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Lnc-ORA interacts with microRNA-532-3p and IGF2BP2 to inhibit skeletal muscle myogenesis

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100376

1083-351X

Rui Cai, Que Zhang, Yingqian Wang, Wenlong Yong, Rui Zhao, Weijun Pang,

RNA modifications and cancer

Skeletal muscle is one of the most important organs of the animal body. Long noncoding RNAs play a crucial role in the regulation of skeletal muscle development via several mechanisms. We recently identified obesity-related lncRNA (lnc-ORA) in a search for long noncoding RNAs that influence adipogenesis, finding it impacted adipocyte differentiation by regulating the PI3K/protein kinase B/mammalian target of rapamycin pathway. However, whether lnc-ORA has additional roles, specifically in skeletal muscle myogenesis, is not known. Here, we found that lnc-ORA was significantly differentially expressed with age in mouse skeletal muscle tissue and predominantly located in the cytoplasm. Overexpression of lnc-ORA promoted C2C12 myoblast proliferation and inhibited myoblast differentiation. In contrast, lnc-ORA knockdown repressed myoblast proliferation and facilitated myoblast differentiation. Interestingly, silencing of lnc-ORA rescued dexamethasone-induced muscle atrophy in vitro. Furthermore, adeno-associated virus 9–mediated overexpression of lnc-ORA decreased muscle mass and the cross-sectional area of muscle fiber by upregulating the levels of muscle atrophy–related genes and downregulating the levels of myogenic differentiation–related genes in vivo. Mechanistically, lnc-ORA inhibited skeletal muscle myogenesis by acting as a sponge of miR-532-3p, which targets the phosphatase and tensin homolog gene; the resultant changes in phosphatase and tensin homolog suppressed the PI3K/protein kinase B signaling pathway. In addition, lnc-ORA interacted with insulin-like growth factor 2 mRNA-binding protein 2 and reduced the stability of myogenesis genes, such as myogenic differentiation 1 and myosin heavy chain. Collectively, these findings indicate that lnc-ORA could be a novel underlying regulator of skeletal muscle development. Skeletal muscle is one of the most important organs of the animal body. Long noncoding RNAs play a crucial role in the regulation of skeletal muscle development via several mechanisms. We recently identified obesity-related lncRNA (lnc-ORA) in a search for long noncoding RNAs that influence adipogenesis, finding it impacted adipocyte differentiation by regulating the PI3K/protein kinase B/mammalian target of rapamycin pathway. However, whether lnc-ORA has additional roles, specifically in skeletal muscle myogenesis, is not known. Here, we found that lnc-ORA was significantly differentially expressed with age in mouse skeletal muscle tissue and predominantly located in the cytoplasm. Overexpression of lnc-ORA promoted C2C12 myoblast proliferation and inhibited myoblast differentiation. In contrast, lnc-ORA knockdown repressed myoblast proliferation and facilitated myoblast differentiation. Interestingly, silencing of lnc-ORA rescued dexamethasone-induced muscle atrophy in vitro. Furthermore, adeno-associated virus 9–mediated overexpression of lnc-ORA decreased muscle mass and the cross-sectional area of muscle fiber by upregulating the levels of muscle atrophy–related genes and downregulating the levels of myogenic differentiation–related genes in vivo. Mechanistically, lnc-ORA inhibited skeletal muscle myogenesis by acting as a sponge of miR-532-3p, which targets the phosphatase and tensin homolog gene; the resultant changes in phosphatase and tensin homolog suppressed the PI3K/protein kinase B signaling pathway. In addition, lnc-ORA interacted with insulin-like growth factor 2 mRNA-binding protein 2 and reduced the stability of myogenesis genes, such as myogenic differentiation 1 and myosin heavy chain. Collectively, these findings indicate that lnc-ORA could be a novel underlying regulator of skeletal muscle development. Skeletal muscle development is regulated by a series of myogenic regulatory factors (MRFs) (1Braun T. Gautel M. Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis.Nat. Rev. Mol. Cell. Biol. 2011; 12: 349-361Crossref PubMed Scopus (361) Google Scholar). The MRF family plays a crucial positive role in skeletal muscle myogenic determination and differentiation during embryogenesis and postnatal myogenesis and includes myogenic differentiation 1 (MyoD), myogenic factor 5, myogenin (MyoG), and myogenic regulatory factor 4 (2Buckingham M. Rigby P.W. Gene regulatory networks and transcriptional mechanisms that control myogenesis.Dev. Cell. 2014; 28: 225-238Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Once MRFs have been activated, many myogenic transcription factors form obligate heterodimers with their coregulators to activate the myoblast differentiation program by regulating the transcription of many genes, including coding and noncoding genes. In addition, myoblast proliferation leads to an increase in the number of nuclei, contributing to muscle growth in some forms of muscle hypertrophy in adults. Skeletal muscle atrophy is controlled by the balance between the protein degradation rate and protein synthesis rate and induced by various stressors, including starvation, denervation, mechanical unloading, inflammation, and aging (3Schiaffino S. Dyar K.A. Ciciliot S. Blaauw B. Sandri M. Mechanisms regulating skeletal muscle growth and atrophy.FEBS J. 2013; 280: 4294-4314Crossref PubMed Scopus (667) Google Scholar). This balance reflects the physiological condition of the muscle fiber, and breakage results in muscular dystrophy (4Bonaldo P. Sandri M. Cellular and molecular mechanisms of muscle atrophy.Dis. Model Mech. 2013; 6: 25-39Crossref PubMed Scopus (598) Google Scholar). Two critical protein degradation pathways, the autophagic–lysosomal and ubiquitin–proteasome systems, are activated during skeletal muscle atrophy (5Jackman R.W. Cornwell E.W. Wu C.L. Kandarian S.C. Nuclear factor-kappaB signalling and transcriptional regulation in skeletal muscle atrophy.Exp. Physiol. 2013; 98: 19-24Crossref PubMed Scopus (44) Google Scholar, 6Jiao J. Demontis F. Skeletal muscle autophagy and its role in sarcopenia and organismal aging.Curr. Opin. Pharmacol. 2017; 34: 1-6Crossref PubMed Scopus (61) Google Scholar). These pathways include many atrophy-related genes, which are regulated by specific transcription factors. Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) are atrophy markers, which represent two of many E3 ubiquitin ligases that are mostly expressed in skeletal muscle. Knockdown of MAFbx prevents skeletal muscle loss during fasting, and the absence of MuRF1 alleviates dexamethasone (Dex)-induced muscle atrophy in mice (7Bodine S.C. Baehr L.M. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1.Am. J. Physiol. Endocrinol. Metab. 2014; 307: E469-484Crossref PubMed Scopus (416) Google Scholar). The PI3K/protein kinase B (AKT) signaling pathway is one of the signaling pathways that regulate mammalian skeletal muscle atrophy (8Wimmer R.J. Russell S.J. Schneider M.F. Green tea component EGCG, insulin and IGF-1 promote nuclear efflux of atrophy-associated transcription factor Foxo1 in skeletal muscle fibers.J. Nutr. Biochem. 2015; 26: 1559-1567Crossref PubMed Scopus (12) Google Scholar). The animal genome contains abundant noncoding RNAs, which serve as regulators of gene expression at the transcriptional, translational, and epigenetic levels. Noncoding RNAs regulate various muscle biological processes (9Jandura A. Krause H.M. The new RNA world: Growing evidence for long noncoding RNA functionality.Trends Genet. 2017; 33: 665-676Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 10Cai R. Sun Y.M. Qimuge N.R. Wang G.Q. Wang Y. Chu G.Y. Yu T.Y. Yang G.S. Pang W.J. Adiponectin AS lncRNA inhibits adipogenesis by transferring from nucleus to cytoplasm and attenuating Adiponectin mRNA translation.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2018; 1863: 420-432Crossref PubMed Scopus (37) Google Scholar, 11Pang W.J. Lin L.G. Xiong Y. Wei N. Wang Y. Shen Q.W. Yang G.S. Knockdown of PU.1 AS lncRNA inhibits adipogenesis through enhancing PU.1 mRNA translation.J. Cell. Biochem. 2013; 114: 2500-2512Crossref PubMed Scopus (62) Google Scholar). Recent studies have confirmed that functional long noncoding RNAs (lncRNAs) are involved in skeletal muscle development, including skeletal muscle cell proliferation, differentiation, injury, atrophy, and regeneration by chromatin remodeling, transcription regulation, and microRNA sponge absorption (12Wang S.S. Jin J.J. Xu Z.Y. Zuo B. Functions and regulatory mechanisms of lncRNAs in skeletal myogenesis, muscle disease and meat production.Cells. 2019; 8: 1107Crossref Scopus (11) Google Scholar, 13Geng T. Liu Y. Xu Y. Jiang Y. Zhang N. Wang Z. Carmichael G.G. Taylor H.S. Li D. Huang Y. H19 lncRNA promotes skeletal muscle insulin sensitivity in part by targeting AMPK.Diabetes. 2018; 67: 2183-2198Crossref PubMed Scopus (34) Google Scholar, 14Wang L. Zhao Y. Bao X. Zhu X. Kwok Y.K. Sun K. Chen X. Huang Y. Jauch R. Esteban M.A. LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration.Cell Res. 2015; 25: 335-350Crossref PubMed Scopus (147) Google Scholar, 15Yu X. Zhang Y. Li T. Ma Z. Jia H. Chen Q. Zhao Y. Zhai L. Zhong R. Li C. Long non-coding RNA Linc-RAM enhances myogenic differentiation by interacting with MyoD.Nat. Commun. 2017; 8: 14016Crossref PubMed Scopus (83) Google Scholar, 16Zhou L. Sun K. Zhao Y. Zhang S. Wang X. Li Y. Lu L. Chen X. Chen F. Bao X. Linc-YY1 promotes myogenic differentiation and muscle regeneration through an interaction with the transcription factor YY1.Nat. Commun. 2015; 6: 10026Crossref PubMed Scopus (96) Google Scholar, 17Jin J.J. Lv W. Xia P. Xu Z.Y. Zheng A.D. Wang X.J. Wang S.S. Zeng R. Luo H.M. Li G.L. Zuo B. Long noncoding RNA SYISL regulates myogenesis by interacting with polycomb repressive complex 2.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E9802-9811Crossref PubMed Scopus (40) Google Scholar, 18Chen X. He L. Zhao Y. Li Y. Zhang S. Sun K. So K. Chen F. Zhou L. Lu L. Wang L. Zhu X. Bao X. Esteban M.A. Nakagawa S. et al.Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity.Cell Discov. 2017; 3: 17002Crossref PubMed Scopus (50) Google Scholar). Although the effects of these lncRNAs in skeletal muscle myogenesis have been partially characterized, the function and regulatory mechanism of obesity-related lncRNA (lnc-ORA) in this process remains elusive. In the present study, we found that lnc-ORA was significantly differentially expressed in skeletal muscle between two important developmental stages. Furthermore, the results indicated that lnc-ORA promoted myoblast proliferation, inhibited myoblast differentiation, and induced muscle atrophy in vitro. Moreover, overexpression of lnc-ORA reduced muscle mass and the cross-sectional area of muscle fibers by upregulating the levels of muscle atrophy–related genes and downregulating the levels of myoblast differentiation–related genes in vivo. Mechanistic investigations showed that lnc-ORA functioned as a sponge for miR-532-3p and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), which activated phosphatase and tensin homolog (PTEN) and attenuated PI3K/AKT signaling, a critical pathway of myogenesis and muscle atrophy. Based on the aforementioned results, our findings provide a novel strategy for the regulation of skeletal muscle development. To confirm whether lnc-ORA is associated with skeletal muscle development, the absolute expression of lnc-ORA in the tibialis anterior, gastrocnemius (GAS), and extensor digitorum longus muscles from 8-week-old and 52-week-old mice was examined. The results showed that the levels of lnc-ORA were much higher in 52-week-old mice than in 8-week-old mice (Fig. 1, A–C). Furthermore, the level of lnc-ORA was the highest at 12 h and then gradually decreased from 24 to 48 h during myoblast proliferation (Fig. 1D). The level of lnc-ORA gradually increased during the early stage and then decreased in the late stage of C2C12 cell differentiation (Fig. 1E), indicating that lnc-ORA was involved in myoblast proliferation and differentiation during muscle formation. To confirm the stability of lnc-ORA in C2C12 cells, a half-life experiment was performed. The results showed that the half-life of lnc-ORA was approximately 12 h, indicating that lnc-ORA is stably expressed in C2C12 cells (Fig. 1F). In addition, based on lnc-ORA–targeted genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. GO term analysis revealed that lnc-ORA participated in the regulation of skeletal muscle development, muscle atrophy, cellular metabolic process, muscle hypertrophy, especially myoblast proliferation and differentiation (Fig. 1G). KEGG analysis indicated that lnc-ORA could regulate biological processes through the PI3K/AKT, peroxisome proliferator–activated receptors, mammalian target of rapamycin, and forkhead box O signaling pathway (Fig. 1H). Collectively, these results imply that lnc-ORA is a potential regulator of skeletal muscle development, partly through the PI3K/AKT signaling pathway. To investigate the role of lnc-ORA in myoblast proliferation, lnc-ORA overexpression and knockdown experiments were carried out in proliferating C2C12 cells. The results showed that overexpression and knockdown of lnc-ORA worked well (Fig. S1, A and B). Overexpression of lnc-ORA increased the number of 5-ethynyl-20-deoxyuridine (EdU)-positive cells (Fig. 2, A and B). A flow cytometry analysis also indicated that overexpression of lnc-ORA increased the number of cells that progressed to S phase (Fig. 2, C and D). In addition, cell count assay showed that overexpression of lnc-ORA increased the total cell number (Fig. S1C). Moreover, overexpression of lnc-ORA increased the mRNA and protein levels of proliferation-related genes, including cyclin E, cyclin D1, and proliferating cell nuclear antigen (Fig. 2, E–G). Furthermore, knockdown of lnc-ORA decreased the number of EdU-positive cells (Fig. 3, A and B), the total cell number (Fig. S1D), the number of cells in S phase (Fig. 3, C and D), and the mRNA and protein levels of proliferation-related genes (Fig. 3, E–G). Taken together, these results indicate that lnc-ORA promotes myoblast proliferation.Figure 3Knockdown of lnc-ORA inhibits myoblast proliferation. A, EdU and DAPI (nuclei) staining analysis 24 h after transfection of silnc-ORA and si-NC in proliferating myoblasts (n = 3). The scale bar represents 200 μm. B, percentage of EdU-positive cells/total cells (n = 3). C, flow cytometry analysis 24 h after transfection of silnc-ORA and si-NC in proliferating myoblasts (n = 3). D, statistical results of flow cytometry (n = 3). E, mRNA levels of cyclin E, cyclin D1, and PCNA 24 h after knockdown of lnc-ORA (n = 3). F, Western blot detection of cyclin E, cyclin D1, and PCNA (n = 3). G, quantitation of protein level in F (n = 3). Data represent the mean ± SD. ∗p < 0.05; ∗∗p < 0.01. DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynyl-20-deoxyuridine; lnc, long noncoding; lnc-ORA, obesity-related lncRNA; PCNA, proliferating cell nuclear antigen; silnc-ORA, siRNA-lnc-ORA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To verify the effect of lnc-ORA on myogenic differentiation, overexpression and knockdown experiments were performed during C2C12 cell differentiation. C2C12 cells were induced by differentiation culture medium (Fig. S2A). After myoblasts were transfected with pcDNA3.1-lnc-ORA or siRNA-lnc-ORA (silnc-ORA) vector, the levels of lnc-ORA markedly changed after myogenic induction (Fig. S2, B and C), showing that overexpression and knockdown of lnc-ORA worked well. Overexpression of lnc-ORA decreased the number of myosin heavy chain (MyHC)-positive cells, the differentiation index, and the multinuclear fusion index (Fig. 4, A–C). Moreover, overexpression of lnc-ORA downregulated the mRNA and protein levels of myogenic markers, including MyoD, MyoG, and MyHC (Fig. 4, D–F). Conversely, knockdown of lnc-ORA increased the number of MyHC-positive cells and the differentiation index, as well as the multinuclear fusion index at day 4 after myogenic induction (Fig. 5, A–C). Knockdown of lnc-ORA upregulated the mRNA and protein levels of these markers (Fig. 5, D–F). Taken together, these results demonstrate that lnc-ORA inhibits myogenic differentiation of myoblasts.Figure 5Knockdown of lnc-ORA promotes myogenic differentiation. A, immunofluorescent staining of MyHC and DAPI (nuclei) on day 4 of differentiation after knockdown of lnc-ORA (n = 3). The scale bar represents 100 μm. B, statistical analysis of the differentiation index in A (n = 3). C, statistical analysis of the fusion index in A (n = 3). D, mRNA levels of myogenic genes on day 4 of differentiation after knockdown of lnc-ORA (n = 3). E, Western blot detection of MyoD, MyoG, and MyHC (n = 3). F, quantitation of protein levels in E (n = 3). Data represent the mean ± SD. ∗p < 0.05; ∗∗p < 0.01. DAPI, 4′,6-diamidino-2-phenylindole; lnc, long noncoding; lnc-ORA, obesity-related lncRNA; MyHC, myosin heavy chain; MyoD, myogenic differentiation 1; MyoG, myogenin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine whether lnc-ORA could influence muscle atrophy, a Dex-induced muscle atrophy model was used in C2C12 cells (Fig. 6A). The level of lnc-ORA was significantly upregulated by the Dex treatment (Fig. 6B), and an increase in the protein levels of MAFbx and MuRF1 also occurred (Fig. 6, C and D). Furthermore, knockdown of lnc-ORA markedly rescued Dex-induced muscle atrophy, as shown by MyHC staining (Fig. 6E). Knockdown of lnc-ORA decreased the levels of MAFbx and MuRF1 and increased the levels of myogenic differentiation factors (Fig. 6, F and G). Therefore, knockdown of lnc-ORA rescued Dex-induced muscle atrophy in vitro, suggesting that lnc-ORA could be a potential therapeutic target for treating muscle atrophy. To investigate the role of lnc-ORA in the regulation of muscle development in vivo, 10-week-old mice were injected with adeno-associated virus-GFP (AAV-GFP) or AAV-lnc-ORA overexpression virus and sacrificed after 8 weeks. Compared with the AAV-GFP control group, the AAV treatment group showed decreased size and mass of the tibialis anterior and GAS muscles (Fig. 7, A and B). Meanwhile, the muscle percentage of AAV-lnc-ORA–injected mice was lower (Fig. 7C). H&E staining showed that the cross-sectional areas of GAS fibers were dramatically smaller in the AAV-lnc-ORA group than in the AAV-GFP group (Fig. 7, D and E). Overexpression of lnc-ORA significantly decreased the expression levels of MyHC and MyoD but increased the expression levels of MAFbx and MuRF1 (Fig. 7, F and G). The PI3K/AKT signaling pathway was inhibited in the GAS muscles of AAV-lnc-ORA mice (Fig. 7, F and H). Together, these results suggest that overexpression of lnc-ORA decreases muscle mass and induces muscle atrophy in vivo. To investigate the molecular mechanism of lnc-ORA in myogenesis, FISH experiments were performed to confirm the subcellular locations of lnc-ORA in myoblasts and myotubes. The results showed that lnc-ORA was predominantly expressed in the cytoplasm of both myoblasts and myotubes (Fig. 8, A and B). This finding indicates that lnc-ORA plays a role in the post-transcriptional regulation mechanism in the cytoplasm. Moreover, we predicted that miR-532-3p with a conserved seed sequence could be adsorbed by lnc-ORA (Fig. S3, A and B) and that PTEN acts as a target of miR-532-3p (Fig. S3C). To confirm this hypothesis, we constructed wildtype and mutant dual luciferase reporters, psi-CHECK 2.0-lnc-ORA or psi-CHECK 2.0-PTEN 3'UTR (Fig. 9, A and B). The dual-luciferase reporter assay revealed that miR-532-3p bound to lnc-ORA transcripts and the PTEN 3′UTR (Fig. 9, C and D). Furthermore, significantly enriched miR-532-3p and lnc-ORA were measured through argonaute 2 RNA immunoprecipitation (RIP) assay (Fig. S3D). Moreover, lnc-ORA was significantly enriched by biotin-labeled miR-532-3p compared with the control or mutated miR-532-3p (Fig. S3E). We have also detected the absolute copy number of miR-532-3p under the different conditions. The results showed that the absolute copy number of miR-532-3p exhibited opposite trends as that of lnc-ORA under different conditions (Fig. S4, A–E). Based on the aforementioned results, we speculated that lnc-ORA acted as a competing endogenous RNA to sponge miR-532-3p. Furthermore, miR-532-3p mimics promoted myogenic differentiation, as shown by immunofluorescence staining of MyHC-positive myotubes (Fig. S5, A and B). Besides, rescue experiments indicated that miR-532-3p attenuated the positive effect of lnc-ORA on myoblast proliferation (Fig. 9, E and F), whereas it rescued the repressive effect of lnc-ORA on myoblast differentiation (Fig. 9, G and H). Moreover, miR-532-3p showed the opposite effect against lnc-ORA on the protein levels of PTEN, phosphorylated PI3K (p-PI3K), and phosphorylated AKT (p-AKT) (Fig. 9, I and J). In contrast, lnc-ORA knockdown reduced the protein level of PTEN and increased the protein levels of p-PI3K and p-AKT (Fig. S6). Therefore, lnc-ORA inhibited myogenic differentiation and promoted muscle atrophy through absorption of miR-532-3p via PTEN/PI3K/AKT signaling pathway.Figure 9Lnc-ORA functions as a ceRNA for miR-532-3p to control myogenesis. A and B, schematic of the double luciferase assay vector. C and D, analysis of the luciferase reporter assay (n = 3). E, EdU and DAPI (nuclei) staining analysis. The scale bar represents 200 μm. F, statistical analysis in E (n = 3). G, immunofluorescent staining of MyHC analysis. The scale bar represents 200 μm. H, statistical analysis in G (n = 3). I, Western blot detection of PTEN and myogenic factors (n = 3). J, statistical analysis in I (n = 3). Data represent the mean ± SD. ∗p < 0.05; ∗∗p < 0.01. ceRNA, competing endogenous RNA; DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynyl-20-deoxyuridine; lnc, long noncoding; lnc-ORA, obesity-related lncRNA; MyHC, myosin heavy chain; PTEN, phosphatase and tensin homolog.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further explore the mechanism by which lnc-ORA regulates myogenesis, we used a biotinylated lnc-ORA probe to perform an RNA pull-down assay, followed by mass spectrometry. Through this analysis, we identified IGF2BP2, an RNA-binding protein, as being bound to lnc-ORA, and we confirmed this interaction by Western blotting (Fig. 10A). Next, RIP was performed in C2C12 cells using an IGF2BP2 antibody. Lnc-ORA was present in the IGF2BP2 RIP sample at a much higher level than in the control IgG RIP sample (Fig. 10B). It has been demonstrated that IGF2BP2 could promote RNA stability. We then used this half-life assay to explore the RNA stability of lnc-ORA. The results showed that the interaction of lnc-ORA and IGFBP2 increased lnc-ORA stability and the ability to sponge miR-532-3p (Fig. 10, C and D). Moreover, lnc-ORA directly bound to IGF2BP2 and negatively regulated the IGF2BP2-mediated stability of myogenesis genes such as MyoD and MyHC (Fig. 10, E and F). Together, we used RNA pull-down experiments to identify IGF2BP2 as an important binding partner of lnc-ORA in the myogenesis process. Based on the aforementioned results, we suggest that lnc-ORA, acting as a miR-532-3p sponge and interacting with IGF2BP2 to control PTEN-mediated PI3K/AKT signaling, inhibits skeletal muscle myogenesis and induces muscle atrophy (Fig. 11).Figure 11Molecular regulatory mechanism of lnc-ORA that inhibits skeletal muscle myogenesis and induces muscle atrophy. Lnc-ORA controls PTEN protein levels and further activates the PI3K/AKT signaling pathway by functioning as ceRNA to sponge miR-532-3p and interact with IGF2BP2. AKT, protein kinase B; ceRNA, competing endogenous RNA; IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; lnc, long noncoding; lnc-ORA, obesity-related lncRNA; PTEN, phosphatase and tensin homolog.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Skeletal muscle is closely associated with physiological function, muscle strength, and metabolic performance, which influence human chronic disease, quality of life, and animal meat production. Recently, several studies have indicated that lncRNAs are implicated in skeletal muscle formation. However, the effect and molecular mechanism of lnc-ORA on myoblast proliferation, differentiation, and muscle atrophy are unknown. In the present study, we found that lnc-ORA was differentially expressed in mouse skeletal muscle with age, functioning as a miR-532-3p sponge and interacting with IGF2BP2 to inhibit myogenesis and induce muscle atrophy, and it could be a novel target for the regulation of skeletal muscle development. LncRNAs have been demonstrated to regulate myoblast proliferation and differentiation, such as Myoparr (19Hitachi K. Nakatani M. Takasaki A. Ouchi Y. Uezumi A. Ageta H. Inagaki H. Kurahashi H. Tsuchida K. Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation.EMBO. Rep. 2019; 20e47468Crossref PubMed Scopus (20) Google Scholar), Linc-smad7 (20Song C.C. Wang J. Ma Y.L. Yang Z.X. Dong D. Li H. Yang J. Huang Y. Plath M. Ma Y. Chen H. Linc-smad7 promotes myoblast differentiation and muscle regeneration via sponging miR-125b.Epigenetics. 2018; 13: 591-604Crossref PubMed Scopus (18) Google Scholar), Lnc-31 (21Ballarino M. Cazzella V. D'Andrea D. Grassi L. Bisceglie L. Cipriano A. Santini T. Pinnarò C. Morlando M. Tramontano A. Bozzoni I. Novel long noncoding RNAs (lncRNAs) in myogenesis: A miR-31 overlapping lncRNA transcript controls myoblast differentiation.Mol. Cell. Biol. 2015; 35: 728-736Crossref PubMed Scopus (64) Google Scholar), and Gm26917 (22Chen Z. Bu N.P. Qiao X.H. Zuo Z.X. Shu Y.H. Liu Z.L. Qian Z. Chen J. Hou Y. Forkhead Box M1 transcriptionally regulates the expression of long noncoding RNAs Snhg8 and Gm26917 to promote proliferation and survival of muscle satellite cells.Stem Cells. 2018; 36: 1097-1108Crossref PubMed Scopus (15) Google Scholar). Our previous study revealed that knockdown of lnc-ORA inhibited preadipocyte proliferation and differentiation (23Cai R. Tang G.R. Zhang Q. Yong W.L. Zhang W.R. Xiao J.Y. Wei C.S. He C. Yang G.S. Pang W.J. A novel lnc-RNA, named lnc-ORA, is identified by RNA-Seq analysis, and its knockdown inhibits adipogenesis by regulating the PI3K/AKT/mTOR signaling pathway.Cells. 2019; 8: 477Crossref Google Scholar). In the current study, we found that overexpression of lnc-ORA promoted myoblast proliferation but inhibited myotube formation. Furthermore, its knockdown had the opposite effect, implying that lnc-ORA is a vital negative regulatory factor of skeletal muscle myogenesis. An increasing number of studies have also indicated that lncRNAs, including lncIRS1 (24Li Z.H. Cai B.L. Abdalla B.A. Zhu X.N. Zheng M. Han P.G. Nie Q.H. Zhang X.Q. LncIRS1 controls muscle atrophy via sponging miR-15 family to activate IGF1-PI3K/AKT pathway.J. Cachexia Sarcopenia Muscle. 2019; 10: 391-410Crossref PubMed Scopus (47) Google Scholar), lncMUMA (25Zhang Z.K. Li J. Guan D. Liang C. Zhuo Z. Liu J. Lu A. Zhang G. Zhang B.T. Long noncoding RNA lncMUMA reverses established skeletal muscle atrophy following mechanical unloading.Mol. Ther. 2018; 26: 2669-2680Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), Atrolnc-1 (26Sun L. Si M. Liu X. Choi J.M. Wang Y. Thomas S.S. Peng H. Hu Z. Long-noncoding RNA Atrolnc-1 promotes muscle wasting in mice with chronic kidney disease.J. Cachexia Sarcopenia Muscle. 2018; 9: 962-974Crossref PubMed Scopus (27) Google Scholar), Pvt1 (27Alessio E. Buson L. Chemello F. Peggion C. Grespi F. Martini P. Massimino M.L. Pacchioni B. Millino C. Romualdi C. Bertoli A. Scorrano L. Lanfranchi G. Cagnin S. Single cell analysis reveals the involvement of the long non-coding RNA Pvt1 in the modulation of muscle atrophy and mitochondrial network.Nucleic Acids Res. 2019; 47: 1653-1670Crossref PubMed Scopus (30) Google Scholar), and SMN-AS1 (28d'Ydewalle C. Ramos D.M. Pyles N.J. Ng S.Y. Gorz M. Pilato C.M. Ling K. Kong L. Ward A.J. Rubin L.L. Rigo F. Bennett C.F. Sumner C.J. The antisense transcript SMN-AS1 regulates SMN expression and is a novel therapeutic target for spinal muscular atrophy.Neuron. 2017; 93: 66-79Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), play a significant role in the regulation of muscle atrophy. In the current study, the level of lnc-ORA markedly increased in a Dex-induced muscle atrophy model, and knockdown of lnc-ORA significantly rescued muscle atrophy by inhibiting MAFbx and MuRF1 and promoting myogenic differentiation factor expression. In addition, skeletal muscle atrophy is defined as a decline in skeletal muscle mass because of muscle cellular shrinkage (29Dumitru A. Radu B.M. Radu M. Cretoiu S.M. Muscle changes during atrophy.Adv. Exp. Med. Biol. 2018; 1088: 73-92Crossref PubMed Scopus (15) Google Scholar, 30Pin F. Novinger L.J. Huot J.R. Harris R.A. Couch M.E. O'Connell T.M. Bonetto A. PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia.FASEB J. 2019; 33: 7778-7790Crossref PubMed Scopus