| Muscle development is an important factor affecting meat production and meat quality.proliferation and differentiation of myoblasts play the most important role in muscle development.Process of myoblasts proliferation and differentiation involves the expression of multiple genes,signal pathways and network regulation are regulated by many cytokines.Although the molecular mechanism of muscle development has been a major research object on developmental biology,so far there is still a considerable number of muscle regulatory factors needed to be identified.MiRNA is the most important single-stranded non-coding small RNA in the tissue,the length of about 18~24 nucleotide.miRNAs are subsequently processed by two RNase III proteins(Drosha and Dicer).In recent years,it has been found that miRNAs play an important role in muscle development,and the study of miRNA mainly focuses on the regulation of downstream target genes,only few studies were performed on the factors that affect the expression of miRNAs.There is little research on muscle development in cattle and the regulation mechanism is still unclear.It has also been reported that circular RNA can act as miRNA molecular sponge to regulate miRNA function,but at present,whether there are circRNAs existing in bovine muscle or not,and the function of circRNA remains unknown.In this study,the expression of miRNAs and circRNAs in bovine muscle tissue were analyzed by high-throughput sequencing and real-time fluorescence quantitative PCR.The function mechanism and the relationship between circRNAs and miRNAs on myoblasts proliferation and differentiation were detected by means of overexpression or interference,real-time fluorescence quantitative PCR,double luciferase gene reporter system,CCK-8,EdU and Flow cytometry.A regulation network of circRNA-miRNA-mRNA were constructed for the mechanism of bovine muscle development,which provides a new theoretical basis for molecular breeding of Qinchuan cattle.Our main results are as follows:1.miR-378a-3p regulates myoblast proliferation,differentiation and apoptosis by targeting HDAC4miR-378a-3p,selected as potential regulatory candidates based on our previous Solexa SBS technology sequence results,has significantly higher expression in muscle tissue than that in other tissues.The results of CCK-8,EdU and qRT-PCR were demonstrated that the proliferation of myoblasts was inhibited by miR-378a-3p,and the expression of cyclinD1 was down-regulated.MiR-378a-3p promoted myoblasts apoptosis via down-regulating the expression of BCL-2 and up-regulating the expression of BAX and caspase-3.Meanwhile,overexpression of miR-378a-3p promotes differentiation and significantly up-regulated the expression of MyoD and MyHC in mRNA and protein levels in myoblasts.Mechanism study indicated that miR-378a-3p promote myoblasts differentiation and inhibits proliferation by inhibiting the protein expression of HDAC4 which is known to promote differentiation and proliferation by repress the MyoD activity.2.miR-107 inhibits proliferation and differentiation of myoblasts by targeting Wnt3amiR-107 is derived from the gene of Pantothenic acid kinase 1(PANK1)on chromosome 26,which is highly conserved to human and mouse sequences and has high level of expression in muscle tissue.Overexpression of miR-107 significantly down-regulated the expression of MyHC,MyoD and MyoG in both mRNA and protein levels,and decreased the cell proliferation index and the expression of proliferation marker gene PCNA.Similarly,overexpression of miR-107 regulates the expression of BCL-2,p53 and caspase9 mRNA and protein translation.TargetScan software predicted that the Wnt3 a gene is a direct target gene for miR-107 and identified by dual luciferase reporter systems.Overexpression of miR-107 significantly reduced the expression of Wnt3 a in both mRNA and protein levels,and the myoblasts differentiation and proliferation were inhibited by RNA interference of Wnt3 a.These results suggested that miR-107 inhibits the differentiation,proliferation and apoptosis of myoblasts by inhibiting the expression of Wnt3 a,and it may act as another molecular marker of bovine breeding.3.Identification of circRNAs in muscle tissue of Qinchuan cattleIn order to study the presence of muscular development related circRNA,the expression of circRNA in different developmental stages of muscle tissue in Qinchuan cattle was identified by Ribo-Zero RNA-Seq,and total 12,981 circRNAs were detected including 2400 circRNAs which were exist in both embryonic and adult muscle.The transcript length of most circRNAs was less than 2000 nucleotides(nt),and the mean length was 822 nt,which was shorter than the transcript of protein-coding genes.Thousands of detected circRNAs were widely distributed in all chromosomes except the Y chromosome in embryonic and adult muscle tissues.We found the number of circRNA reads located in the chromosome increased with the increase of chromosome length.We found 828 circRNAs were significantly different between the embryonic stage and adult stage libraries,of which 624 were up-regulated and 204 down-regulated.This results indicated that circRNA may play an important role in muscle development.To identify specific circRNAs displaying development-related changes in abundance,17 differentially expressed circRNAs were randomly selected and detected by qRT-PCR.The results were consistent with RNA-Seq and we focused on the most down-regulated circRNA,circRNA42,which may has important function in muscle development.4.circLMO7 regulating the proliferation,differentiation and apoptosis of primary myoblasts by sponging miR-378a-3pCircLMO7 is derived from LMO7 gene,the full length has 2,312 nt and is composed of nine exons.The expression of circLMO7 in muscle was significantly higher than other tissues.A binding site of miR-378a-3p was predicted in circLMO7 sequence by RNAhybrid software and overexpression of circLMO7 could significantly reduce the expression of miR-378a-3p.CCK-8,EdU and qRT-PCR results showed that circLMO7 promoted myoblasts proliferation and up-regulated the expression of proliferation marker gene cyclinD1.Also circLMO7 inhibits the apoptosis of myoblasts through promoting the expression of BCL2 mRNA and protein,and inhibition of BAX and caspase9 mRNA expression and protein translation.BCL2,BAX and caspase9 are apoptosis markers.Meanwhile,circLMO7 significantly inhibits the expression of MHC,MyoG and MyoD and the production of myotubes in myoblasts,suggesting that circLMO7 suppresses the myoblasts differentiation.These results are contrast to the effects of overexpression of miR-378a-3p on cell proliferation and differentiation,and overexpression of circLMO7 could significantly reduce the expression of miR-378a-3p and enhance the expression of HDAC4,suggesting that circLMO7 promotes differentiation and inhibits proliferation of myoblasts by competitive binding to miR-378a-3p.Our study accumulates valuable fundamental data and provides new insights for revealing the molecular mechanism of muscle development in Qinchuan cattle,and adds new evidence for non-coding RNAs study.Meanwhile,this study provide basic theoretical foundations for molecular breeding and resource protection of excellent beef cattle in China. |
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