Study On Expression Profile Of MiR-432-5p In Different Tissues Of Xuhuai Goat And Its Related Function

Micro RNAs(miRN As)are a group of highly conserved single-stranded noncoding small RN As that are about 21~25 nt in length.miRN As do not encode proteins,but they can regulate life activity by targeted inhibiting or degrading mRNA.miRNAs are distributed in the body widely and play a certain regulatory role in skeletal muscle proliferation and differentiation.The results of transcripto me sequencing of goat muscle show that miR-432-5p is highly express in muscle,we analyze the expression of miR-432-5p in different tissues of embryonic and adult Xuhuai goat,find that the expression of miR-432-5p has the organization diversity and growth development stage diversity,we suspect that it may regulate the growth of skeletal muscle.Therefore,we constructed the highly efficient overexpression vector of miR-432-5p,transfected the miR-432-5p overexpression vector,miR-432-5p mimics,negative control(NC),miR-432-5p inhibitors and anti-negative control(Anti-NC)into C2C12 cells to investigate the effects of miR-432-5p on the proliferation and differentiation of C2C12 cells with RT-qPCR and Western blot technology.We predicted four potential target genes through target gene prediction software and verifyd relationship between these four target genes and miR-432-5p with the dual-luciferase reporter gene experiment,the main results are as follows: 1.miR-432-5p expression analysis of different tissuesThrough the analysis of the relative expression of miR-432-5p in different tissues of Xuhuai goats,it was found that the expression of miR-432-5p was the highest in the leg muscle of fetal goat,followed by the longissimus dorsi.The expression leve l of miR-432-5p in cardiac and leg muscle was higher in adult sheep,and the expression of miR-432-5p in muscle tissue of fetal goat was significantly higher than that of adult goat.The results showed that miR-432-5p has the organization diversity and gro wth development stage diversity.2.The construction and validation of pcDNA3.1(+)-miR-432-5pWe amplified the precursor miR-432-5p sequence and ligated it to pcDNA3.1,then the expression of miR-432-5p was detected after transfected into 293 T cell by RT-qPCR.Compared with the control group,the expression of miR-432-5p was significantly up-regulated in the experimental group.The results showed that pcDNA3.1(+)-miR-432-5p vector could be expressed efficiently in cells and could be used in subsequent experiments 3.miR-432-5p promotes the proliferation of C2C12 cellsThe results of RT-qPCR showed that the expression of miR-432-5p and K i67 increased with cell proliferation under normal growth,we hypothesized that miR-432-5p could promote the proliferation of C2C12 cells.In order to verify this hypothesis,pc DNA3.1(+)-miR-432-5p,miR-432-5p mimics,negative control(NC),miR-432-5p inhibitors,anti-negative control(Anti-NC)were transfected into C2C12 cells.The results showed that the expression of K i67 mRN A and protein were significantly increased after transfection with pc DNA3.1(+)-miR-432-5p and miR-432-5p mimics,which indicated that miR-432-5p promoted the expression of K i67 Gene.The expression of K i67 mRNA was significantly down-regulated after transfection with miR-432-5p Inhibitors.The above results show that miR-432-5p can promote the proliferation of C2C12 cells.4.miR-432-5p inhibits the differentiation of C2C12 cellsThe pc DNA3.1(+)-miR-432-5p,miR-432-5p mimics,negative control(NC),miR-432-5p inhibitors and anti-negative control(anti-NC)were transfected into C2C12 cells respectively and induced C2C12 to differentiate using DMEM medium containing 2% horse serum.The changes of cell growth status and morphology were observed under microscope.The total cell RNA and total protein were extracted for RT-qPCR and western blot to inspect the expression of cell differentiation marker genes MyoG,Myf5,Mef2 C.Morphological observation showed that C2C12 cells had obvious myotubes at the 4th day.Fluorescence quantitative PCR and Western blot showed that the expression of MyoG,Myf5,Mef2 c mRNA and protein was significantly reduced after overexpression miR-432-5p.The expression of MyoG,Myf5 and Mef2 c increased significantly after transfection with miR-432-5p inhibitors.The results showed that miR-432-5p inhibited the differentiation of C2C12 cells.5.The prediction and verification of miR-432-5p target genesmiR-432-5p target genes were predicted by Targetscan.Because there is no goat sequence in the database,we used sequence of cattle to predict miR-432-5p target genes,and then the predicted candidate genes with DAVID online Software for gene annotation,to further study the genes which possibly have an effect on the proliferation and development of muscle cells.Four target genes were screened: SORT1,TGFBR2,GJC1,and BC L2.The wild type and mutant type of dual-luciferase reporter gene vectors of these four potential genes were constructed and transfected into 293 T cells with miR-432-5p mimics respectively.According to the changes of fluorescence values,SORT1 and TGFBR2 were MiR-432-5p target genes.In order to further verify the target relationship of SORT1 and TGFBR2 between miR-432-5p,we transfected miR-432-5p mimics and negative contro l(NC)into C2C12 cells respectively.Western blot results also proved that SORT1 and TGFBR2 were the target genes of miR-432-5p.