Screening And Identification Of Key CircRNAs During C2C12 Myoblasts Differentiation

Skeletal muscle growth and development is a process that is precisely regulated by many genetic factors,such as myogenic regulatory factors,myogenic enhancement factors,Pax family protein,and myostatin,In addition,non-coding RNAs such as microRNA and lncRNA also play an important role in this process.The circular RNA is an RNA molecule that does not contain 3' end poly(A)tail and 5' end cap structure and is covalently bonded to a closed circular structure.CircRNA can be used as a molecule sponge for miRNA or RNA binding proteins that participate in the regulation of gene expression.CircRNAs are ubiquitous in organisms and play a regulatory role at both the transcriptional and post-transcriptional levels.Their highly conserved,specific,stable,biologically functional regulatory roles,and disease relevance have gradually gained attention in recent years and have become research hotspots.However,there are few studies on the role of circRNA in skeletal muscle development.In this study,We used the mouse C2C12 myoblast cell line model to detect and analyze the expression of circRNAs in different stages of differentiation by RNA-seq.Screening the key circRNAs that regulate the differentiation of C2C12 cells for further verification and analysis,revealing the regulatory role of circRNAs in the differentiation of C2C12 cells.The main results were as follows:1.Sequencing results were systematically analyzed.We detected a total of 37,751 circRNAs,Among them,36,724 weren't found in the circBase database,It were unknown circRNAs;The expression of circRNAs was dynamically changed in three different differentiation phases.To lay the foundation for studying the regulation mechanism of circRNA in the myogenesis process.2.By analyzing the characteristics of circRNA,the results showed that the number of circRNA expression from the same host gene,It was related to the structure and chromosomal location of this gene,while the expression abundance of circRNA was low correlated with the expression of the host gene,indicating that the expression regulation of circRNA was not only due to its host gene determines that there are other regulatory factors involved in it;Through the GO annotations of the host genes of the differentially expressed circRNAs,we found that the host genes that are downregulated during differentiation are clustered into biological processes,the majority of groups were related to cell cycle,such as the regulation of chromosome separation,mitotic cell cycle process,and nuclear division,these were consistent with the phenomenon that myoblasts exit the cell cycle after differentiation,whereas the expression of upregulated circRNAs corresponds to the host gene in the molecular function,The functional process is mainly clustered to the binding function,which is consistent with the function of circRNA as a molecular sponge of miRNA and RNA binding proteins.3.We screened the circRNAs ifferentially expressed at different stages of differentiation,selected 10 differentially expressed circRNAs,analyzed the miRNAs interaction to constructed circRNA-mi RNA interactions network.The results showed that the same circRNA can interact with many different miRNAs,and some miRNAs that play an important role in the myogenesis,such as miR-133,miR-24 and miR-23 a,we speculate that circRNA may act as a molecular sponge with miRNAs to regulate the myogenesis process;We randomly selected 10 circRNAs to design the diliverge primers verified the circRNA juction region and verified the sequence of the back spilicing sites,We verified the authenticity of the circRNAs,We used q RT-PCR to detected the relative expression levels of the circRNAs.The results were consistent with the sequencing results and confirmed the reliability of the sequencing results.According to the sequencing results and combined with the information of the circRNAs,we designed the forward primers that obtain the full length sequences of five circRNAs,which will prepare for the subsequent construction of overexpression vectors.4.We used FISH to detect subcellular localization of circRNAs that were differentially expressed,the results showed that both the circRNAs are located in the cytoplasm,we speculated the place where circRNAs functioned was in the cytoplasm.and the fluorescence intensity of the two circRNAs is different in same excitation light was used,CircRNA.23599 was clearly visible and circRNA.27822 was weaker,which is also consistent with the RNA-seq results.This study laid a foundation for exploring the regulatory mechanism of circRNA in the process of myogenesis for future,and also provided a more powerful data support for further supplementing and perfecting the regulatory mechanism and molecular mechanism during the growth and development of skeletal muscle.