Regulation Of MiR-193b-3p On Proliferation And Differentiation Of Goat Skeletal Muscle Satellite Cells Through Targeting IGF2BP1

MiRNA is a type of non-coding RNA with a length of 22bp.It participates in the regulation of a series of life activities such as growth,development and metabolism.Muscle growth plays an important role in livestock breeding,which is an indicator to improve the economic benefits of livestock.miRNAs have been reported to participate in the process of muscle cell proliferation,differentiation,apoptosis,etc.Through our previous sequencing experiments,we found that miR-193b-3p was closely related to the development of goat skeletal muscle.However,its effects on specific mechanism of goat skeletal muscle satellite cells(SMSCs)remains unclear.This study predicted that insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1)is the target gene of miR-193b-3p using FINDTAR3 and BIBIServ2,and this result was confirmed by dual luciferase reporter system.Moreover,The expression of miR-193b-3p and target gene IGF2BP1 at different stages of proliferation and differentiation of goat SMSCs were detected,and we analysed the mRNA and protein levels of those target genes and SMSRs proliferation differentiation markers,as well as the effects on phenotypes,responding to overexpression and suppression of miR-193b-3p.Our main results are as follows:1.RT-qPCR was used to detect expression patterns of miR-193b-3p in goat embryos on the 75th embryonic day and different tissues of goat on the third postnatal day.miR-193b-3p was found to be highly enriched in longissimus dorsi muscle of goats at both two stages,while its expression was especially higher in the embryonic dorsomedial dorsal muscle than in heart,liver,spleen,lung,and kidney,although higher in lamb liver than in muscle on the third postnatal day.In addition,the expression level gradually increased during the development of embryos.In vitro experiments showed that the highest expression levels were found on the 7th day after differentiation in SMSCs.These results suggested that miR-193b-3p plays an important role in the development of muscle tissue and cells.2.In goat SMSCs,overexpression of miR-193b-3p significantly up-regulated the mRNA levels of the proliferation marker genes Pax7 and PCNA(P<0.05),which was further confirmed by WB detection of the Pax7 protein level.Using EdU for further cell counting,we found that the number of newly added cells in the experimental group overexpressing miR-193b-3p was twice as high as that of the control group.Meanwhile,CCK-8 cell proliferation assay also showed that overexpression caused a significant increase in OD values compared with control group NC(P<0.05).However,inhibiting miR-193b-3p leaded to a decreasing trend(P>0.05)of proliferative marker genes,while the number of newly added cells decreased.The results of CCK-8 showed that the OD value was significantly lower than that of control group under 24-hour inhibition(P<0.05)..These results indicated that miR-193b-3p can promote the proliferation of goat SMSCs.3.In goat SMSCs,the mRNA levels of differentiation markers MyoG and MyoD increased significantly(P<0.05)in response to overexpression of miR-193b-3p.The detection of MyoG protein level further validated miR-193b-3p as a marker for differentiation.As we found in immunohistochemistry,we also found that the number of differentiated myotubes after overexpression miR-193b-3p was twice as high as that of control group.In contrast,after inhibiting miR-193b-3p in SMSCs,the mRNA levels of differentiation marker genes was decreased(P>0.05),and the number of myotubes was approximately one-half that of control group,indicating that miR-193b-3p could promote the differentiation of goat SMSCs.4.Using FINDTAR3 and BIBIServ2 softwares,we found that there was a miR-193b-3p binding site in the 3’UTR region of Insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1).Further verification of the dual luciferase reporter system showed that the fluorescence value of the experimental group(3’UTR target fragment with IGF2BP1)was significantly higher than that of control group(P<0.05),and the fluorescence value decreased significantly(P<0.05)after mutating the binding site of IGF2BP1 and miR-193b-3p.The results of FISH showed that IGF2BP1 mRNA colocalized with miR-193b-3p in the cytoplasm.These results indicated that IGF2BP1 is the target gene of miR-193b-3p,and miR-193b-3p promotes the expression of IGF2BP1.5.We used qPCR to compare the expression of miR-193b-3p and IGF2BP1 in goat cells and tissues.The mRNA of IGF2BP1 also reached the highest level in the 7th day after differentiation of goat SMSCs,which was consistent with the aforementioned trend of miR-193b-3p.Overexpression of miR-193b-3p caused a significant increase of IGF2BP1 expression in SMSCs(P<0.05),while decreased IGF2BP1 the expression of mRNA after miR-193b-3p inhibition(P>0.05).At different stages of goat embryonic and postnatal development,IGF2BP1 and miR-193b-3p were highly expressed in muscle.These results further suggested that miR-193b-3p cooperates and promotes IGF2BP1 expression.6.Furthermore,overexpression of IGF2BP1 in SMSCs significantly up-regulated the expression of miR-193b-3p,the proliferation marker Pax7,and the differentiation marker genes MyoG and MyoD(P<0.05).Quantification of Pax7 and MyoG protein levels confirmed the changes in mRNA levels.On the contrary,after inhibiting IGF2BP1,the proliferation and differentiation marker genes of SMSCs showed a decreasing trend(P>0.05),showing that IGF2BP1 has the same effect on proliferation and differentiation of SMSCs as miR-193b-3p.7.Co-transfection of Overexpressed miR-193b-3p and interfered IGF2BP1 vector in SMSCs showed that the proliferation marker genes Pax7,PCNA and target gene IGF2BP1 were all decreased compared with control group(P<0.05),while the differentiation marker genes MyoG,MyoD didn’t declined.EdU results showed that the number of new nuclei in the co-transfection group was reduced by half compared to control group,revealing that the effect of miR-193b-3p on the proliferation of SMSCs was mainly achieved through the IGF2BP1 gene.8.In order to reveal the role of miR-193b-3p in promoting IGF2BP1 mRNA,we operated ATCD experiments,and the half-life of mRNA of IGF2BP1 was found to decrease after exogenous addition of miR-193b-3p mimic,suggesting that miR-193b-3p has cytoplasmic effects on IGF2BP1 mRNA.Degradation.In summary,miR-193b-3p and IGF2BP1 are enriched in goat muscle tissue and SMSCs cells.Both miR-193b-3p and IGF2BP1 can promote the proliferation and differentiation of goat SMSCs.IGF2BP1 is a target gene of miR-193b-3p which is positively regulated by miR-193b-3p.Since miR-193b-3p can unstabilize the mRNA of IGF2BP1,We speculated that miR-193b-3p may promote IGF2BP1 at the transcriptional level.