Study On The Regulation Mechanism Of MicroRNA-143 In The Proliferation And Myogenic Differentiation Process Of Bovine Skeletal Muscle Satellite Cells

Controlled collectively by a variety of regulatory factors and pathways,the growth and development of skeletal muscle is an extremely complex biological process.Skeletal muscle satellite cells play a key role in muscle differentiation and muscle injury repair.Previous studies had shown that non-coding RNAs including micro RNAs(mi RNAs)and lnc RNAs(long non-coding RNAs)act as a critical role in muscle developmental differentiation.On the basis of prior research,the regulation mechanism of mi R-143 on the differentiation of bovine skeletal muscle satellite cells was studied deeply by the myogenic differentiation induced model of bovine skeletal muscle satellite cells in vitro.Then,the target genes of mi R-143 was predicted and verified to analyze its possible signal pathways.In addition,the interaction between mi R-143 and lnc RNAs was identified and analyzed.Furthermore,the in vivo experiments were conducted to analyze whether mi R-143 had a modest effect on muscle development and to provide a reference for the study of non-coding RNA regulatory mechanisms in bovine muscle development.1.In our previous study,we performed a mi RNAs microarray to analyze the expression of mi RNAs in bovine skeletal muscle satellite cells myogenic differentiation.It was found that mi R-143 was highly expressed in bovine skeletal muscle satellite cells and was significantly up-regulated in the process of bovine myogenic differentiation.These results suggested that mi R-143 was likely to play a role in bovine myogenic differentiation.To investigate the function of mi R-143 in bovine skeletal muscle satellite cells differentiation,we used q RT-PCR to detect the expression pattern of mi R-143 during the differentiation of bovine skeletal muscle cells.These target genes of mi R-143 were predicted by bioinformatics and IGFBP5 was confirmed to be directly regulated by mi R-143 using a dual-luciferase reporter assay.Overexpression of mi R-143 led to decreased level of IGFBP5 protein and restrained cell proliferation and differentiation.Downregulation of mi R-143 resulted in increased levels of IGFBP5 protein and restrained cell proliferation but improved differentiation.Furthermore,when IGFBP5 expression was inhibited by transfecting with si RNA,bovine skeletal muscle satellite differentiation process was inhibited,which indicates that IGFBP5 plays an important role in bovine skeletal muscle cells differentiation.Our study demonstrates mi R-143 can regulate proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5.2.In view of the regulation of non-coding RNA in myoblast differentiation,we analyzed the differentially expressed lnc RNA during myoblast differentiation by RNA-seq technique in our previous study.We found some Lnc RNAs may interacted with mi R-143.In order to explore the relationship between mi R-143 and its interacted lnc RNA and its mechanism in the process of myoblast differentiation,two predicted highly expressed lnc RNA lnc RNA-HZ5 and lnc RNA-HZ8 were chosed for further research.The expression changes of these lnc RNAs during myoblast differentiation were detected by q RT-PCR.The result shows that lnc RNA-HZ5 was directly regulated by mi R-143 using dual-luciferase assay.The expression of lnc RNA-HZ5 was significantly down-regulated when mi R-143 was over expressed.It was found that inhibition of lnc RNA-HZ5 expression using si RNA can down-regulate mi R-143 and up-regulate the protein level of mi R-143 target gene IGFBP5,thereby promoting the differentiation of muscle satellite cells.The results of this study show that mi R-143 can regulate the myogenic differentiation of bovine muscle satellite cells by interacting with lnc RNA-HZ5.3.To investigate whether mi R-143 regulates myoblasts differentiation in vivo,our study performed a futher research on the effect of mi R-143 on myofibers of denevated muscle atrophy in mouse.As it was difficult to carry out in vivo experiments in cattle,an amyotrophia mouse model of sciatic nerve resection was established,and mi R-143 mimics or mi R-143 inhibitors were injected to denevated gastrocnemius.Then,the expression of mi R-143 was detected by q RT-PCR,the wet weight of gastrocnemius and the cross section diameter of the muscle fibers were compared between the atrophying skeletal muscle and control.The results showed that the diameter of myofiber injected with mi R-143 mimics is significantly lower than that of control group while the diameter of myofiber injected with mi R-143 inhibitors is significantly higher than that of control group.The results showed that mi R-143 could affect the development of muscle fibers.Inhibition of mi R-143 significantly increased the diameter of muscle fibers and delayed the process of denervation of muscle atrophy.The results indicated that mi R-143 plays a crucial role in regulating muscle development in vivo.In this paper,the regulation mechanism of mi R-143 on bovine myogenic differentiation was investigated by the myogenic differentiation induced model in vitro.The results suggested that mi R-143 can target IGFBP5 to regulate its expression and then to regulate the proliferation and myoblastic differentiation of bovine skeletal muscle cells.Also,mi R-143 can influence the myoblast differentiation process of bovine muscle satellite cells by interacting with lnc RNA-HZ5.The in vivo experiments showed that mi R-143 played a role in regulating muscle development.Taken together,the mechanism of mi R-143 on the proliferation and differentiation of bovine skeletal muscle cells was illustrated in this paper.Our study can provide a reference for the non-coding RNA research of bovine muscle development and differentiation,and offer new ideas and useful clues for meat quality improvement and muscle damage repair.