The Role Of Mir-29and Mir-195/497in Skeletal Muscle Growth And Development

microRNAs (miRNAs) are eukaryotic non-coding small RNAs. They generally inhibit translation or promote mRNA degradation to regulate expression of genes, and participate in multiple different physiological procedures. MiRNAs play important roles in skeletal muscle growth and development. In order to identify key miRNAs in skeletal muscle development, our previous study used microarray to detect the miRNAs expression profiling of longissimus dorsi muscle from33-day,65-day fetuses and adult pigs. Finally,140differentially expressed miRNAs were identified. Among all identified miRNAs, miR-29and miR-195/497were steadily up-regulated during porcine skeletal muscle growth. The studies indicate that miR-29plays a role in muscle development, but the clamant role of miR-29in skeletal muscle development and the conclusive mechanism still need further investigation. There was few studies on the role of miR-195/497in skeletal muscle development. In the present study, mice and C2C12myoblasts, which are capable to differentiate into myotubes after induction, were used as in vivo and in vitro model to uncover the concrete role of miR-29and miR-195/497in myoblasts proliferation and differentiation during skeletal muscle development, and novel targets of miR-29and miR-195/497were identified. According to the results, we concluded that:(1) miR-29was up-regulated during skeletal muscle development in pigs and mice. Q-PCR results showed that miR-29expression in longissmus dorsi muscle was higher in E95d fetuses and adult pigs in comparison of E50d fetuses, which was in line with previous microarray data. miRNA-29a/b/c expression was detected in hindlimb muscle from mice of different ages (2d,2w,4w,6w, and12w), in which miR-29expression was lowest in2d mice compared to other group while miR-29expression was increased gradually in2w,4w, and6w mice and was highest in12w mice. It was63.96,77.54and96.72fold comparing to that in2d mice. Northern Blot result showed that the expression pattern was in consistent with with the Q-PCR result. miR-29c up-regulated expression was also detected in C2C12differentiated cells at0,1,2,4, and6days.(2) miR-29repressed C2C12myoblasts proliferation. After C2C12cells were transfected with pooled miR-29, flow cytometry analysis was performed and the results revealed that more cells were arrest in G0/G1stage (p<0.01), less cells were presented at S stage (p<0.01), and the Cell Proliferation Index was lower than NC transfected group (p<0.01). The cell growth dynamics monitored by real-time xCELLigence system showed that pooled miR-29transfected C2C12cells behaving a lagging growth pattern compared to control cells. C2C12cells were transfected with miR-29b/c over-expression vector, and subsequently the proliferating cells were stained by EdU. It was turned out that the dual-positive cells in miR-29b/c over-expressed group were much fewer than that in the control group (p<0.001).(3) miR-29promoted C2C12myoblasts differentiating into myotubes. The Immunofluorescence results reveled that the differentiation program in pooled miR-29transfected C2C12cells was faster than that in NC transfected cells, and pooled miR-29transfected cells formed more myotubes (p<0.001). The Q-PCR result showed that the expression of MCK, MyHC, and myogenin in pooled miR-29transfected cells was higher than that in NC transfected cells (p<0.05).(4) miR-29directly target Akt3by binding its3’UTR. After TargetScan predicting and screening, Akt3was chosen to be the candidate target of miR-29. The specific binding of miR-29and Akt33’UTR was confirmed by Dual-Luciferase reporter system. According to Western Blot result the endogenous Akt3protein was repressed by miR-29.(5) Akt3promoted proliferation and inhibited differentiation. After C2C12cells were transfected with siAkt3, more cells were arrested in G0/G1stage (p<0.01), fewer cells were presented at S stage (p<0.001), and the Cell Proliferation Index was lower than that in NC transfected group (p<0.01). It indicated that Akt3could promote C2C12myoblasts proliferation. Immuno fluorescence results reveled that differentiation program in Akt3over-expressed C2C12cells was slower than that in the control group, and Akt3over-expressed myoblasts formed fewer myotubes (p<0.001). The Q-PCR result demonstrated that the expression of MCK, MyHC, and myogenin in Akt3over-expressed group was lower than that in control group (p<0.05; p<0.001).(6) Over-expressing Akt3could rescue the proliferation inhibition, and attenuate the differentiation promotion by miR-29in C2C12myoblasts. The Cell Proliferation Index was decreased by miR-29(p<0.001), whereas co-transfection with Akt3-3.1decreased cells population arrest in G0/G1stage (p<0.01), increased the cells presented at S stage (p<0.05), and finally the Cell Proliferation Index was rescued by Akt3(p<0.01). The expression of myogenic marker gene was raised by miR-29, whereas co-transfection with Akt3-3.1decreased the raised expression of myogenic marker gene (p<0.05; p<0.001).(7) A T/C substitution was discovered in primary transcript of ssc-miR-29b-2/29c cluster, and further Q-PCR results demonstrated that it could influence the miR-29c expression in porcine longissimus dorsi muscle; Besides, miR-29c could also bind Sus scrofa YY1in its3’UTR, which indicated that targeting YY1by miR-29was one of potential pathway to modulate skeletal muscle in pigs.(8) miR-195/497was up-regulated during skeletal muscle development in mice. Q-PCR technique was used to detect the expression of miR-195/497in hindleg muscle from different ages (2d,2w,4w, and6w) mice. It is demonstrated that the expression of miR-195/497in hindleg muscle from2d-mice was the lowest compared that to other groups; it increased gradually in hindleg muscle from2w-and4w-mice; the expression of miR-195/497in hindleg muscle from6w-mice was highest, and it was up to10.70and4.38fold compared to2d-mice. Besides, After C2C12cells were transfected with miR-497, the flow cytometry analysis results revealed that more cells were arrested in G0/G1stage (p<0.05), fewer cells were presented at S stage (p<0.05), and the Cell Proliferation Index was lower than NC transfected group (p<0.05).Thus, our study concluded that targeting Akt3is a key pathway for miR-29modulates skeletal muscle growth and myogenic differentiation. Besides, we found the evidences to prove that miR-195/497directly participate in skeletal muscle development.