Influence Of MiR-103 On The Differentiation Of Bovine Skelletal Muscle Satellite Cells

Growth and development of skeletal muscle directly affects the animal meat yield and meat quality. As in adult muscle-derived stem cells, satellite cells are widely used for in vitro studies of skeletal muscle development. Proliferation and differentiation of satellite cells is a complex biological processes are regulated by multiple transcription factors and cell signaling molecules. Recent research findings, micro RNA on the proliferation and differentiation of satellite cells have regulatory role. After this laboratory for bovine skeletal muscle satellite cells during differentiation of micro RNA complete high-throughput sequencing and found high levels of mi R-103 expression and significant difference in the change process in satellite cell differentiation, but the proliferation of satellite cells and regulatory mechanisms of differentiation is not clear.We first bovine skeletal muscle satellite cells as test object, the satellite cells were induced to differentiate in vitro, using real-time quantitative PCR technique to detect the satellite in the process of cell differentiation in the expression pattern of endogenous mi R-103. And then use the method of transient transfection, the exogenous over-expression of mi R-103 or mi R-103 inhibitor vector transfected into satellite cells, up-regulated or down-regulated after the expression of mi R-103 by EDU function testing and morphological changes were observed by immunofluorescence experiments and statistical analysis of satellite cells. While the use of real-time quantitative PCR and Western blot were quantitatively analyzed to detect the expression levels of the late satellite cell differentiation marker genes are affected Myo G. Bioinformatics software to predict the possible role of mi R-103 gene m RNA CCNE1 3’UTR. After the reporter gene experiments by dual luciferase, quantitative real-time quantitative PCR and Western blot analysis to verify the regulatory role of mi R-103 CCNE1.The results obtained are as follows:1. In the process of differentiation of bovine skeletal muscle satellite cells, the expression of mi R-103 increases gradually, and in the differentiation of the first six days maximum.2. The build successful overexpression of mi R-103 or mi R-103 inhibitor vector was transfected into bovine skeletal muscle satellite cells proliferation by EDU immunofluorescence assay to detect and can be observed in the expression of mi R-103 is upregulated the proliferation of satellite cells was inhibited differentiation level has been improved. And when mi R-103 expression is down-regulated levels of satellite cell proliferation has been improved, the level of differentiation is inhibited. While the use of real-time quantitative PCR and Western blot were quantitatively analyzed to detect the expression levels of the late satellite cell differentiation marker genes are affected Myo G. Found after expression of mi R-103 to improve significantly increase the amount of expression Myo G contrary after mi R-103 activity was inhibited, Myo G expression level also will be lowered. These results indicate that, mi R-103 can promote the differentiation of satellite cells.3. In the dual luciferase reporter assay, confirming the mi R-103 can be combined CCNE1 the 3’UTR by specific, reducing CCNE13’UTR dual luciferase recombinant plasmid luciferase activity. In real-time quantitative PCR and Western blot analysis of the results can be seen seen CCNE expression of mi R-103 can be lowered at the mRNA and protein levels. Change change and mi R-103 are CCNE1 in satellite cell differentiation corresponds CCNE1 and downregulation can promote differentiation of satellite cells. Therefore, as a target gene can be drawn CCNE1 ofmi R-103.In summary, in the course of the development of satellite cells, mi R-103 affect the proliferation and differentiation of bovine skeletal muscle satellite cells by the downregulation of CCNE1. We identify mi R-103 promotes the differentiation of satellite cells and their regulation mechanisms this provide a theoretical basis for the subsequent cultivation of transgenic cattle.