Study On The Regulation Of Yak MiR-487b And MiR-433 In C2C12 Proliferation And Differentiation

The normal development of skeletal muscle is the basis for the body to maintain a series of important life activities.The proliferation and differentiation of myoblasts plays a key role in the development of skeletal muscle.Therefore,the study of the regulatory network of myoblast proliferation and differentiation is an important field in developmental biology.The proliferation and differentiation of myoblasts are regulated by a cascade of transcription factors and miRNAs.miRNAs is a class of small non-coding RNA of 18-24 nucleotides in length,and combines with target mRNA to down-regulate target gene expression,and participates in the regulation of a variety of biological processes.Based on previous high-throughput sequencing of miRNAs in the longissimus muscle of Datong yak fetus and adult yak,we found that miR-487 b and miR-433 were significantly higher in the longissimus dorsi of fetal bovine than that of adult yaks.We inferred that miR-487 and miR-433 may exert potential roles in skeletal muscle growth and development.Of the established research results showed that miR-487 b and miR-433 as tumor suppressor genes inhibit the proliferation,migration and invasion of many tumors,however,what's the function of miR-487 b and miR-433 in skeletal muscle development is rarely reported.Therefore,our study used murine myoblast cell line(C2C12)as cell model to explore the roles of miR-487 b and miR-433 in the proliferation and differentiation of myoblasts,which provides a theoretical basis for the further improvement of skeletal muscle development regulatory network.In this study,miR-487 b and miR-433 were over-expressed or inhibited by transfection of miRNA mimics or inhibitors,to study the function of yak miR-487 b and miR-433 in C2C12 proliferation and differentiation.Dual-luciferase reporter assay was used to confirm that PITX2 is the target gene of miR-487 b and to further clarify the regulation mechanisms of miR-487 b in the development process of skeletal muscle.The following is the main results of our study:(1)The mature sequences of yak miR-487 b and miR-433 were obtained by cloning sequencing analysis.Conservative analysis showed that the mature sequences of miR-487 b and miR-433 were highly conserved among species.The results of mouse tissue expression profiles showed that miR-487 b and miR-433 were highly expressed in skeletal muscle.(2)The expression levels of miR-487 b and miR-433 in C2C12 gradually upregulated.After over-expression of miR-487 b and miR-433,respectively,the expression levels of PCNA,CCNB1 and MKI67 mRNA were significantly increased,and the expression of PCNA protein was significantly increased.After CCK-8 assay,compared with the control group,over-expression of miR-487 b and miR-433 significantly promoted cell proliferation.On the contrary,after inhibiting the expression of miR-487 b and miR-433,the expression levels of PCNA,CCNB1 and MKI67 were down-regulated,and the proliferation activity of C2C12 cells was significantly decreased.Together,miR-487 b and miR-433 enhanced C2C12 myoblasts proliferation.(3)After successfully establishing the differentiation system of C2C12 myoblasts in vitro,we found that the expression of miR-487 b and miR-433 increased with the prolonged induction of differentiation.After over-expression of miR-487 b and miR-433,the myogenic marker genes MYOD,MYOG and MYHC were significantly reduced at mRNA and protein levels.On the contrary,the level of MYOD,MYOG and MYHC increased in both transcriptional and protein levels after inhibiting the expression of miR-487 b and miR-433.The results showed that miR-487 b and miR-433 suppress C2C12 myoblasts differentiation.(4)The online prediction software found that PITX2 is a potential gene of miR-487 b.The dual-luciferase reporter assay verified that miR-487 b directly targeted of the 3' UTR of PITX2 to suppress its expression,which may involved in the regulation of skeletal muscle growth and development.