Mechanism Of CircAIDA Regulating Bovine Myoblast Development And Copy Number Variation Of Its Target CLCN2 Gene

With the improvement of beef consumption in China,beef production is far from meeting the increasing demand.It is urgent to solve key issues such as the regulation mechanism of cattle muscle growth and development,and promote the application of molecular breeding and genetic improvement.In recent years,many studies have shown that circular RNA(circRNA),as a new key candidate factor,plays an important role in muscle development.However,the effects of circRNA on the bovine skeletal muscle development and the related molecular mechanisms still need to be studied in depth.In this study,based on previous sequencing data in the laboratory,circAIDA,which is differentially expressed between embryonic and adult cattle muscles with a higher expression level,was screened as the research object.The molecular experiments and biochemical analysis were used to explore the authenticity of circAIDA and its effect on the proliferation,differentiation and apoptosis of bovine myoblasts.Through the dual-luciferase reporter system and functional reconstruction experiments,it was verified that circAIDA targeted the mi R-29a/CLCN2 axis,thereby affecting bovine myoblast development.Furthermore,copy number variation(CNV)was found on CLCN2 gene and this study used RT-q PCR technology to analyze the possible association between the copy number variation of CLCN2 gene and growth traits in four Chinese cattle breeds and look for molecular markers.The main results are as follows:1.circAIDA affects the proliferation,differentiation and apoptosis of bovine myoblastscircAIDA is composed of exons 2 to 6 of its maternal gene AIDA.Sanger sequencing,cross-jiont primer amplification and RNase R treatment experiments proved the authenticity of circAIDA.Constructed overexpression vector pc DNA2.1-circAIDA and designed interference fragment si-circAIDA were separately transfected into bovine myoblasts.Using RT-q PCR,Western blot,CCK-8 and Ed U experiments,it was found that circAIDA could promote the proliferation,inhibit the differentiation and apoptosis of bovine myoblasts.2.circAIDA can act as a mi R-29 a molecular spongeFirst,circAIDA was subjected to nucleocytoplasmic localization,and the results showed that it was mainly expressed in the cytoplasm,which means that circAIDA might play a role by adsorbing mi RNA.The online software RNAhybrid predicted that there was a mi R-29 a binding site on the circAIDA sequence,and then the dual-luciferase reporter system was used to verify the interaction between the two.In terms of function,using RT-q PCR,Western blot,CCK-8 and Ed U assays,we found that mi R-29 a inhibited cell proliferation,promoted differentiation and apoptosis in bovine myoblasts,while part of its effects could be abolished by co-transfection with circAIDA.These above experiments demonstrated that circAIDA could play a role during the development of bovine myoblasts by sponging mi R-29 a.3.circAIDA targets mi R-29 a to activate CLCN2 gene to regulate bovine myoblast developmentIt was predicted by the Targetscan website that CLCN2 gene might be the target gene of mi R-29 a.The dual-luciferase reporter system was performed to verify the targeting relationship between mi R-29 a and the 3’UTR region of CLCN2 gene,which could be relieved by circAIDA to a certain extent.Here,we explored the function of CLCN2 gene using RT-q PCR,Western blot,CCK-8 and Ed U assays.It was found that interference with CLCN2 gene expression could inhibit proliferation as well as promote apoptosis and differentiation of bovine myoblasts,which corresponded to the function of mi R-29 a.The interaction between circAIDA,mi R-29 a and CLCN2 gene was further demonstrated by RT-q PCR.In addition,a cardiotoxin induced mouse muscle injury model was constructed to verify the function of circAIDA in vivo.The above results indicated that circAIDA could regulate the proliferation,differentiation and apoptosis of bovine myoblasts by competitively binding mi R-29 a to release the inhibition of CLCN2.In addition,circAIDA could weaken muscle regeneration in vivo.4.The distribution of CLCN2 gene CNVs in cattle populations and its correlation with growth traitsThe CLCN2 gene is not only a target gene of mi R-29 a but also has copy number variations.To further investigate the effect of the CLCN2 gene on growth and development in beef cattle,this study used a total of 573 individuals from four Chinese cattle populations(Yunling cattle,Xianan cattle,Qinchuan cattle and Pinan cattle)for identifying there were two copy number variation regions(CNV1: 3,600 bp;CNV2: 4,800 bp)of the CLCN2 gene.The CNV1 mutation in YL cattle population was significantly associated with cannon circumference and hip girth(P<?0.01).And the CNV2 mutation in XN cattle population had a significant effect on body slanting length,chest girth and body weight(P?<0.05).In YL cattle,the association analysis of CLCN2 gene CNV1&CNV2combination with growth traits including cannon circumference,hip girth and body weight was significant(P<?0.01 or P?<0.05).In addition,q PCR analysis showed that the CNV type of CNV1 affected the expression of CLCN2 gene to a certain extent.This study explored the function of circAIDA,mi R-29 a,and CLCN2 gene during proliferation,differentiation and apoptosis of bovine myoblasts and the target regulatory relationship of all three.Meanwhile,the significant association between CNVs of the target gene CLCN2 and several growth traits of Chinese cattle was revealed,which could be potential molecular markers for selection of growth traits and provide a theoretical basis for the future development and molecular breeding of Chinese beef cattle.