W, Su G, Wei J, Li P, Huang R. MiR-696 J. miRNA-1290 promotes aggressiveness in pancreatic ductal regulates C2C12 cell proliferation and differentiation by targeting CNTFRα. adenocarcinoma by targeting IKK1. Cell Physiol Biochem. 2018;51:711–28. Int J Biol Sci. 2017;13:413–25. 41. Xiao X, Yang D, Gong X, Mo D, Pan S, Xu J. miR-1290 promotes lung 18. Cheng M, Gao T, Xi F, Cao C, Chen Y, Zhao C, Li Q, Yu W. adenocarcinoma cell proliferation and invasion by targeting SOCS4. Dexmedetomidine ameliorates muscle wasting and attenuates the Oncotarget. 2018;9:11977–88. alteration of hypothalamic neuropeptides and inflammation in 42. Jin JJ, Liu YH, Si JM, Ni R, Wang J. Overexpression of miR-1290 contributes endotoxemic rats. PLoS One. 2017;12:e0174894. to cell proliferation and invasion of non small cell lung cancer by targeting 19. Powers SK, Kavazis AN, McClung JM. Oxidative stress and disuse muscle interferon regulatory factor 2. Int J Biochem Cell Biol. 2018;95:113–20. atrophy. J Appl Physiol. 1985;102(2007):2389–97. 43. Yan L, Cai K, Sun K, Gui J, Liang J. MiR-1290 promotes proliferation, 20. Piccirillo R, Demontis F, Perrimon N, Goldberg AL. Mechanisms of muscle migration, and invasion of glioma cells by targeting LHX6. J Cell Physiol. growth and atrophy in mammals and Drosophila. Dev Dyn. 2014;243:201– 2018;233:6621–9. 44. Ma Q, Wang Y, Zhang H, Wang F. miR-1290 contributes to colorectal cancer 21. Endo T. Molecular mechanisms of skeletal muscle development, cell proliferation by targeting INPP4B. Oncol Res. 2018;26:1167–74. regeneration, and osteogenic conversion. Bone. 2015;80:2–13. 22. Braun T, Gautel M. Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis. Nat Rev Mol Cell Biol. 2011;12:349– Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in 23. Plant PJ, Brooks D, Faughnan M, Bayley T, Bain J, Singer L, Correa J, Pearce published maps and institutional affiliations. D, Binnie M, Batt J. Cellular markers of muscle atrophy in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2010;42:461–71. 24. Foletta VC, White LJ, Larsen AE, Léger B, Russell AP. The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy. Pflugers Arch. 2011;461:325–35. 25. Comai G, Tajbakhsh S. Molecular and cellular regulation of skeletal myogenesis. Curr Top Dev Biol. 2014;110:1–73. 26. Jia L, Li YF, Wu GF, Song ZY, Lu HZ, Song CC, Zhang QL, Zhu JY, Yang GS, Shi XE. MiRNA-199a-3p regulates C2C12 myoblast differentiation through IGF-1/AKT/mTOR signal pathway. Int J Mol Sci. 2013;15:296–308. 27. Sanchez AM, Candau RB, Bernardi H. FoxO transcription factors: their roles in the maintenance of skeletal muscle homeostasis. Cell Mol Life Sci. 2014;71: 1657–71. 28. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD. Akt/ http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation

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Copyright © The Author(s) 2021
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10.1186/s13395-021-00262-9
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Abstract

Background: Sarcopenia is a common skeletal disease related to myogenic disorders and muscle atrophy. Current clinical management has limited effectiveness. We sought to investigate the role of miR-1290 in myoblast differentiation and muscle atrophy. Methods: By transfecting miR-1290 into C2C12 cells, we investigated whether miR-1290 regulates myogenesis and myotube atrophy via AKT/P70 signaling pathway. MHC staining was performed to assess myoblast differentiation. Differentiation-relatedMHC,Myod, andMyogprotein levels, and atrophy-related MuRF1 and atrogin-1 were explored by western blot. An LPS-induced muscle atrophy rat model was developed. RT-PCR was conducted to analyze miR-1290 serum levels in muscle atrophy patients and normal controls (NCs). Results: The miR-1290 transfection increased MHC-positive cells and MHC, Myod, and Myog protein levels in the miR- 1290 transfection group, demonstrating that miR-1290 promoted C2C12 myoblast differentiation. Myotube diameter in the miR-1290 transfection group was higher than in the TNF-α-induced model group. Western blot analysis showed decreased MuRF1 and atrogin-1 levels in the miR-1290 transfection group compared with the model group, demonstrating that miR-1290 protected against myoblast cellular atrophy. Luciferase assay and western blot analysis showed that miR-1290 regulation was likely caused by AKT/p70/FOXO3 phosphorylation activation. In the LPS-induced muscle atrophy rat model, miR-1290 mimics ameliorated gastrocnemius muscle loss and increased muscle fiber cross- sectional area. Clinically, miR-1290 serum level was significantly decreased in muscle atrophy patients. Conclusions: We found that miR-1290 enhances myoblast differentiation and inhibits myotube atrophy through Akt/ p70/FoxO3 signaling in vitro and in vivo. In addition, miR-1290 may be a potential therapeutic target for sarcopenia treatment. Keywords: miR-1290, Myoblast differentiation, Atrophy, Akt/P70/FoxO3 * Correspondence: 200100798@163.com; zhengyongjun@fudan.edu.cn Ji Che and Cuidi Xu contributed equally to this work. Department of Pain, Huadong Hospital, Shanghai Key Laboratory of Clinical Geriatric Medicine, Fudan University, No. 221, West YanAn Rd, Shanghai 200040, P.R. China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. Che et al. Skeletal Muscle (2021) 11:6 Page 2 of 12 Background (Thermo Fisher Scientific, CA, USA). TNF-α (AF-315- Skeletal muscle is a crucial component of the human 01A) was obtained from PeproTech. Protein extraction body, occupying about 40–50% of body mass [1]. Skel- Kit, BCA Protein Assay kit, Alexa Fluor 488-labeled goat etal muscle has exercise, metabolism, and endocrine anti-mouse IgG, and Nuclear and Cytoplasmic Protein functions [2] and participates in most activities of the Extraction kit were purchased from Beyotime Institute human body. Abnormal skeletal muscle causes a marked of Biotechnology (Beyotime, Haimeng, China). Trizol decrease in quality of life and presents a great medical was purchased from Sigma-Aldrich (St. Louis, MO, burden on the population, especially elders [3]. Muscle USA). Lipofectamine 2000 (Invitrogen, Waltham, MA, atrophy is a common skeletal disease characterized by USA) was purchased from Invitrogen. The miR-1290 muscle wasting or loss of muscle fibers. The causes of mimic and negative control were synthesized by Gene- muscle atrophy include metabolic diseases or toxins, Pharma (Shanghai, China). Akt inhibitor GDC-0068 such as diabetes, AIDS, toxic drugs, and insufficient (#HY-15186) was purchased from MedChemExpress kidney function [4]. To date, clinical strategies to treat (Suite Q, NJ, USA). Primary antibodies against myosin skeletal muscle atrophy have included surgery, such as heavy chain (MHC) (sc-376157, 1:500), Akt (sc-5298; 1: free muscle transfer; as well as pharmacology interven- 500), P-P70 (sc-8416; 1:500), and P70(sc-8418; 1:500) tion; rehabilitation, such as radiation, muscle stimula- were purchased from Santa Cruz Biotechnology (Dallas, tion, and manual therapy; and myogenic stem cell TX, USA). FOXO3A (10849-1-AP, 1:1000), MyoG transplantation. However, despite numerous advances in (67082, 1:1000), MyoD (18943-1-AP, 1:1000), Lamin the past, current clinical strategies have not achieved full B1(12987-1-AP, 1:5000), and GAPDH (60004-1-Ig, 1: muscle function restoration [5]. Furthermore, the above 5000) were obtained from Proteintech (Wuhan, China). treatments are often accompanied by side effects [6]. MuRF1 (ab183094; 1:5000) and atrogin-1 (ab168372; 1: Commonly, muscle atrophy can be divided into de- 5000) were purchased from Abcam (Cambridge, MA, creased muscle protein synthesis and increased proteoly- USA). Antibodies specific for p-AKT were obtained from sis [6]. The decreased regenerative capacity of the Cell Signaling Technology (Danvers, MA, USA). Second- muscle plays crucial roles in the complex process of ary antibodies were provided by Proteintech (Wuhan, muscle atrophy. Myogenesis initializes the proliferation China). All other chemicals not mentioned were of ana- and differentiation of myoblasts into muscle cells, and lytic grade. consequently the fusion of muscle cells into myotubes, which in turn leads to the maturation of muscle fibers Cell culture [7]. miRNAs are noncoding single-stranded RNAs that The mouse myoblast C2C12 cells were provided by Cya- can block translation or elicit degradation of target genes gen Biosciences, Inc., and cultured in DMEM containing [8]. At present, targeting miRNAs is regarded as a new 1% penicillin-streptomycin and 10% fetal bovine serum therapeutic approach for muscle activities, such as MiR- following the American Type Culture collection instruc- 133 [9], miR-222 [10], and MiR-204-5p [11]. A special tions. Cells were maintained in an incubator at 37 °C miRNA, miR-1290, has been reported to be associated with 5% CO for regular maintenance. For the differenti- with nasopharyngeal development [12], lung cancer bio- ation stage, we changed pre-confluent C2C12 cultures markers [13], and pulmonary fibrosis [14]. However, its from high-serum (10% FBS) to low-serum conditions role in the regulation of C2C12 myogenic differentiation (2% HS) to induce cell cycle exit, commitment to myo- and myotube atrophy, as well as potential-related mech- genic differentiation and fusion between myoblasts to anisms, has not been elucidated. form multinucleated myotubes. Normally, during C2C12 In this study, we demonstrated that miR-1290 en- differentiation, myotube density increased sharply 5 days hanced C2C12 myogenic differentiation and inhibited after confluence. For the differentiation study, C2C12 myotube atrophy in vitro by targeting FOXO3 via the cells with miR-1290 mimic were transfected with Lipo- AKT/P70 pathway. Furthermore, miR-1290 ameliorated fectamine 2000 and set as the transfection group, while the loss of gastrocnemius muscle and increased muscle the C2C12 cells were set as the control group. For the cell area in an LPS-induced muscle atrophy rat model. C2C12 myotube atrophy study, miR-1290 mimic was Additionally, miR-1290 was decreased in muscle atrophy transfected into C2C12 cells by Lipofectamine 2000 fol- patients. lowing the manufacturer’s instructions. After 24 h, TNF- α (20 ng/ml) was added. The C2C12 cells were cultured Materials and methods for 24 h. After transfection of miR-1290 mimic, cell dif- Materials ferentiation was induced by substituting the regular Phosphate-buffered saline (PBS), Dulbecco’s Modified maintenance medium with the differentiation medium Eagle’s Medium (DMEM), penicillin-streptomycin, and (DMEM + 2% horse serum). fetal bovine serum were provided by Thermo Fisher Che et al. Skeletal Muscle (2021) 11:6 Page 3 of 12 C2C12 cell differentiation assay and 10 s at 95 °C by Applied Biosystems® StepOne RT- The MHC immunofluorescence (IF) assay was employed PCR system. The housekeeping gene U6 was run and used to investigate the differentiation role of miR-1290 in as an internal standard for RT-PCR assay with primer: C2C12 cells [15]. C2C12 cells were cultured in six-well (forward 5′-GGAACGATACAGAGAAGATTAGC-3′, plates with prepared coverslips [16]. After adding miR- reverse 5′-TGGAACGCTTCACGAATTTGC-G-3′). 1290 mimic, the C2C12 cells on coverslips were carefully washed using PBS two times, then incubated in 0.25% Western blot Triton X-100 for 15 min. Subsequently, the C2C12 cells Western blotting was performed to explore the related were blocked using blocking reagent, incubated with pri- protein activities in the C2C12 cells. In brief, C2C12 mary anti-MHC overnight, then incubated in Alexa cells after exposure were harvested, and protein of the Fluor 488-labeled goat anti-mouse IgG for 2 h. Finally, cells was isolated. The protein concentration was esti- the cells were counterstained by DAPI for 10 min. The mated by BCA kits and adjusted. Subsequently, protein images were processed using Image Pro Plus 6.0. The ra- was separated and transferred to PVDF membrane. The tio of MHC-positive myotube area versus the total area PVDF membrane was blocked using blocking reagent was observed. and incubated with primary antibodies separately over- night at 4 °C. Then, the PVDF membrane was subjected Giemsa staining and measurement of myotube diameters to incubation with desired secondary anti-rabbit anti- C2C12 myotube diameter was measured as previously body. GAPDH was used as the internal control. The in- reported. Cells were first washed with PBS twice and tensity was analyzed by ChemDoc. fixed in 4% paraformaldehyde for 10 min, after which the myotubes were stained with 10% Giemsa solution RNA interference for 40 min and then observed by an optical microscope The siRNA was transfected into C2C12 cells by Lipofec- (Leica). For each condition, six pictures were randomly tamine 2000 following the manufacturer’s instructions. taken from each well of the six-well plates. The diame- After transfection, the cells were cultured with growth ters of three different sites in each myotube were mea- medium or induction medium for differentiation for 4 sured using ImageJ software, and at least 100 myotubes days. si-FoxO3-1: TGGACGACCTGCTGGATAA, si- in one well were measured. FoxO3-2: CCGAGAACCTCATGGACGA. Dual-luciferase reporter assay Animal experimental design To verify whether FOXO3 was the direct target gene of All animal experiments were approved and followed by miR-1290, luciferase analysis was performed. FOXO3 the Institutional Animal Care and Use Committee of 3’UTR psiCHECK-2 vector containing miR-1290 pre- Fudan University. Sprague Dawley (SD) rats (150–160 g, dicted seed match site or mutant site were purchased 6 weeks old, specific pathogen-free (SPF), healthy, and from HANBIO (Shanghai, China). HEK293T cells were all males) were obtained from Shanghai Sippr BK La- cultured in 12-well plates and co-transfected with lucif- boratory Animals, Ltd., and housed in Fudan University erase reporter vector and miR-1290 using the Lipofecta- at a temperature of 20–25 °C, humidity of 40% ± 5%, mine 2000 following the guidance of a previous report and in a 12-h light/12-h dark cycle environment at SPF [17]. After 48 h of co-culture, the C2C12 cells were har- grade. Rats were provided with free access to adequate vested and washed twice using PBS. The dual-luciferase pellets, and diet and water were SPF grade. A total of 20 assays were employed to explore the fluorescence inten- rats were distributed in two different groups. All rats sity. Luciferase activity was assessed via a Dual- were injected with LPS to establish the atrophy model Luciferase Reporter Assay System (Promega), and the group, and 10 were set as the control group and intra- data were normalized to the control, which was the β- muscularly injected with miR-NC, while 10 were set as gal luciferase activity levels, before data calculation. the treatment group with miR-1290 mimic. LPS (50 μg/ kg) dissolved in saline was injected intraperitoneally into Qualitative real-time polymerase chain reaction (RT-PCR) the rat to induce atrophy following previously described RNA was isolated by Trizol (Takara) and obtained using methods [18]. The miR-NC and mir-1290 mimic were a miRNeasy Mini kit (QIAGEN) in accordance with the administrated 7 days after model establishment. After manufacture manual. Reverse transcription was per- last administration, rats in all groups were euthanized formed using a TaqMan microRNA cDNA Synthesis kit after anesthesia, the gastrocnemius muscles were har- (MT006) in accordance with the manufacturer’s instruc- vested, and the gastrocnemius muscle weight (GW) and tions. Each qPCR reaction solution was prepared using gastrocnemius muscle weight/body weight (GW/BW) ra- 50 ng of cDNA, TB Green® qRT-PCR (1×) in a total vol- tio were recorded. Then, the gastrocnemius was fixed ume of 20 μl with 40 cycles of 5 s at 95 °C, 20 s at 60 °C, using 10% paraformaldehyde, embedded, and cut into Che et al. Skeletal Muscle (2021) 11:6 Page 4 of 12 slices at 5 μm. The slices were stained using hematoxylin Effect of miR-1290 on C2C12 cell proliferation in vitro and eosin (HE), then imaged using a microscope (MF31, As shown in Fig. 2a, miR-1290 transfection led to an in- Mshot, Guangzhou, China). The myofiber cross- crease in the MHC-positive area in the MHC group sectional area was determined following a previous compared with the control group. This result hinted that report. miR-1290 transfection increased differentiation in the C2C12 cells. In addition, the western blot results vali- Clinical serum sample preparation dated that miR-1290 transfection enhanced the protein Serum samples were obtained from 12 knee osteoarth- levels of MHC, MyoD, and MyoG (Fig. 2c). These results ritis patients with muscle atrophy and 6 patients without revealed that differentiation in the miR-1290 transfection muscle atrophy as normal controls. For all patients, group was promoted compared with the group with nor- lower limb muscle strength was measured by a manual mal C2C12 cells. dynamometer, and lean mass was also measured. Protective effect of miR-1290 in myotube atrophy in vitro Analysis of data To further explore the effect of miR1290 on muscle at- Cell experiments were repeated three times. The results rophy, we treated C2C12 myotubes with TNF-α for 24 h were shown as mean ± standard deviation (SD) and de- to establish a muscle atrophy model. Giemsa staining termined using one-way analysis of variance (ANOVA) (Fig. 3a and b) was used to measure cell diameter. Com- to analyze statistical significance. P value < 0.05 was con- pared with the normal control group (21.14 ± 1.07 μm), sidered statistically significant. All statistical analyses myotube diameters in the TNF-α stimulation group were performed using statistical software (SPSS22.0, (15.11 ± 1.06 μm) were significantly decreased. However, Chicago, IL, USA) and are shown by GraphPad Prism myotube diameters increased in the miR-1290 transfec- 6.0 (GraphPad Software Inc., San Diego, CA, USA). tion group (19.55 ± 1.26 μm). Furthermore, western blot analysis showed that the MuRF1 levels were increased in Results the TNF-α stimulation group, but decreased in the miR- MiR-1290 expression is negatively correlated with muscle 1290 mimic group (Fig. 3c). Atrogin-1 showed similar atrophy trends (Fig. 3c). These results demonstrated the protect- Through analyzing the serum expression of miR-1290 ive effect of miR-1290 in myotube atrophy in myoblast via RT-PCR, we concluded that the miR-1290 level of C2C12 cells. muscle atrophy patients was markedly lower compared with normal controls (Fig. 1a). To further assess the as- Protective effect of miR-1290 against myotube atrophy sociation between miR-1290 and the severity of muscle in vivo atrophy, we divided human subjects into three groups To validate the protective effect in vivo, LPS was according to muscle strength (Q1: > 21 kg; Q2: 20–21 employed to create a muscle atrophy model. The miRNA- kg; Q3: < 20 kg) and lean mass (Q1: > 37,000; Q2: 34, 1290 mimic injection attenuated the decrease in gastro- 000–37,000; Q3: < 34,000): Q1, Q2, and Q3 (Fig. 1b and cnemius muscle weight (GW), body weight (BW), and the c). The degree of miR-1290 reduction showed a negative gastrocnemius muscle weight/body weight (GW/BW) ra- correlation with the severity of atrophy. tio (Fig. 4b–d). Moreover, HE staining revealed that LPS Fig. 1 The correlation between miR-1290 and knee osteoarthritis patient with muscle atrophy. a RT-PCR result of miR-1290’s difference in serum. b and c The muscle strength and lean mass of different severity of muscle atrophy. The data was normalized to U6 and statistical difference among different group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 Che et al. Skeletal Muscle (2021) 11:6 Page 5 of 12 Fig. 2 The effect of miR-1290 mimic transfection on C2C12 cells. a and b The MHC staining of C2C12 myoblast with miR-1290 or miR-NC and quantification of MHC area (scale bar, 50 μm). c–f The western blot analysis and quantification of MHC, MyoD, and MyoG after transfection of miRNAs. GAPDH was used as an internal control for western blot analysis. The statistical difference among miR-1290 transfection group and control group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 induced a marked decrease in the muscle fiber cross- 1290-binding site. Results showed that miR-1290 overex- sectional area, but miRNA-1290 mimic injection increased pression notably inhibited luciferase reporter activity from the muscle fiber cross-sectional area (Fig. 4e and f). the vector containing the WT FOXO3 3’UTR, but mutated FOXO3 did not change significantly (Fig. 5b). Moreover, MiR-1290 regulates C2C12 myogenesis and myotube after transfecting miR-1290 mimics in C2C12 cells, we atrophy by directly targeting FOXO3 found gradually increasing FOXO3 expression levels in the To investigate the molecular mechanisms by which miR- cytoplasm and decreasing levels in the nucleus (Fig. 5c). To 1290 mediates C2C12 myogenic regulation and muscle atro- confirm whether FOXO3 affected myoblasts, siFOXO3 was phy, we hypothesized that miR-1290 may promote myo- transfected into C2C12 myoblasts. Western blot analysis genic differentiation and inhibit myotube atrophy by directly showed that siFOXO3 inhibited FOXO3 expression success- targeting FOXO3. To explore whether miR-1290 directly fully (Fig. 5e). The MHC-positive cell areas were markedly targets FOXO3, we purchased wild-type (WT) and mutant increased upon FOXO3 knockdown (Fig. 5g), and protein (MUT) 3’UTR psi-CHECK2, which contained the miR- levels of MyoD and MyoG were also increased (Fig. 5i). Che et al. Skeletal Muscle (2021) 11:6 Page 6 of 12 Fig. 3 The effect of miR-1290 mimic transfection on C2C12 cells under TNF-α. a and b Giemsa staining and myotube diameters among all groups. c–e The western blot analysis and quantification of MuRF1 and atrogin-1 after overexpression of miR-1290 in TNF-α-induced atrophy. GAPDH was used as an internal control for western blot analysis. The statistical difference among miR-1290 transfection group and other groups were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 Taken together, these results suggestthatmiR-1290 pro- factor in an inactive state in the cytoplasm. As shown in motes myoblast differentiation by activating AKT/p70 Fig. 6c, the phosphorylation protein levels of AKT and pathway-mediated FOXO3 phosphorylation, which conse- P70 were increased in the miR-1290 transfection group, quently prevents FOXO3 nuclear translocation. which confirmed that miR-1290 may promote myoblast differentiation via AKT/P70/FOXO3 signaling pathway Role of protein kinase B (AKT)/P70/FOXO3 signaling modulation. Next, we added AKT inhibitor (GDC-0068) pathway in miR-1290’s effect on myogenic differentiation to C2C12 myotubes. Western blot analysis showed de- It is known that activation of AKT induces FOXO3 creased expression of MyoD and MyoG, as well as de- phosphorylation, which maintains the transcription creased MHC measured by IF (Fig. 6a and c). However, Che et al. Skeletal Muscle (2021) 11:6 Page 7 of 12 Fig. 4 Effect of miR-1290 on muscle atrophy rat model. a The typical gastrocnemius muscle after injecting miR-NC and miR-1290 respectively in LPS-induced atrophy mice model. b The gastrocnemius muscle weight (GW) ratio after injecting miR-NC and miR-1290 respectively in LPS- induced atrophy mice model. c and d The body weight and the gastrocnemius muscle weight/body weight (GW/BW) ratio after injecting miR- NC and miR-1290 respectively in LPS-induced atrophy mice model. e HE staining after injecting miR-NC and miR-1290 respectively in LPS-induced atrophy mice model. f The muscle fiber cross-sectional area after injecting miR-NC and miR-1290 respectively in LPS-induced atrophy mice model. The statistical difference among miR-1290 mimic group and NC groups were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 miR-1290 transfection did not increase the expression of Role of protein kinase B (AKT)/P70/FOXO3 signaling MHC, MyoD, and MyoG after GDC-0068 treatment. pathway in miR-1290’s effect on myotube atrophy Furthermore, after adding GDC-0068, miR-1290 did not To explore the effect of miR-1290 on muscle atrophy, we increase the phosphorylation of AKT and P70 (Fig. 6c). measured myotube diameters after treating TNF-α- In C2C12 myoblasts, we observed increased FOXO3 induced C2C12 myotubes with miR-1290 and/or GDC- protein in the cytoplasm and a decreased level in the nu- 0068 (Fig. 7a). As shown in Fig. 7c, miR-1290 significantly cleus after miR-1290 transfection. However, combination increased the phosphorylation of both AKT and P70 and treatment of miR-1290 and GDC-0086 did not increase decreased the expression of MuRF1 and atrogin-1 even FOXO3 in the cytoplasm or decrease FOXO3 in the nu- after TNF-α treatment, while this ability disappeared after cleus of C2C12 myoblasts (Fig. 6c). Taken together, we GDC-0086 treatment. Moreover, miR-1290 transfection conclude that miR-1290 promotes myogenesis by acti- did not activate FOXO3 phosphorylation or promote vating AKT/P70 signaling-mediated FOXO3 nuclear translocation of FOXO3 from the nucleus to the cyto- translocation. plasm after inhibiting the AKT pathway (Fig. 7c). Che et al. Skeletal Muscle (2021) 11:6 Page 8 of 12 Fig. 5 The intrinsic mechanism of miR-1290’s effect. a Bioinformatics analysis was performed to predict the miR-1290-binding seed sequence in the 3’UTR of FoxO3. b The luciferase result of miR-1290 and FOXO3. c and d The western blot analysis and quantification to determine FoxO3 levels in cytoplasm and nucleus in miR-1290-transfected C2C12 myoblast. e and f The knockdown efficiency of FoxO3-specific siRNA was confirmed by western blot. g and h MHC staining was performed after FoxO3 knockdown (scale bar, 50 μm). i and j.The expression of MyoD and MyoG were analyzed by western blot. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and other groups were considered significant at the levels of * P < 0.05, **P <0.01, or ***P <0.001 Discussion The proliferation and differentiation of muscle cells This study was designed to investigate the role of miR- are important biological processes in skeletal myogen- 1290 in C2C12 myoblast cells. In this study, miR-1290 esis, and foremost factors against muscle atrophy [19]. expression accelerated the differentiation of C2C12 cells. When proliferation and differentiation are insufficient, The AKT/P70/ FoxO3 signaling pathway played crucial muscle atrophy occurs when the rate of protein degrad- roles in the myoblast differentiation Importantly, the ation in body tissue exceeds the rate of protein synthesis. promoted differentiation and protective effect against at- The differentiation stage of myoblast cells is a complex rophy were validated using a rat atrophy model. process regulated by specific transcription factors [20]. Che et al. Skeletal Muscle (2021) 11:6 Page 9 of 12 Fig. 6 MiR-1290 activates AKT/P70/FoxO3 signaling pathways during myoblast differentiation. a MHC staining performed after treated miR-1290/ miR-NC with or without GDC-0068 (scale bar, 50 μm). b MHC-positive areas/total areas were quantified using Harmony 4.1 software (n = 6). c–f The western blot analysis and quantification of phosphorylated and all forms of AKT and P70, MyoG, and MyoD, after transfecting miR-1290/miR- NC with or without GDC0068. GDC-0068 inhibited miR-1290-activated phosphorylation of AKT and P70 in C2C12 myoblasts. Western blot to analyze FoxO3 expression levels in cytoplasm and nucleus of C2C12 myoblasts. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and inhibitor group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 First, proteins related to the cell cycle, such as cyclin D1, study, TNF-α downregulated the myoD genes and protein cyclin E, and CDK4, can promote cell proliferation in nor- levels. However, the MyoD and MyoG genes, and protein mal and cancer cells [17]. Meanwhile, transcription factors levels were enhanced after miR-1290 transfection. There- such as MYF5 (Myogenic factor 5), MYF6 (Myogenic fac- fore, miR-1290 promoted the differentiation process. tor 6), MyoD (Myogenic differentiation antigen), MyoG Several proteins and signaling pathways participate in (Myogenin), and MRF4 (Myogenic regulatory factor 4), the regulation of the myoblast differentiation process, also called the myogenic regulatory factor family, can such as AKT, p70, AMP-activated protein kinase regulate differentiation into myotubes [21, 22]. The pro- (AMPK), FoxO3, IGF-1, and TGF-β [24, 25]. AKT and inflammatory cytokine TNF-α elicits muscle proteolysis its downstream p70 can receive upstream stimuli, such and blocks myogenesis by destabilizing MyoD [23]. In this as IGF-1, during myoblast differentiation and muscle Che et al. Skeletal Muscle (2021) 11:6 Page 10 of 12 Fig. 7 Role of Protein kinase B (AKT)/P70/FOXO3 signaling pathway in effect of miR-1290 on myotube atrophy. a Giemsa staining was performed to calculate myotube diameters for TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 treatments. b Cell diameters of five groups were measured. c–f Western blot analysis and quantification of phosphorylated and all forms of AKT and P70 of C1C12 myotubes for TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 treatments. Western blot was performed to analyze the expression of MuRF1 and atrogin-1 in TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 groups. After treatment with miR-1290/miR-NC or with GDC-0068, FoxO3 expression levels in cytoplasm and nucleus of C2C12 myotubes were examined by western blot. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and inhibitor group were considered significant at the levels of *P < 0.05, **P < 0.01, or ***P < 0.001 mass maintenance and activation [26]. A previous report YY1. Consequently, the AKT pathway regulated energy indicated that phosphorylation of AKT/mTOR blocked homeostasis processes in myoblasts, including intracellu- FoxO3 translocation to the nucleus, and therefore, lar ATP synthesis, citrate synthase activity, glycolysis, muscle atrophy was inhibited [27]. The mTOR phos- mitochondrial DNA copy maintenance, gene transcrip- phorylation also elicited the downstream targets such as tion, and protein translation, which are linked to energy Che et al. Skeletal Muscle (2021) 11:6 Page 11 of 12 metabolism [28]. In this study, an AKT inhibitor, GDC- alpha; HE: Hematoxylin–eosin; SD: Standard deviation; WT: Wild-type; MYF5: Myogenic factor 5; MYF6: Myogenic factor 6; MyoD: Myogenic 0068, decreased the MHC-positive ratio compared with differentiation antigen; MRF4: Myogenic regulatory factor 4; AMPK: AMP- the miR-1290 transfection group, and downregulated the activated protein kinase; IGF-1: Insulin-like growth factor 1; TGF- MyoD and MyoG genes and proteins levels, thus validat- β: Transforming growth factor-β; mTOR: Mammalian target of rapamycin; ROS: Reactive oxygen species; IKK1: IkB kinase complex; SOCS4: Suppressor of ing the crucial role of AKT in miR-1290’s regulatory ef- cytokine signaling 4; INPP4B: Inositol polyphosphate 4-phosphatase B fect on C2C12 cells. The transcription factor FOXO3 is one member of the human FoxO family [29]. FOXO3 is Acknowledgements widely expressed in skeletal muscle tissue and partici- Not applicable pates in numerous cellular responses such as autophagy, Authors’ contributions apoptosis, stem cell homeostasis, and ROS diminishing (I) Conception and design: JC, CX, XW, and YZ; (II) Administrative support: YZ; [30]. FOXO3 activation in the nucleus accelerates the (III) Provision of study materials or patients: YM, QH, PJ, XW, and YW; (IV) upregulation of Mafx and Murf1 mRNA [31], as well as Collection and assembly of data: JC and CXu; (V) Data analysis and interpretation: JC and CX; (VI) Manuscript writing: all authors; (VII) Final MAFbx and HDAC6 proteins [32]. In this study, FOXO3 approval of manuscript: all authors. was found to be the target of miR-1290. MiR-1290 is a miRNA that often contributes to cancer Funding development. MiR-1290 accelerated cell proliferation This work was supported by grants from Shanghai Key disciplines program of Health and Family Planning (No: 2017ZZ02010) and the National Key R&D and invasion in the pathological processes of gastric can- Program of “Strategic Advanced Electronic Materials” (No: 2017YFB0403803). cer [33], esophageal squamous cell carcinoma [34], lung cancer [13], pancreatic carcinoma [35], and colorectal Availability of data and materials cancer [36]. Therefore, miR-1290 was recognized as a The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. cancer biomarker [37], and thus a potential therapeutic target of anti-cancer compounds [38]. Additionally, miR- Declarations 1290 expression enhanced the production of pulmonary fibrosis markers. Most importantly, miR-1290 acceler- Ethics approval and consent to participate The clinical study was approved by the Ethics Committee of Huadong ated the differentiation of hepatocyte-like cells from Hospital (approval code: 2016K049), this study was carried out in accordance mesenchymal stem cells [39]. This report hinted at the with the World Medical Association’s Declaration of Helsinki. All participants possibility of miR-1290’s effect on myoblast cells. In this were informed about the objectives of the study, and they all signed the Informed Consent Form. study, we found that the miR-1290 mimic successfully All animal care and experimental procedures were approved by and promoted myoblast cell differentiation and exerted a performed following the standards of the Animal Welfare Committees of protective effect against muscle atrophy in vivo. Previous Fudan University in Shanghai, China [No.202006007S]. reports revealed that targets of miRNA contained the BY Consent for publication targeting nuclear factor I/X [34], IkB kinase complex Not applicable (IKK1) [40], suppressor of cytokine signaling 4 (SOCS4) [41], interferon regulatory factor 2 [42], LHX6 [43], and Competing interests inositol polyphosphate 4-phosphatase B (INPP4B) [44]. The authors declare that they have no competing interests. Our study is the first to reveal that FOXO3, a critical Author details skeletal muscle myogenesis regulation protein, is the tar- 1 Department of Pain, Huadong Hospital, Shanghai Key Laboratory of Clinical get of miR-1290. Geriatric Medicine, Fudan University, No. 221, West YanAn Rd, Shanghai 200040, P.R. China. Department of Osteoporosis and Bone Disease, Huadong Hospital, Research Section of Geriatric Metabolic Bone Disease, Shanghai Conclusion Geriatric Institute, Shanghai, China. In summary, the present study revealed the role of miR- Received: 16 November 2020 Accepted: 28 February 2021 1290 in myoblast cell differentiation and muscle atrophy. The miR-1290 mimics accelerated C2C12 cell differenti- ation. The AKT/P70/ FoxO3 signaling pathway plays References 1. Mafakheri S, Flörke RR, Kanngießer S, Hartwig S, Espelage L, De Wendt C, crucial roles in the effect of miR-1290 in myogenesis. Schönberger T, Hamker N, Lehr S, Chadt A, Al-Hasani H. AKT and AMP- The promoted differentiation and protective effects on activated protein kinase regulate TBC1D1 through phosphorylation and its atrophy were validated using a rat atrophy model. interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP. J Biol Chem. 2018;293:17853–62. Abbreviations 2. Romagnoli C, Pampaloni B, Brandi ML. Muscle endocrinology and its AIDS: Acquired immunodeficiency syndrome; miRNAs: MicroRNAs; relation with nutrition. Aging Clin Exp Res. 2019;31:783–92. FOXO3: Forkhead Box O3; AKT: Protein kinase B; PBS: Phosphate-buffered 3. Waltz TB, Fivenson EM, Morevati M, Li C, Becker KG, Bohr VA, Fang EF. saline; DMEM: Dulbecc o’s Modified Eagle’s Medium; MHC: Myosin heavy Sarcopenia, aging and prospective interventional strategies. Curr Med chain; MyoG: Myogenin; MuRF1: Muscle-specific RING Finger 1; Chem. 2018;25:5588–96. IF: Immunofluorescence; qPCR: Real-time polymerase chain reaction; 4. Li J, Chan MC, Yu Y, Bei Y, Chen P, Zhou Q, Cheng L, Chen L, Ziegler O, SD: Sprague Dawley; SPF: Specific pathogen-free; NC: Negative control; Rowe GC, Das S, Xiao J. miR-29b contributes to multiple types of muscle BW: Body weight; GW: Gastrocnemius weight; TNF-α: Tumor necrosis factor atrophy. Nat Commun. 2017;8:15201. Che et al. Skeletal Muscle (2021) 11:6 Page 12 of 12 5. Rao VK, Kapp D, Schroth M. Gene therapy for spinal muscular atrophy: an mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can emerging treatment option for a devastating disease. J Manag Care Spec prevent muscle atrophy in vivo. Nat Cell Biol. 2001;3:1014–9. Pharm. 2018;24:S3–s16. 29. Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, 6. Cao RY, Li J, Dai Q, Li Q, Yang J. Muscle atrophy: present and future. Adv Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL, Schiaffino S, Sandri M. Exp Med Biol. 2018;1088:605–24. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab. 2007;6: 458–71. 7. Sabourin LA, Rudnicki MA. The molecular regulation of myogenesis. Clin Genet. 2000;57:16–25. 30. Morris BJ, Willcox DC, Donlon TA, Willcox BJ. FOXO3: a major gene for human longevity--a mini-review. Gerontology. 2015;61:515–25. 8. Bhaskaran M, Mohan M. MicroRNAs: history, biogenesis, and their evolving 31. Nakao R, Abe T, Yamamoto S, Oishi K. Ketogenic diet induces skeletal role in animal development and disease. Vet Pathol. 2014;51:759–74. muscle atrophy via reducing muscle protein synthesis and possibly 9. Zhang J, Hua C, Zhang Y, Wei P, Tu Y, Wei T. KAP1-associated transcriptional activating proteolysis in mice. Sci Rep. 2019;9:19652. inhibitory complex regulates C2C12 myoblasts differentiation and 32. Wang J, Gao X, Ren D, Zhang M, Zhang P, Lu S, Huan C, Yao Y, Zheng L, mitochondrial biogenesis via miR-133a repression. Cell Death Dis. 2020;11: Bao Z, Zhou J. Triptolide induces atrophy of myotubes by triggering IRS-1 degradation and activating the FoxO3 pathway. Toxicol In Vitro. 2020;65: 10. Gan M, Shen L, Liu L, Guo Z, Wang S, Chen L, Zheng T, Fan Y, Tan Y, Jiang D, Li X, Zhang S, Zhu L. miR-222 is involved in the regulation of genistein 33. Huang J, Shen M, Yan M, Cui Y, Gao Z, Meng X. Exosome-mediated transfer on skeletal muscle fiber type. J Nutr Biochem. 2020;80:108320. of miR-1290 promotes cell proliferation and invasion in gastric cancer via 11. Houzelle A, Dahlmans D, Nascimento EBM, Schaart G, Jörgensen JA, NKD1. Acta Biochim Biophys Sin (Shanghai). 2019;51:900–7. Moonen-Kornips E, Kersten S, Wang X, Hoeks J. MicroRNA-204-5p modulates 34. Mao Y, Liu J, Zhang D, Li B. MiR-1290 promotes cancer progression by mitochondrial biogenesis in C2C12 myotubes and associates with oxidative targeting nuclear factor I/X(NFIX) in esophageal squamous cell carcinoma capacity in humans. J Cell Physiol. 2020;235:9851–63. (ESCC). Biomed Pharmacother. 2015;76:82–93. 12. Chen X, Huang Y, Shi D, Nie C, Luo Y, Guo L, Zou Y, Xie C. LncRNA ZNF667- 35. Tavano F, Gioffreda D, Valvano MR, Palmieri O, Tardio M, Latiano TP, Piepoli AS1 promotes ABLIM1 expression by adsorbing micro RNA-1290 to A, Maiello E, Pirozzi F, Andriulli A. Droplet digital PCR quantification of miR- suppress nasopharyngeal carcinoma cell progression. Onco Targets Ther. 1290 as a circulating biomarker for pancreatic cancer. Sci Rep. 2018;8:16389. 2020;13:4397–409. 36. Li H, Zhang H, Lu G, Li Q, Gu J, Song Y, Gao S, Ding Y. Mechanism analysis 13. Wu Y, Wei J, Zhang W, Xie M, Wang X, Xu J. Serum exosomal miR-1290 is a of colorectal cancer according to the microRNA expression profile. Oncol potential biomarker for lung adenocarcinoma. Onco Targets Ther. 2020;13: Lett. 2016;12:2329–36. 7809–18. 37. Imaoka H, Toiyama Y, Fujikawa H, Hiro J, Saigusa S, Tanaka K, Inoue Y, Mohri 14. Guan S, Wu Y, Zhang Q, Zhou J. TGF-β1 induces CREB1-mediated miR-1290 Y, Mori T, Kato T, Toden S, Goel A, Kusunoki M. Circulating microRNA-1290 upregulation to antagonize lung fibrosis via Napsin A. Int J Mol Med. 2020; as a novel diagnostic and prognostic biomarker in human colorectal cancer. 46:141–8. Ann Oncol. 2016;27:1879–86. 15. D'Andrea P, Civita D, Cok M, Ulloa Severino L, Vita F, Scaini D, Casalis L, 38. Zhou W, Wang S, Ying Y, Zhou R, Mao P. miR-196b/miR-1290 participate in Lorenzon P, Donati I, Bandiera A. Myoblast adhesion, proliferation and the antitumor effect of resveratrol via regulation of IGFBP3 expression in differentiation on human elastin-like polypeptide (HELP) hydrogels. J Appl acute lymphoblastic leukemia. Oncol Rep. 2017;37:1075–83. Biomater Funct Mater. 2017;15:e43–53. 39. Zhou X, Cui L, Zhou X, Yang Q, Wang L, Guo G, Hou Y, Cai W, Han Z, Shi Y, 16. Liang T, Zhou B, Shi L, Wang H, Chu Q, Xu F, Li Y, Chen R, Shen C, Schinckel Han Y. Induction of hepatocyte-like cells from human umbilical cord- AP. lncRNA AK017368 promotes proliferation and suppresses differentiation derived mesenchymal stem cells by defined microRNAs. J Cell Mol Med. of myoblasts in skeletal muscle development by attenuating the function of 2017;21:881–93. miR-30c. FASEB J. 2018;32:377–89. 40. Ta N, Huang X, Zheng K, Zhang Y, Gao Y, Deng L, Zhang B, Jiang H, Zheng 17. Wang H, Shi L, Liang T, Wang B, Wu W, Su G, Wei J, Li P, Huang R. MiR-696 J. miRNA-1290 promotes aggressiveness in pancreatic ductal regulates C2C12 cell proliferation and differentiation by targeting CNTFRα. adenocarcinoma by targeting IKK1. Cell Physiol Biochem. 2018;51:711–28. Int J Biol Sci. 2017;13:413–25. 41. Xiao X, Yang D, Gong X, Mo D, Pan S, Xu J. miR-1290 promotes lung 18. Cheng M, Gao T, Xi F, Cao C, Chen Y, Zhao C, Li Q, Yu W. adenocarcinoma cell proliferation and invasion by targeting SOCS4. Dexmedetomidine ameliorates muscle wasting and attenuates the Oncotarget. 2018;9:11977–88. alteration of hypothalamic neuropeptides and inflammation in 42. Jin JJ, Liu YH, Si JM, Ni R, Wang J. Overexpression of miR-1290 contributes endotoxemic rats. PLoS One. 2017;12:e0174894. to cell proliferation and invasion of non small cell lung cancer by targeting 19. Powers SK, Kavazis AN, McClung JM. Oxidative stress and disuse muscle interferon regulatory factor 2. Int J Biochem Cell Biol. 2018;95:113–20. atrophy. J Appl Physiol. 1985;102(2007):2389–97. 43. Yan L, Cai K, Sun K, Gui J, Liang J. MiR-1290 promotes proliferation, 20. Piccirillo R, Demontis F, Perrimon N, Goldberg AL. Mechanisms of muscle migration, and invasion of glioma cells by targeting LHX6. J Cell Physiol. growth and atrophy in mammals and Drosophila. Dev Dyn. 2014;243:201– 2018;233:6621–9. 44. Ma Q, Wang Y, Zhang H, Wang F. miR-1290 contributes to colorectal cancer 21. Endo T. Molecular mechanisms of skeletal muscle development, cell proliferation by targeting INPP4B. Oncol Res. 2018;26:1167–74. regeneration, and osteogenic conversion. Bone. 2015;80:2–13. 22. Braun T, Gautel M. Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis. Nat Rev Mol Cell Biol. 2011;12:349– Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in 23. Plant PJ, Brooks D, Faughnan M, Bayley T, Bain J, Singer L, Correa J, Pearce published maps and institutional affiliations. D, Binnie M, Batt J. Cellular markers of muscle atrophy in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2010;42:461–71. 24. Foletta VC, White LJ, Larsen AE, Léger B, Russell AP. The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy. Pflugers Arch. 2011;461:325–35. 25. Comai G, Tajbakhsh S. Molecular and cellular regulation of skeletal myogenesis. Curr Top Dev Biol. 2014;110:1–73. 26. Jia L, Li YF, Wu GF, Song ZY, Lu HZ, Song CC, Zhang QL, Zhu JY, Yang GS, Shi XE. MiRNA-199a-3p regulates C2C12 myoblast differentiation through IGF-1/AKT/mTOR signal pathway. Int J Mol Sci. 2013;15:296–308. 27. Sanchez AM, Candau RB, Bernardi H. FoxO transcription factors: their roles in the maintenance of skeletal muscle homeostasis. Cell Mol Life Sci. 2014;71: 1657–71. 28. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD. Akt/

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