| Micro RNAs are a class of 22-24 nt endogenous non-coding RNAs that play an important regulatory role in the development of skeletal muscle.In this study,the high-throughput sequencing of sheep skeletal muscle micro RNAs was combined with real-time quantitative PCR detection of mi R-452 expression in 8 fetuses and adult Hu sheep.It was found that mi R-452 was related to skeletal muscle development.So select to explore the expression and function of mi RNA-452 in skeletal muscle.In this study,the constructed pc DNA3.1(+)-mi R-452,pc DNA3.1(+)and synthetic mi R-452 chemical mimetics(NC,mimics,Anti-NC,inhibitors)were transfected into C2C12 cells,respectively.The purpose is to examine the effect of mi R-452 on proliferation and differentiation of cells.Comprehensive predictive and analytical software was used to screen out seven mi R-452 target genes that might be involved in the growth and development of skeletal muscles,in which ANGPT1,CACNB4 and RB1 was verified the target gene of mi R-452 by dual luciferase assays.Interfering with the expression of endogenous gene ANGPT1 and exploring the effect on the proliferation and differentiation of C2C12 cells.The main results of this study are as follows:1.Expression pattern of mi R-452 in fetus and adult Hu sheepAnalysis the expression of mi R-452 in 8 tissues(heart,liver,spleen,lung,kidney,intestine,pectoral and fat)of the fetal and adult in Hu sheep,mi R-452 expression significantly difference in pectoral of fetus and adult sheep,and the expression of mi R-452 is high in pectoral of fetus sheep.2.Successfully constructed pc DNA3.1(+)-mi R-452 vectorDNA was extracted from myocardium of Hu sheep and the precursor of mi R-452 was amplified.The target mi R-452 and vector pc DNA3.1 were digested and ligated,and the successfully inserted pc DNA3.1(+)-mi R-452 was transfected into 293 T for their expression efficiency.Real-time quantitative PCR analysis showed that pc DNA3.1(+)-mi R-452 vector can successfully express mi R-452.3.mi R-452 promotes proliferation of C2C12 cellsIn this experiment,the constructed pc DNA3.1(+)-mi R-452,pc DNA3.1(+)and synthetic mi R-452 chemical mimetics(NC,mimics,Anti-NC,inhibitors)were transfected into C2C12 cells,respectively.The analysis showed that by increasing the expression of endogenous mi R-452,the m RNA expression levels of Ki67,Pax7,and CDK1 cells increased,and the Pax7 and CDK1 protein levels increased,indicating that mi R-452 promoted the expression of Ki67,Pax7,and CDK1 genes.Inhibiting the expression of endogenous mi R-452,the expression levels of Ki67,Pax7,and CDK1 m RNAs were significantly down-regulated,and the protein expressions of Pax7 and CDK1 were also significantly decreased.It was shown that inhibition of mi R-452 can inhibit the proliferation of C2C12 cells.The CCK-8 experiment found that overexpression of mi R-452 increased the number of proliferating cells.The Ed U experiment found that overexpression of mi R-452 increased the proportion of Ed U-positive cells.It is further explained that mi R-452 promotes the proliferation of C2C12 cells.4.mi R-452 inhibits differentiation of C2C12 cellsThe constructed pc DNA3.1(+)-mi R-452,pc DNA3.1(+)and synthetic mi R-452 chemical mimetics(NC,mimics,Anti-NC,inhibitors)were transfected into C2C12 cells,respectively.The total RNA and total protein of the cells was used to detect the m RNA and protein expression levels of Myo G,Myf5,Mef2 c at days 1,3,5 and 7.The results showed that m RNA and protein expression of Myo G,Myf5,and Mef2 c decreased significantly after endogenous mi R-452 expression on days 1,3,5,and 7.After endogenous mi R-452 expression was inhibited,the m RNA and protein expression levels of Myo G,Myf5 and Mef2 c increased significantly.The results showed that mi R-452 inhibited the differentiation of C2C12 cells.5.Verify that ANGPT1,CACNB4,and RB1 are mi R-452 target genesTarget genes for mi R-452 with muscle development were predicted using online prediction software Targetscan and analysis software DAVID,and seven target genes were initially screened: ANGPT1,CACNB4,RB1,BCL2,BTG2,MBNL1,and PAX5.By constructing a dual luciferase reporter gene vector(wild-type and mutant),and co-transfected with mi R-452 mimics or NC into H293 T cells,the fluorescence value of renilla/firefly was analyzed and compared,and it was found that the fluorescence ratio of ANGPT1,CACNB4 and RB1 decreased.In C2C12 cells,at the m RNA and protein level,the targeting relationship between ANGPT1,CACNB4 and RB1 genes and mi R-452 was further verified.MRNA results showed that ANGPT1 and RB1 are target genes of mi R-452;Western Blot results show that ANGPT1 and CACNB4 are mi R-452 target genes.6.Si RNA interferes with ANGPT1,promotes C2C12 proliferation,inhibits C2C12differentiationThe si-ANGPT1 and si-NC were transfected into C2C12 cells,cultured in proliferation medium for 24 h and 48 h respectively,and cultured in differentiation medium for 3 days.The cell proliferation marker genes Pax7,CDK1 and differentiation marker gene Myo G,Myf5,Mef2 c were detected by Western Blot.The results showed that the interference with ANGPT1,the expression of the proliferative marker genes Pax7 and CDK1 was up-regulated,and the expression levels of the differentiation marker genes Myo G,Myf5 and Mef2 c were down-regulated.Ed U test results showed that interference with ANGPT1 increased the proportion of Ed U-positive cells.These results indicate that interference with ANGPT1 promotes C2C12 cell proliferation to inhibit C2C12 differentiation.This is the same as the overexpression of mi R-452,indicating that ANGPT1 is the target gene for mi R-452.It was further proved that mi R-452 promotes C2C12 proliferation while inhibits C2C12 differentiation by targeting ANGPT1. |
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