Identification And Functional Analysis Of CircRNA Related To Skeletal Muscle Development In Mice

The development of skeletal muscle is regulated by inheritance and epigenetic modification.The relative genes mainly included myogenic regulatory factor family,myocyte growth factor family,Pax gene family and myostatin gene.They play an important role in the regulation of skeletal muscle development.In addition,with the development of biotechnology and bioinformation technology,more and more noncoding genes have gradually been excavated,including Long noncoding RNAs,microRNAs and the highlights of CircRNAs.Circular RNA is a kind of noncoding RNAs which exists widely in wild life,which is formed by splicing of pre-mRNA.CircRNAs are mainly formed by the circulation of exons while some of introns.Many studies showed that circular RNAs can act as sponges,which can act as circular inhibitors of miRNA.It can also play a role in the regulation of miRNA targets as well as posttranscriptional regulation,and further participate in the regulation of body functions.However,CircRNAs associated with skeletal muscle development was rarely reported.This study takes C2C12 as the model,two kinds of CircRNAs which related to muscle development--Circ2196 and Circ2179 are screened by microarray.And then the effects on cell proliferation and differentiation of C2C12 cells were studied.The results showed that Circ2179 can promote cell differentiation,Circ2196 can inhibit cell proliferation.In order to study its mechanism,cell localization experiment was conducted and found out that the two CircRNAs were located in the cytoplasm,which laid the foundation for the study of the CircRNAs' sponge mechanism.Through the bioinformatics analysis,Circ2196 was predicted to have miRNAs binding sites with miR133 a,miR133b and miR133 c,Circ2196 was proved to target with miR133 a,miR133b and miR133 c by the Luciferase Reporter Gene Assay.The cell proliferation experiments turned out that Circ2196 can inhibit the activity of these three miRNAs.After knocking down Circ2196,the activity of these three miRNAs increased.Previous studies have shown that miR133 a and miR133 b can inhibit the proliferation of cells by inhibiting SRF protein.The results of western blotting showed that the expression of SRF protein was significantly decreased after the knockdown of Circ2196,and it increased significantly after the overexpression of Circ2196.The results were consistent with the previous studies on the relationship between Circ2196 and miR-133.This study revealed that Circ2196 inhibited the proliferation of C2C12 myoblasts and studied the mechanism of Circ2196,which promoted the expression of SRF protein by inhibiting miRNA-133 and then inhibited the proliferation of C2C12,which plays an important role in the further study of Circ2196 in the regulation of muscle development.