Study On MiR-193b-3p Promotes Goat SMSCs Proliferation By Targeting Positively Regulating IGF2BP1

MicroRNAs(miRNAs),about 20～24 nt long,are a class of small non-coding single strand RNAs with regulatory functions.Traditionally,miRNAs are thought to inhibit genes’ expression by binding to the 3′ UTR of target mRNAs in the cytoplasm to degrade mRNA or inhibit translation,but this theory is difficult to explain the miRNAs’ role in promoting gene expression.A growing number of studies have found that enhancer-mediated transcriptional promotion is an important mechanism for miRNAs to regulate gene expression positively.These kinds of miRNAs,which can be found in the nucleus and have the function of activating gene expression,are called nuclear activated miRNAs(NamiRNAs).Previous studies in our laboratory found that miR-193b-3p could target positively regulate IGF2BP1,and then promote goat skeletal muscle satellite cells(SMSCs)proliferation.In order to investigate whether miR-193b-3p functions as a NamiRNA and its mechanism,this study use goat SMSCs as the experitmental material,we analyzed the differential expressed mRNAs between miR-193b-3p overexpression(n=4)and control groups(n=4)based on RNA-seq results,and then verify whether miR-193b-3p is a NamiRNA by nucleoplasm separation,RT-qPCR and dual luciferase reporting system techniques.Finally,the effects of miR-193b-3p on proliferation and apoptosis of SMSCs were studied by RT-qPCR,flow cytometry analysis,immunofluorescence staining and other techniques.The main results obtained are as follows:1.A total of 489,496,104 double-end clean reads with a read length of 150 nt were obtained from 8 sequencing libraries,yielding a total of approximately 72.13 Gb data.Among them,97.09%～97.37% of clean reads can be matched to the goat genome(GCA＿001704415.1).miR-193b-3p up-regulated 264 genes and down-regulated 207 genes.The up-regulated mRNA expression trend was verified by RT-qPCR.GO enrichment analysis found that up-regulated genes can be enriched in the insulin-like growth factor binding pathway,and IGF2BP1 in this pathway was predicted to have a binding site with miR-193b-3p.2.RT-qPCR results showed that miR-193b-3p was distributed both in the nucleus and cytoplasm of goat SMSCs,and the distribution ratio in the nucleus gradually decreased with cell differentiation(differentiation 0 days: 7.47%,differentiation 7 days: 2.68%);pc-DNA3.1(+)plasmid successfully overexpressed miR-193b-5p(P < 0.05),and miR-193b-5p promote the expression of miR-193b’s neighboring genes(PARN)and IGF2BP1(P < 0.01),the expression of miR-193b-3p was not significantly affected(P >0.05),however;In addition,RT-qPCR results indicate that miR-193b-3p can promote the expression of its neighboring genes(MKL2,PARN and BFAR)and target gene IGF2BP1(P<0.05);The gene sequence of miR-193 b was found to have enhancer activity by the dual luciferase reporter system.These results confirm that miR-193b-3p is a NamiRNA.3.EdU immunofluorescence test showed that both miR-193b-3p and its target gene IGF2BP1 can promote the proliferation of goat SMSCs;Flow cytometry analysis found that overexpression of miR-193b-3p increased the proportion of G2 phase goat SMSCs(P< 0.01).This results further verify that miR-193b-3p promote cell proliferation.4.RT-qPCR was used to detect the effect of miR-193b-3p on apoptosis in goat SMSCs.It was found that miR-193b-3p had no effect on the expression of apoptosis marker gene Caspase1(P > 0.05);TUNEL test further verified that miR-193b-3p has no influence on cell apoptosis.Act D test found that miR-193b-3p can down-regulate the stability of IGF2BP1 mRNA.This indicate that miR-193b-3p may promote the proliferation of goat SMSCs by up-regulaing IGF2BP1.5.RT-qPCR results showed that miR-193b-3p tended to inhibite its adjacent genes(MKL2,PARN and BFAR)and IGF2BP1 expression in mouse myoblast(C2C12);EdU immunofluorescence test showed that miR-193b-3p inhibited C2C12 proliferation,flow cytometry analysis also found that the proportion of G2 phase cells decreased after overexpression of miR-193b-3p.This indicate that miR-193b-3p has different functions in different cells.The above research shows that miR-193b-3p is a NamiRNA,which promote the proliferation of goat SMSCs by targeting positively regulating IGF2BP1.Our results provide a reference for comprehensively revealing the molecular mechanism of miRNAs in the development of goat skeletal muscle.