miR-127 Regulated the Proliferation and Differentiation of Sheep Skeletal Myoblasts and Its Transcription Factor PAX3 Screening
The aim of this study was to investigate the effects of miR-127 on the skeletal myoblast proliferation and differentiation in sheep,and to screen for transcription factors that regulate the expression of miR-127 by identifying its upstream core promoter region.In this study,primary skeletal muscle myoblasts of small-tailed Han fetal sheep were used as experimental materials.After overexpression or inhibition of miR-127,the RT-qPCR,EdU staining,flow cytometry analysis,and immunofluorescence staining were used to detect the effects of miR-127 on the proliferation,apoptosis and differentiation of sheep myoblasts.The bioinformatics analysis and dual luciferase reporting assay were used to predict and identify the sheep miR-127 core promoter region and its transcription factors.The results showed that overexpression of miR-127 in sheep myoblasts promoted the expression of cell proliferation related genes PCNA and CDK4(P＜0.01),and inhibited the expressions of cell apoptosis related genes Caspase3 and BAX(P＜0.05).EdU results showed that the positive rate of sheep myoblasts was significantly increased after miR-127 overexpression(P＜0.05).Flow cytometry analysis showed that overexpression of miR-127 significantly increased the proportion of S and G2 phase myoblasts and decreased the apoptosis of myoblasts.In the cell differentiation assay,overexpression of miR-127 significantly increased the mRNA expression levels of myoblast differentiation related genes MYOD1 and MYHC(P＜0.05),and significantly increased the MYHC positive myotube area(P＜0.05).The opposite result was found after miR-127 inhibition.In order to further reveal the regulatory factors regulating the expression of miR-127,the dual luciferase activity assay showed that the 1 500-1 800 bp(miR-127-P6)region upstream of miR-127 had the highest activity,which was inferred to be the core promoter region of miR-127.Bioinformatics predictions revealed a site in the core promoter region of miR-127 that bound to the transcription factor PAX3.Overexpression of the transcription factor PAX3 in sheep myoblasts significantly increased the promoter activity and the expression of miR-127(P＜0.01).This study showed that sheep miR-127 significantly promoted the proliferation and differentiation of skeletal muscle myoblasts and reduced their apoptosis,which participated in their development process.Further studies showed that 1 500-1 800 bp upstream was the core promoter region of sheep miR-127,and the transcription factor PAX3 positively regulated the transcription of miR-127.This study provided a theoretical reference for further exploring the molecular mechanism of sheep muscle growth and development.
sheepmyoblast proliferation and differentiationmiR-127transcription factor PAX3
万方数据
维普
