| Long noncoding RNAs(lncRNAs)are initially discovered as transcripts out of junk DNA regions of our genome,but more and more studies have confirmed that lncRNAs are emerging as novel family of regulatory factor,which is widely involved in various biological processes,including regulation of skeletal muscle development.Our preliminary study work manifested that lncRNA TCONS＿00135123(linc-MD1)was positively correlated with Myo G and other myocyte differentiation marker genes based on the lncRNA sequencing data(SRR3567144)of Jianzhou Da’er goat skeletal muscle,but it’s mechanism was still unclear.In this study,we first analyzed the sequence characteristics of linc-MD1 and examined the expression profiles of linc-MD1 in different tissues and different phase of Jianzhou Da’er goat embryos.In addition,we explored the roles and mechanisms of linc-MD1 and miR-133a-3p in goat SMSCs differentiation.The main results were shown as follows:(1)The sequence alignment between goat linc-MD1 and mouse linc-MD1 was aligned by DNAMAN software revealed that goat linc-MD1 is highly homologous to a part of the mouse linc-MD1 sequence and the homologous sequence contains a binding site for muscle-specific miR-133a-3p.The prediction of protein coding ability indicates that goat linc-MD1 does not have protein coding ability.(2)The expression of linc-MD1 was significantly higher in goat skeletal muscle tissues than in other non-muscle tissues(P<0.01).In the developmental expression,the expression level of linc-MD1 was the highest at 60 days of embryos,and it increased first and then decreased.(3)It was predicted that there may be a binding site of miR-133a-3p on the sequence of linc-MD1 through analysis by RNA22 and RNAhybrid software.The dual luciferase reporter system assay and the Ag O2-RIP experiment confirmed the interaction of goat linc-MD1 and miR-133a-3p.These results indicated that linc-MD1 is the target gene of miR-133a-3p.Through nuclear separation experiments,it was found that linc-MD1 is mainly localized in the cytoplasm.(4)In the process of goat SMSCs differentiation,the expression of miR-133a-3p showed an up-regulated trend.Through overexpression and inhibition experiment of miR-133a-3p,we found that after miR-133a-3p overexpression,the expression of Myo D,Myo G and MAML1/Dlx3 were significantly down-regulated(P<0.01),while the expression of goat linc-MD1 was significantly down-regulated(P<0.05).After the expression of miR-133a-3p was inhibited,the expression of Myo D,Myo G and MAML1/Dlx3 were significantly up-regulated(P<0.05),while the expression of linc-MD1 was significantly up-regulated(P<0.05).Theses results indicated that miR-133a-3p can inhibit the differentiation of SMSCs,and miR-133a-3p targeting MAML1/Dlx3 affects the expression level of MAML1/Dlx3 and participates in the regulation of differentiation of goat SMSCs.The result also proved that linc-MD1 can play a functional role in the differentiation of goat SMSCs by interacting with miR-133a-3p.(5)As goat SMSCs differentiated,the expression of linc-MD1 showed an up-regulated trend.After linc-MD1 overexpression,the expression of miR-133a-3p was significantly down-regulated(P<0.01),while the expression of Myo D,Myo G and MAML1 were significantly up-regulated(P<0.05).These results indicated that linc-MD1 can bind miR-133a-3p by acts as a competing endogenous RNA,alleviate the inhibitory effect of miR-133a-3p on its target gene MAML1,thereby increasing the expression of MAML1 gene and promoting the differentiation of goat SMSCs.(6)After overexpression of linc-MD1,the expression of SRF was significantly increased compared with the control group(P<0.05).Through overexpression and inhibition of miR-133a-3p,the expression of SRF m RNA was no effect.It has been reported in the goat SMSCs that miR-133a-3p can inhibit the expression of the target gene SRF protein level.Therefore,we hypothesized that linc-MD1 can bind miR-133a-3p by acts as a competing endogenous RNA,alleviate the inhibitory effect of miR-133a-3p on it’s target gene SRF,thereby increasing the expression of SRF protein in cells and promoting the differentiation of goat SMSCs.In conclusion,linc-MD1 promoted the differentiation of goat SMSCs and miR-133a-3p inhibited the differentiation of goat SMSCs.We hypothesized that linc-MD1 can bind miR-133a-3p by acts as a competing endogenous RNA,alleviate the inhibitory effect of miR-133a-3p on its target gene MAML1/SRF,thereby increasing the expression of MAML1/SRF in cells and promoting the differentiation of goat SMSCs.Our results provide a useful resource for better understanding muscle related lncRNAs in goat. |
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