Study On MiR-214 Regulating Chicken Myoblast Proliferation And Differentiation

The proliferation and differentiation of myoblasts is an important stage in the process of muscle development.In this stage,microRNAs(miRNAs)play an important role.Based on the previous research of the research group,this study selected miR-214 as the starting point for studying the growth performance of broilers,exploring the biological functions of miR-214 and the mechanism of miR-214 on the proliferation and differentiation of chicken primary myoblasts(CPMs).To further understand the function of miR-214 in CPMs,overexpression and interference of miR-214 in CPMs were performed,and then transcriptome sequencing was performed.The results of this study are as follows:(1)RT-PCR found that the expression level of miR-214 was relatively stable during the embryonic chicken muscle development from 12 to 18 embryonic age,and the expression level was extremely significantly up-regulated at 18 embryonic age(P<0.01),indicating that miR-214 was involved in chicken embryos Muscle development.(2)CPMs were induced to differentiate in vitro.As the induction time increased,the expression level of miR-214 continued to increase,indicating that miR-214 was involved in the differentiation process of CPMs.(3)Through RT-PCR,flow cytometry,CCK8,EDU detection of CPMs after overexpression and interference with miR-214,the results show that miR-214 can inhibit the proliferation of CPMs;use RT-PCR,Westernblot,indirect immunofluorescence technology to detect overexpression And interfere with miR-214 to induce differentiation of CPMs,the results show that miR-214 can promote the differentiation of CPMs.(4)According to the previous sequencing results of the research group and the prediction of the miRDB database,it was found that TRMT61A may be a potential target of miR-214,and the RT-PCR and dual luciferase reporter system test confirmed that miR-214 can target and regulate the expression of TRMT61A gene.Through the TRMT61A overexpression test,it is found that the TRMT61A gene can promote the proliferation and differentiation of CPMs.(5)Overexpression and interference of CPMs,and then transcriptome sequencing:After overexpression of miR-214,141 differential genes were obtained,of which 38 were up-regulated genes and 103 were down-regulated genes;the biological processes with a higher enrichment score in GSEA-GO include biological processes related to the development of CPMs,including muscle fibers(Myofibril),muscle fiber assembly(myofibril assembly),sarcomere(I band)and other processes;among the pathways with higher enrichment scores in GSEA-KEGG,Type Ⅱ diabetes mellitus,insulin secretion),Nitrogen metabolism and other pathways;through protein network interaction,key genes MYH1D,LDB3,VAV3,CACNG1,CDKN1C,MYOM2 were screened out.After interfering with miR-214,74 differential genes were obtained,of which 43 were up-regulated and 31 were down-regulated.The biological processes with higher enrichment scores in GSEA-GO were hormone biosynthetic process and acid secretion.secretion)and other material metabolism processes;among the pathways with a higher enrichment score in GSEA-KEGG,there are Apoptosis-multiple species,Fat digestion and absorption,and P53 signaling pathway(p53 signaling pathway)and many other pathways;protein network interaction screened out the key genes NROB2,KCNJ2,CDKN2B,FOS.