Study On The Regulation Mechanism Of MicroRNA-128 In The Proliferation And Myogenic Differentiation Process Of Bovine Skeletal Muscle Satellite Cells

Some studies indicated that microRNAs(miRNAs) play essential roles in muscle cell proliferation and differentiation. However, fewer researches were focused on bovine muscle miRNAs and its functions in muscle satellite cell proliferation and differentiation. In this study, we established a bovine skeletal muscle satellite cells myogenic differentiation model. And through the study of relevant literatures, with microRNA-128(miR-128) as the research object, studied on the regulation mechanism of microRNA-128 in the proliferation and myogenic differentiation process of bovine skeletal muscle satellite cells. Through these studies, the regulation mechanism of microRNA-128 in the proliferation and myogenic differentiation process of bovine skeletal muscle satellite cells will be clarified, which can provide theoretical support for further research on mechanism of miRNAs regulating muscle proliferation and differentiation, and offer new view for beef cattle breeding.1. In this study, bovine skeletal muscle satellite cells were isolated by collagenase and trypsinase from muscle tissues. Then the satellite cells were purified and induced differentiate into myotubes. The muscle satellite cells and myotubes were identified by RT-PCR and immunofluorescent staining technology. The results showed that more than 93 percent of the muscle satellite cells expressed the marker genes paired box protein 7(Pax7), and the myotubes expressed the marker genes myosin heavy chain(MHC). Our results indicated that high purity skeletal muscle satellite cells were successfully isolated and it can provide cell source for muscle development and bovine muscle quality improvement study.2. In this study, we investigated the biological functions of miR-128 in bovine skeletal muscle cell development. Using a dual-luciferase reporter assay, we confirmed that miR-128 regulates the Sp1 gene. Sp1 gene plays an important role in the process of proliferation and differentiation of bovine muscle cells. We regulated the expression levels of miR-128, and tested the expression of Sp1, MyoD and CDKN1 A protein levels, to study the regulation mechanism of microRNA-128 in the proliferation and myogenic differentiation process of bovine skeletal muscle satellite cells. Increasing Sp1 protein levels and promoted muscle satellite cell differentiation but suppressed proliferation, reducing Sp1 protein levels and inhibited muscle satellite cell proliferation and differentiation.3. In this study, we established an amyotrophia model of sciatic nerve resection. 5 nmol miR-128 mimics or 10 nmol miR-128 inhibitors were injected to denevated gastrocnemius. The results showed that the diameter of myofiber injected with miR-128 mimics is significantly lower than that of control group while the diameter of myofiber injected with miR-128 inhibitors is significantly higher than that of control group. The results indicated that miR-128 plays an important role in muscle damage repairing and it can regulate muscle differentiation in vivo.