Role Of MiR-181a-5p And Endoplasmic Reticulum Stress In The Regulation Of Myogenic Differentiation

Myogenic differentiation process involves multiple pathways, accumulating evidences indicate that miRNAs and endoplasmic reticulum stress (ERS) play critical roles in myoblast differentiation. Researches have shown that miR-181a-5p increased the expression of myogenic differentiation related genes MyoD, MyoG by targeting Hox-A11, and promoted myogenic differentiation. In addition, endoplasmic reticulum stress (ERS) promoted apoptosis through ERS-mediated apoptosis signaling, which selectively eliminated vulnerable cells, and finally facilitated differentiation. Thus we speculate that miR-181a-5p and ERS have synergistic effect on promoting myogenic differentiation, but need further experimental verification.Here, we analyzed the expression pettern of miR-181a-5p during C2C12 cells differentiation. C2C12 cells were transfected with miRNA mimics or inhibitor to up-regulate or down-regulate miR-181a-5p, and explored the role of miR-181a-5p in the regulation of C2C12 cells proliferation, differentiation, ERS, and muscle fiber types. Thapsigargin (Tg) was used to induce ERS, we analyzed the effect of ERS on the expression of endogenous miR-181a-5p and myogenic differentiation related genes in C2C12 cells. Finally, we explored the role of miR-181a-5p in regulating apoptosis in the Tg pretreated C2C12 cells and porcine muscle fibroblasts (PMFs), respectively. And we further detected the targets relationship between GRP78 and ssc-miR-181a-5p through dual luciferase report system in pigs.The main results are as follows:(1) The seed sequences of miR-181 a-5p and its binding sequences in GRP?8 were highly conserved in different species, and miR-181a-5p was strongly up regulated during C2C12 cells differentiation.(2) Over-expression of miR-181a-5p promoted C2C12 cells differentiation, the target gene GRP78 was reduced, but not significantly (P> 0.05). The mRNA expression of myogenic differentiation related genes Myod, MyoG, Myf5, Myf6 and ERS-mediated apoptosis signaling related genes ATF6, Casp12, CHOP, EIF-2a were increased (P< 0.01 or P< 0.05); inhibition of miR-181a-5p blocked myogenic differentiation, and markedly increased the mRNA expression of target gene GRP78 (P< 0.01). Myogenic differentiation related genes Myod, MyoG, Myf5, Myf6 and ERS-mediated apoptosis signaling related genes ATF6, Casp12, CHOP, EIF-2a were significantly reduced (P< 0.01 or P< 0.05). The expression levels of apoptosis suppressor gene BCL2 were markedly down-regulated both overexpression or inhibition of miR-181a-5p (P< 0.01 or P<0.05).(3) Over-expression of miR-181a-5p markedly increased the mRNA expression levels of type ? muscle fiber related gene MyHc ?a and decreased the mRNA expression levels of type ? muscle fiber related gene MyHc ?(P< 0.01 and P< 0.05), as a result, over-expression of miR-181a-5p increased the type ? muscle number; inhibition expression of miR-181a-5p markedly decreased the mRNA expression levels of type ? muscle fiber rerated gene MyHc ?? (P< 0.01), as a result, inhibition expression of miR-181 a-5p increased the type ? muscle number.(4) Over-and inhibition expression of miR-181a-5p did not significantly influence the indexes of the C2C12 cells compared to their controls (P> 0.05).(5) C2C12 cells were treated with 0.1 ?M Tg, the mRNA expression of ERS-mediated apoptosis signaling related genes ATF6, Caspl2, CHOP, EIF-2a and GRP78 were markedly increased at 24 h (P< 0.01), indicated that ERS was induced successfully. Then we detected that both miR-181 a-5p and myogenic marker genes Myf5, Myf6, MyoD were significantly up regulated (P<0.01).(6) Over-expression of miR-181a-5p in Tg pretreated C2C12 cells, the number of nuclear fragmentations were increased (Hoechst staining), and significantly decreased the cell viability (CCK-8 assay) (P< 0.01). The expression levels of ER stress-mediated apoptosis signaling related genes ATF6. Casp12, CHOP and EIF-2a were markedly increased with target gene GRP78 and apoptosis suppressor gene Bcl2 down-regulation in over-expression group (RT-qPCR) at 24 h (P< 0.01). However, the number of nuclear fragmentations, cell viability and these genes expression levels were opposite in inhibition expression group compared to the over-expression group.(7) Pig GRP78 luciferase reporter plasmid was constructed successfully, we comfirmed that pig GRP78 was a target of ssc-miR-181a-5p using the dual luciferase reporter assay system, and the mRNA levels of the target gene was unregulated, prompted that it may regulate its target gene GRP78 at the protein levels.(8) PMFs were isolated and cultured in vitro, over-expression of ssc-miR-181a-5p in Tg pretreated PMFs, the number of nuclear fragmentations were increased. the cell viability was significantly decreased (P< 0.01). and the mRNA expression of the target gene GRP78 was markedly reduced (P< 0.01). Furthermore. ER stress-mediated apoptosis signaling related genes ATF6, EIF-2a were markedly increased (P< 0.01) at 24 h. On the contrary, the number of nuclear fragmentations, cell viability and these genes expression levels were opposite in inhibition expression group compared to the over-expression group.