MiR-96-5p Induced by Palmitic Acid Suppresses the Myogenic Differentiation of C2C12 Myoblasts by Targeting FHL1.
ABSTRACT: Skeletal myogenesis is a multi-stage process that includes the cell cycle exit, myogenic transcriptional activation, and morphological changes to form multinucleated myofibers. Recent studies have shown that saturated fatty acids (SFA) and miRNAs play crucial roles in myogenesis and muscle homeostasis. Nevertheless, the target molecules and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study investigated the critical role played by miR-96-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) significantly reduced FHL1 expression and inhibited the myogenic differentiation of C2C12 myoblasts but induced miR-96-5p expression. The knockdown of FHL1 by siRNA stimulated cell proliferation and inhibited myogenic differentiation of myoblasts. Interestingly, miR-96-5p suppressed FHL1 expression by directly targeting the 3'UTR of FHL1 mRNA. The transfection of an miR-96-5p mimic upregulated the expressions of cell cycle-related genes, such as PCNA, CCNB1, and CCND1, and increased myoblast proliferation. Moreover, the miR-96-5p mimic inhibited the expressions of myogenic factors, such as myoblast determination protein (MyoD), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C), and myosin heavy chain (MyHC), and dramatically impeded differentiation and fusion of myoblasts. Overall, this study highlights the role of miR-96-5p in myogenesis via FHL1 suppression and suggests a novel regulatory mechanism for myogenesis mediated by miRNA in a background of obesity.
Project description:Skeletal myogenesis is a complex process that is finely regulated by myogenic transcription factors. Recent studies have shown that saturated fatty acids (SFA) can suppress the activation of myogenic transcription factors and impair the myogenic differentiation of progenitor cells. Despite the increasing evidence of the roles of miRNAs in myogenesis, the targets and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study examined the implications of SFA-induced miR-183-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) drastically reduced myogenic transcription factors, such as myoblast determination protein (MyoD), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C), and inhibited FHL1 expression and myogenic differentiation of C2C12 myoblasts, accompanied by the induction of miR-183-5p. The knockdown of FHL1 by siRNA inhibited myogenic differentiation of myoblasts. Interestingly, miR-183-5p inversely regulated the expression of FHL1, a crucial regulator of skeletal myogenesis, by targeting the 3'UTR of FHL1 mRNA. Furthermore, the transfection of miR-183-5p mimic suppressed the expression of MyoD, MyoG, MEF2C, and MyHC, and impaired the differentiation and myotube formation of myoblasts. Overall, this study highlights the role of miR-183-5p in myogenic differentiation through FHL1 repression and suggests a novel miRNA-mediated mechanism for myogenesis in a background of obesity. [BMB Reports 2020; 53(11): 605-610].
Project description:Skeletal myogenesis is essential for the maintenance of muscle quality and quantity, and impaired myogenesis is intimately associated with muscle wasting diseases. Although microRNA (miRNA) plays a crucial role in myogenesis and relates to muscle wasting in obesity, the molecular targets and roles of miRNAs modulated by saturated fatty acids (SFA) are largely unknown. In the present study, we investigated the role of miR-320-3p on the differentiation of myogenic progenitor cells. Palmitic acid (PA), the most abundant dietary SFA, suppressed myogenic factors expression and impaired differentiation in C2C12 myoblasts, and these effects were accompanied by CFL2 downregulation and miR-320-3p upregulation. In particular, miR-320-3p appeared to target <i>CFL2</i> mRNA directly and suppress the expression of CFL2, an essential factor for filamentous actin (F-actin) depolymerization. Transfection of myoblasts with miR-320-3p mimic increased F-actin formation and nuclear translocation of Yes-associated protein 1 (YAP1), a key component of mechanotransduction. Furthermore, miR-320-3p mimic increased myoblast proliferation and markedly impeded the expression of MyoD and MyoG, consequently inhibiting myoblast differentiation. In conclusion, our current study highlights the role of miR-320-3p on CFL2 expression, YAP1 activation, and myoblast differentiation and suggests that PA-inducible miR-320-3p is a significant mediator of muscle wasting in obesity.
Project description:MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle. Using miRNA sequencing and bioinformatics analysis, we previously found that the miRNA miR-664-5p is significantly differentially expressed in longissimus dorsi muscles of Rongchang pigs. However, the molecular mechanism by which miR-664-5p regulates myogenesis remains unclear. In this study, using flow cytometry, 5-ethynyl-2'-deoxyuridine staining, and cell count and immunofluorescent assays, we found that cell-transfected miR-664-5p mimics greatly promoted proliferation of C2C12 mouse myoblasts by increasing the proportion of cells in the S- and G<sub>2</sub>-phases and up-regulating the expression of cell cycle genes. Moreover, miR-664-5p inhibited myoblast differentiation by down-regulating myogenic gene expression. In contrast, miR-664-5p inhibitor repressed myoblast proliferation and promoted myoblast differentiation. Mechanistically, using dual-luciferase reporter gene experiments, we demonstrated that miR-664-5p directly targets the 3'-UTR of serum response factor (<i>SRF</i>) and <i>Wnt1</i> mRNAs. We also observed that miR-664-5p inhibits both mRNA and protein levels of <i>SRF</i> and <i>Wnt1</i> during myoblast proliferation and myogenic differentiation, respectively. Furthermore, the activating effect of miR-664-5p on myoblast proliferation was attenuated by <i>SRF</i> overexpression, and miR-664-5p repressed myogenic differentiation by diminishing the accumulation of nuclear ?-catenin. Of note, miR-664-5p's inhibitory effect on myogenic differentiation was abrogated by treatment with Wnt1 protein, the key activator of the Wnt/?-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls <i>SRF</i> and canonical Wnt/?-catenin signaling pathways in myogenesis.
Project description:MicroRNAs are a class of 18-22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and ?-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation.
Project description:Skeletal myogenesis is a highly ordered and complex biological process that is mediated by numerous regulatory factors. In previous studies, it has been demonstrated that microRNAs (miRs) and long non?coding RNAs (lncRNAs) serve key roles in skeletal myogenesis. The present study showed that the expression levels of miR?23a?5p showed a dynamic change from decrease to increase during C2C12 myoblast proliferation and differentiation. Functional analysis using 5?ethynyl?2'?deoxyuridine proliferation and Cell Counting Kit?8 detection assays indicated that overexpression of miR?23a?5p significantly promoted C2C12 myoblast proliferation compared with the negative control. In addition, in C2C12 myoblasts transfected with miR?23a?5p mimics, increased expression levels of regulators associated with cell proliferation (Cyclin E, CCND1 and Cyclin B) were observed compared with the negative control. By contrast, overexpression of miR?23a?5p decreased the expression levels of specific?myogenesis factors (MyoD, MyoG and Myf5) and decreased C2C12 myoblast differentiation. Luciferase activity assays indicated that miR?23a?5p suppressed the luciferase activity of lncDum. Further analysis demonstrated that miR?23a?5p not only showed an opposite expression level pattern compared with lncDum, which was first increased and then decreased, but also had an opposite effect on the proliferation and differentiation of C2C12 myoblasts compared with lncDum which inhibited cell proliferation and promoted cell differentiation. Taken together, these results indicated that miR?23a?5p may mediate the proliferation and differentiation of C2C12 myoblasts, which may be involved in lncDum regulation.
Project description:Skeletal muscle is an important metabolic organ of the body, and impaired skeletal muscle differentiation can result in a wide range of metabolic diseases. It has been shown that microRNAs (miRNAs) play an important role in skeletal muscle differentiation. The aim of this study was to investigate the role of mmu-miR-324-5p in the differentiation of C2C12 myoblasts and lipid droplet deposition in myotubes for future targeted therapies. We found that mmu-miR-324-5p was highly expressed in mouse skeletal muscle. Overexpression of miR-324-5p significantly inhibited C2C12 myoblast differentiation while promoting oleate-induced lipid accumulation and ?-oxidation in C2C12 myoblasts. Conversely, inhibition of mmu-miR-324-5p promoted C2C12 myoblast differentiation and inhibited lipid deposition in myotubes. Mechanistically, mmu-miR-324-5p negatively regulated the expression of long non-coding Dum (lncDum) and peptidase M20 domain containing 1 (Pm20d1) in C2C12 myoblasts. Reduced lncDum expression was associated with a significant decrease in the expression of myogenesis-related genes. Knockdown of mmu-miR-324-5p increased the levels of lncDum and myogenesis-related gene expression. Following oleate-induced lipid deposition in C2C12 myoblasts, overexpression of mmu-miR-324-5p decreased the expression of Pm20d1 while increasing the expression of mitochondrial ?-oxidation and long-chain fatty acid synthesis-related genes. In conclusion, we provide evidence that miR-324-5p inhibits C2C12 myoblast differentiation and promotes intramuscular lipid deposition by targeting lncDum and Pm20d1, respectively.
Project description:The proliferation, apoptosis, and differentiation of myoblasts are essential processes in skeletal muscle development. During this developmental process, microRNAs (miRNAs) play crucial roles. In our previous RNA-seq study (accession number GSE62971), we found that miR-16-5p was differentially expressed between fast and slow growth in chicken. In this study, we report that miR-16-5p could inhibit myoblast proliferation, promote myoblast apoptosis, and repress myoblast differentiation by directly binding to the 3' UTR of SESN1, which is also differentially expressed. Overexpression of SESN1 significantly promoted the proliferation, inhibited apoptosis, and induced differentiation of myoblasts. Conversely, its loss of function hampered myoblast proliferation, facilitated myoblast apoptosis, and inhibited myoblast differentiation. Interestingly, we found SESN1 could regulate p53 by a feedback mechanism, thereby participating in the regulation of p53 signaling pathway, which suggests that this feedback is indispensable for myoblast proliferation and apoptosis. Altogether, these data demonstrated that miR-16-5p directly targets SESN1 to regulate the p53 signaling pathway, and therefore affecting myoblast proliferation and apoptosis. Additionally, SESN1 targets myogenic genes to control myoblast differentiation.
Project description:Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3'UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3'UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.
Project description:BACKGROUND: miRNA profiling performed in myogenic cells and biopsies from skeletal muscles has previously identified miRNAs involved in myogenesis. RESULTS: Here, we have performed miRNA transcriptome profiling in human affinity-purified CD56+ myoblasts induced to differentiate in vitro. In total, we have identified 60 miRNAs differentially expressed during myogenic differentiation. Many were not known for being differentially expressed during myogenic differentiation. Of these, 14 (miR-23b, miR-28, miR-98, miR-103, miR-107, miR-193a, miR-210, miR-324-5p, miR-324-3p, miR-331, miR-374, miR-432, miR-502, and miR-660) were upregulated and 6 (miR-31, miR-451, miR-452, miR-565, miR-594 and miR-659) were downregulated. mRNA transcriptome profiling performed in parallel resulted in identification of 6,616 genes differentially expressed during myogenic differentiation. CONCLUSIONS: This simultaneous miRNA/mRNA transcriptome profiling allowed us to predict with high accuracy target genes of myogenesis-related microRNAs and to deduce their functions.
Project description:The differentiation and regeneration of skeletal muscle from myoblasts to myotubes involves myogenic transcription factors, such as myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF). In addition, post-transcriptional regulation by miRNAs is required during myogenesis. Here, we provide evidence for novel mechanisms regulating MRTF-A during myogenic differentiation. Endogenous MRTF-A protein abundance and activity decreased during C2C12 differentiation, which was attributable to miRNA-directed inhibition. Conversely, overexpression of MRTF-A impaired differentiation and myosin expression. Applying miRNA trapping by RNA affinity purification (miTRAP), we identified miRNAs which directly regulate MRTF-A via its 3'UTR, including miR-1a-3p, miR-206-3p, miR-24-3p and miR-486-5p. These miRNAs were upregulated during differentiation and specifically recruited to the 3'UTR of MRTF-A. Concomitantly, Ago2 recruitment to the MRTF-A 3'UTR was considerably increased, whereas Dicer1 depletion or 3'UTR deletion elevated MRTF-A and inhibited differentiation. MRTF-A protein expression was inhibited by ectopic miRNA expression in murine C2C12 and primary human myoblasts. 3'UTR reporter activity diminished upon differentiation or miRNA expression, whereas deletion of the predicted binding sites reversed these effects. Furthermore, TGF-? abolished MRTF-A reduction and decreased miR-486-5p expression. Our findings implicate miR-24-3p and miR-486-5p in the repression of MRTF-A and suggest a complex network of transcriptional and post-transcriptional mechanisms regulating myogenesis.