| miRNAs are a class of endogenous small molecules with a length of about 22~24 nt,which is highly conserved in evolution.They have emerged as key regulators of skeletal muscle development,while the knowledge of their functions in the molecular network regulating muscle development remain poorly understood.Our previous transcriptome sequencing identified the miRNAs expressed in bovine longissimus thoracis tissue,of which miR-101 was highly enriched in both fetal and adult bovine longissimus thoracis.This research used RT-qPCR,Western Blot,gene cloning and cell transfection technique to explore the role of miR-101-1 on myoblast differentiation as well as the target genes of miR-101-1.The reseach contents included the expression profiles of bovine miR-101-1 in different tissues(heart,liver,spleen,lung,kidney,longissimus muscle and fat tissues)at different developmental stages.We studied the effect of miR-101-1 over-expression on muscle differentiation.And we also examined the relatonshiop between miR-101-1 and its predicted target genes with dual-luciferase constructs.The main results were as follows:(1)The analysis of the expression profiles of bovine miR-101-1 in different tissues at multiple developmental stages of Qinchuan cattleWe obtained the expression profiles of bovine miR-101-1 in different tissues(heart,liver,spleen,lung,kidney,longissimus muscle and fat tissues)at multiple developmental stages of Qinchuan cattle by RT-qPCR.We found miR-101-1 was a miRNA widely expressed.It was detected in all tissues except fatal fat tissues.The expression of miR-101-1 in fatal cattle was lower than calf and adult cattle.But miR-101-1 was detected in the skeletal muscle at different developmental stages,and it was abundantly expressed in calf cattle.It showed that miR-101-1 has a regulatory role on the muscle development of Qinchuan cattle.(2)The construction of the over-expression vector of miR-101-1We cloned the precursor sequence of bovine miR-101-1,which connected with pcDNA3.1(+)to form a recombinant plasmid.Then we transfected the recombinant plasmid into 293 T cells,and found that the miR-101 expression in cells transfected with the recombinant plasmid significantly increased(P<0.001),compared with that in cells transfected with control plasmid and in NC cells.These demonstrated that the over-expression vector of miR-101-1 was successfully established and can be used for subsequent exprements.(3)The effect of miR-101-1 over-expression on differentiation of C2C12 cell linesThe over-expression vector of miR-101-1 was transfected into C2C12 cells and induced cell differentiation.We detected the expression level of myogenic marker genes MyOD,MyOG and MyHC during the differentiation by RT-qPCR and Western Blot technology.The results showed that the expression level of myogenic marker genes was markedly decline(P<0.05)when transfected with exogenous miR-101-1,compared with the control without transfecting exogenous miR-101-1.These results indicated that miR-101-1 can decline the expression of these genes and negatively regulates C2C12 myoblast differentiation.(4)Identification of the target genes of miR-101-1We precideted the target genes of miR-101-1 using TargetScan,we found the APP,ACVR2 B,GJA1 and RXRB genes are potential targets.Then we successfully constructed the wild-type and mutant-type recombinant vector of these target genes with the pGL3-control vector.The over-expression vector of miR-101-1 was cotransfected into 293 T cells with the 3’UTR dual-luciferase vectors of its targeting genes,then we determined the activity changes of the dual-luciferase vectors.The result showed that,compared with the control,the dual-luciferase activity of APP gene and ACVR2 B gene are markedly declined(P<0.001).This indicated that APP and ACVR2 B are the target genes of miR-101-1.miR-101-1 may influence cell differentiation by decreasing the expression level of these two target genes. |
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