Expression Profiling Of Mir-660 And Its Related Functions In The Development Of Bovine Skeletal Muscle

MicroRNAs are a series of the most commonly researched non-coding RNAs in life science.The existing literature reveals that microRNA plays an important role in animal skeletal muscle development.Here,we screened microRNAs with different expression in time and space from our previous high-throughput sequencing results,and the microRNA expression profiles of Qinchuan cattle during different developmental stages(fetal calves,calves),different tissues(leg muscle,heart,liver,spleen,lung,kidney,stomach and intestine)were obtained by RT-qPCR technique Analysis.miR-660 was eventually selected as the subject to study its effect on skeletal muscle development.In this study,miR-660 was overexpressed or disrupted by miR-660 mimics or miR-660 inhibitor in mouse myoblast cell line(C2C12)which was then induced to proliferate and differentiate.RT-qPCR and Westerrn Blot were used to measure mRNA and protein to investigate the effect of miR-660 on the proliferation and differentiation of skeletal muscle.Dual-Luciferase Reporter Assay went for demonstrating that miR-660 directly targeted the 3'-UTR of Rho guanine nucleotide exchange factor 12(ARHGEF-12).Furthermore,we found an inverse relationship between the expression of miR-660 and ARHGEF12 in both gain-and loss-of-function studies: overexpression of miR-660 declined the mRNA and protein expressions of ARHGEF12 in C2C12 cells differentiation,however,knockdown of miR-660 had completely opposite results.In addition,we explored the possibility of target genes involved in the Rho A/Rho A kinase signaling pathway.The main results of this study are as follows:(1)conservation analysis of related sequences showed that the microRNA binding site of mature miR-660 and its target gene ARHGEF12 3 'UTR were highly conserved among different species,such as humans and cattle.(2)The expression of miR-660 spectrum showed that the miR-660 in different developmental stages in Qinchuan cattle(fetal calves,calves)and different tissues(leg muscle,heart,liver,spleen,lung,kidney,stomach and intestine)expressed ubiquitously,and the expression of miR-660 in fetal bovine and bovine skeletal muscle weight were significantly different(P < 0.01),indicating that miR-660 belongs to nonspecific muscle-related microRNAs.(3)RNA oligonucleotides,including miR-660 mimics,negative control(NC),2'-O-methylated miR-660 inhibitors,anti-negative control(anti-NC)were respectively transfected into the C2C12 cells with Lipofectamine 2000 following the manufacturer's instructions.Cells were collected for RT-qPCR and Western Blot analysis against markers of proliferation and differentiation.The results showed that miR-660 could promote the growth and Inhibit differentiation.(4)With the 3'UTR of ARHGEF12,CDH13,ETV1,GNAS,HIF1 A,SDC1 and TPM4 cloned into the XbaI site in luciferase reporter vector pGL3-control,luciferase reporter vectors were successfully constructed.The result of Dual-Luciferase reporters demonstrated miR-660 could directly target ARHGEF12.Furthermore,up-regulated miR-660 repressed the ARHGEF12 mRNA and protein expression in C2C12 cells differentiation.Conversely,inhibition of miR-660 significantly up-regulate ARHGEF12 m RNA and protein expression,further confirming that ARHGEF12 is a target gene of miR-660.(5)ARHGEF12 was analyzed with gene ontology and its highly enrichment in RhoA/ROCK pathway was confirmed by KEGG databases.So we speculated that miR-660 may target ARHGEF12 to suppresse myoblast differentiation through regulating the RhoA/ROCK pathway.