Molecular Mechanisms Of MiR-455-5p In Regulating Myogenic Differentiation Of C2C12 Myoblast

microRNA-455-5p(miR-455-5p)is a widely conserved member of the microRNA family,which is expressed in most plants and animals.In mice,miR-455-5p is located at 4B3;4 on chromosome 4,and it is encoded by Col27a1(collagen type XXVII alpha1 chain).It has been reported that miR-455-5p is associated with human diseases such as preeclampsia,Alzheimer's disease,multiple sclerosis and cancer.In addition,miR-455-5p is involved in cartilage development and adipogenesis.However,little is known about the function of miR-455-5p in skeletal muscle.In this study,C2C12 cells were used as models to explore the role of miR-455-5p in skeletal muscle cell proliferation and myogenic differentiation.In the first part of the study,we first explored the function of miR-455-5p during C2C12 proliferation;Secondly,we optimized the conditions of myogenic differentiation of C2C12 cells,and obtained a better myogenic differentiation condition;Finally,we studied the miR-455-5p function in C2C12 myogenic differentiation.miR-455-5p mimics or miR-455-5p inhibitor were transfected into C2C12 cells,respectively,and then CCK8,EDU and cell cycle experiments were used to detect the proliferation and apoptosis of C2C12 cells.The results showed that overexpression and inhibition of miR-455-5p did not affect the proliferation and apoptosis of C2C12 cells.C2C12 cells were inoculated into 24-well plates with number of 3.75×10~4,5.00×10~4 and 7.00×10~4,respectively.Then,2%,5%and 10%horse serum were added into the culture medium to induce myogenic differentiation.The results showed that 5.00×10~4 C2C12 cells combined with 5%horse serum are best for the myogenic differentiation.Furthermore,miR-455-5p mimics or miR-455-5p inhibitor were used to overexpress or inhibit miR-455-5p,and immunofluorescence staining was performed when C2C12 differentiated for 2,4,6 and 8 days,and the length,diameter and fusion rate of the myotubes were counted.The results showed that the overexpression and inhibition of miR-455-5p did not affect the results of myogenic differentiation at 2 days;But at 4 days of myogenic differentiation,the overexpression of miR-455-5p promoted the differentiation of myotubes,while the inhibition of miR-455-5p led to the failure of myotube differentiation and the formation of short tubules;At 6 and 8 days of myogenic differentiation,the overexpression of miR-455-5p promoted the length,diameter,fusion rate of myotubule and the expression of muscle-specificity genes.While the inhibition of miR-455-5p resulted in a significant decrease in the length,diameter,fusion rate of myotubule and expression of muscle-specific genes.In conclusion,the overexpression and inhibition of miR-455-5p did not affect the proliferation and apoptosis of C2C12 cells.Overexpression of miR-455-5p promoted the myogenic differentiation of C2C12 cells,and even caused the hypertrophy of myotubes.miR-455-5p inhibition will lead to the failure of myogenic differentiation of C2C12.In the second part,in order to further explore the molecular mechanism of miR-455-5p in regulating the myogenic differentiation of C2C12 cells,double luciferase assay was used and we find that Mylip is the target gene of miR-455-5p.Subsequently,we explored the function of Mylip in the myogenic differentiation of C2C12 cells.Finally,we use functional recovery experiments to further verify the regulatory of miR-455-5p on Mylip.We first identified potential target genes Mylip,Add3,Taf4a,Luc7l3,Brd1,Gdap2,Dcaf5 and Irf2 through target gene prediction website,and then we constructed double luciferase vectors to carry out double luciferase experiments.The results showed that Mylip was the target gene of miR-455-5p.To further verify the above results,we overexpressed or inhibited the expression of miR-455-5p to detected Mylip expression at 2,4,6 and 8 days after myotube differentiation.RT-qPCR and immunoblotting showed that Mylip transcription and protein levels decreased significantly when miR-455-5p was overexpressed.While,when miR-455-5p was inhibited,Mylip transcription and protein levels increased significantly,which further proved that Mylip was the target gene of miR-455-5p.Subsequently,we explored the function of Mylip in myogenic differentiation of C2C12 cells.Myogenic differentiations were observed for 2,4,6 and 8 days after Mylip knocked down or overexpressed.The results showed that Mylip knockdown or overexpression did not affect myogenic differentiation at 2 days.At 4 days of myogenic differentiation,Mylip knockdown increased the myotube length,while Mylip overexpression decreased the myotube length.Mylip knockdown or overexpression did not affect the diameter and fusion rate of myotubes.Furthermore,at 6 and 8 days of myogenic differentiation,Mylip knockdown significantly increased the length,fusion rate of myotubes and the expression of muscle-specific genes,while overexpression of Mylip resulted in a significant decrease in the length,fusion rate of myotubes and the expression of muscle-specific genes.In order to further clarify the regulatory relationship between miR-455-5p and Mylip,we overexpressed Mylip when miR-455-5p was overexpressed.And the overexpression of Mylip can partly or completely rescue the myotube hypertrophy caused by the overexpression of miR-455-5p.In conclusion,Mylip knockdown promotes myotube differentiation,while Mylip overexpression inhibits myotube differentiation.miR-455-5p regulates myotube differentiation by regulating the expression of Mylip.In the third part of the study,in order to find the signaling pathways that miR-455-5p and Mylip involved in,we used TMT-labeled quantitative proteomics technology to analyze the mass spectrometry of C2C12 cells which overexpressed miR-455-5p and differentiated for 6 days,and then we validated the screened potential signaling pathways.By analyzing the data of mass spectrometry,113 differential proteins were obtained,including 51 up-regulated proteins and 62 down-regulated proteins.In order to find out the possible signaling pathways that miR-455-5p and Mylip may participate in,we performed GO and KEGG analysis,and finally enriched the RhoA/ROCK signaling pathway.To clarify whether miR-455-5p and Mylip are involved in RhoA/ROCK signaling pathway,we first overexpressed or inhibited miR-455-5p to detect the specific gene expression involved in the RhoA/ROCK signaling pathway.The results showed that the overexpression or inhibition of miR-455-5p did not affect the expression of RhoA and Rock,but significantly changed the expression of Ppp1r12b and Ppp1r12c,which are the members of MLCP family.Then,we knocked down or overexpressed Mylip to detect the specific genes of RhoA/ROCK signaling pathway.Interestingly,the expression of RhoA and Rock did not change significantly,while the expression of Ppp1r12c changed significantly.In addition,knockdown of Ppp1r12c leads to the failure of myotube differentiation.In summary,miR-455-5p regulates Ppp1r12c through the target gene Mylip,and further regulates the myogenic differentiation of C2C12 cells.