Study On The Regulation Of LncRNA Lnc₅94 In Goat Skeletal Muscle Satellite Cell Proliferation And Differentiation

LncRNAs are a family of non-coding RNA greater than 200 nt.They are involved in cell proliferation,differentiation and apoptosis through multiple biological processes,such as epigenetic regulation,transcriptional level,and post-transcriptional regulation.In the previous study,it was found that Inc₅94 was highly correlated with skeletal muscle differentiation.However,the mechanism underpinning was still unclear.In this study,we analyzed the spatiotemporal expression of lnc₅94 in embryonic goat.The binding of miR-133a-3p and lnc₅94 was validated by dual-luciferase reporter assay system and fluorescence in situ hybridization.RT-qPCR was used to detect the expression pattern of lnc₅94,miR-133a-3p and FGFR1 in SMSCs during proliferation and differentiation.The function of lnc₅94 was investigated in vitro.The main results are as follows.?1?The protein coding ability of lnc₅94 was predicted by online software CPAT and iSeeRNA,and the results showed that it is predicted as noncoding.The results of RT-qPCR indicated that lnc₅94 was widely expressed in six tissues of goat at E60,and the expression level of lnc₅94 was significantly higher in longissimus dorsi than in other tissues?P<0.01?.In the temporal expression,the expression level of lnc₅94 was the highest at E60?P<0.01?.?2?Lnc₅94 was predicted to contain binding site of miR-133a-3p.The wild-type and mutant vectors containing the binding slte were successfully constructed and used in dual-luciferase reporter assay system.The results showed that the luciferase activity of wild-type group was significantly reduced when comparing to mutant and NC group?P<0.05?.?3?By separating the nucleus and cytoplasm of SMSCs and RT-qPCR,it was found that Inc₅94 was mainly expressed in cytoplasmic?P<0.05?.Fluorescence in situ hybridization experiments showed that Inc₅94 and miR-133a-3p were co-expressed in the cytoplasm.?4?By using online softwares TargetScan,we screened out FGFR1 was a putative target of miR-133a-3p.The expression of lnc₅94,miR-133a-3p,FGFR1 during SMSCs differentiation was significantly up-regulated?P<0.05?.?5?FGFR1 and myogenic differentiation marker gene MyoD,MyoG were respectively down-regulated 1.6,1.5 and 1.8 fold?P<0.01?comparing with the control group in SMSCs overexpressing lnc₅94,and the expression of miR-133a-3p did not change significantly?P>0.05?.FGFR1,MyoD,MyoG and miR-133a-3p were respectively down-regulated 4,1.6,7.7,2.7 times?P<0.01?comparing with the control group in SMSCs inhibiting Inc₅94.?6?The effect of lnc₅94 on proliferation of SMSCs was detected by CCK-8 assay.The growth curve of transfected cells was plotted according to the OD value.It was found that overexpression or inhibition of lnc₅94 had no significant effect on cell proliferation comparing with NC control group?P>0.05?.?7?Overexpression of miR-133a-3p,the expression of lnc₅94 and FGFR1 was significantly down-regulated compared with the control group?P<0.01?.In summary,we identified lnc₅94 was a IncRNA.It was mainly expressed in the cytoplasm,and regulated the differentiation of goat SMSCs possibly via miR-133a-3p and FGFR1.