The Role And Regulatory Mechanism Of MiR-127-3p In C2C12 Myoblasts Proliferation And Differentiation

Micro RNAs(mi RNAs) are small non-coding RNA that play important roles in regulating skeletal muscle growth and development. In the present study, we first established tissue distribution of mi R-127-3p in adult goat and analyzed expression difference of mi R-127-3p between goat breeds with different muscle phenotypes(Boer vs. Wushan black goats) to claritfy the relationship between mi R-127-3p and muscle phenotypes. And then proliferating and differentiatiing C2C12 myoblasts were transfected with lentivirus vectors of mi R-127-3p(mimic and inhibitor), q PCR were used to detect the dynamic expression level of myogenic marker genes Myo D, Myo G and Myosin cultured at the day 3 and 5. Meanwhile, we detected and analyzed morphology changes of C2C12 myoblasts in different cultured conditions to elucidate overexpression and inhibitor of mi R-127-3p have different influence on C2C12 myoblasts proliferation and differentiation. Besides, verification and analysis of experiments in vitro were carried out the myogenic effects of mi R-127-3p. In order to illustrate the myogenic regulatory mechanism of mi R-127-3p, we constructed dual-luciferase vectors of mi R-127-3p, Vamp2 and Sept7 were the direct target gene of mi R-127-3p by dual-luciferase vectors system, which further reveal the myogenic regulatory mechanism of mi R-127-3p on C2C12 myoblasts proliferation and differentiation.The main results are following:1. Mi R-127-3p was extensively expressed in tissues of goats. Mi R-127-3p was was abundantly expressed in muscle, spleen, heart, and skin, but weakly expressed in lung, kidney, and liver in Boer and Wushan black goats. Especially, mi R-127-3p was significantly higher expressed in muscle in the muscular goats(Boer goats) than the control(Wushan black goats).2. Mi R-127-3p were expressed increasingly during the proliferation of C2C12 cells. Compared with the negative control, C2C12 cells transfected with mi R-127-3p mimic inhibited C2C12 myoblast proliferation, and decreased the m RNA level of Myo D, Myo G, and Myosin at day 3 and day 5 in growth medium(GM). Whileinterference of mi R-127-3p promoted cell proliferation, and increased the m RNA expression of Myo D, Myo G, and Myosin.3. Mi R-127-3p were expressed increasingly during the C2C12 myoblast differentiation. In contrast to the negative control, cells were transfected with mi R-127-3p mimic increased the fusion index of C2C12 myoblasts, as well as promoted the m RNA level of Myo D, Myo G and Myosin at day 3 and day 5 in differentiation medium(DM). While interference of mi R-127-3p inhibited myotube formation by reducing the fusion index, as well as supressed expression of Myo D,Myo G and Myosin.4. Co-transfection of the dual-luciferase vectors for Vamp2 or Sept7 into the293 T cells with the mi R-127-3p mimic demonstrated mi R-127-3p could effectively inhibited expression of Vamp2 and Sept7 by combinating their 3′UTR. When lentivirus vectors of mi R-127-3p(mimic and inhibitor) were transfected in C2C12 myoblasts, mi R-127-3p greatly supressed expression of Vamp2 and Sept7. These results clearly indicated that mi R-127-3p modulated the C2C12 myoblasts proliferation and differentiation by regulating its target genes Vamp2 and Sept7 during myogenesis.