The Regulation Of LncRNA-AK017263 On Myoblast Proliferation And Differentiation

The regulation mechanisms of skeletal muscle growth and development are one of the central issues in animal science.Skeletal muscle is mainly composed of muscle fibers.The interaction of intrinsic and epigenetic mechanisms controls muscle fiber production at various stages of development.Long non-coding RNAs(lncRNAs)are defined sequences as more than 200 nt and no potential protein coding ability.And they perform many functions through various mechanisms.At present,a variety of lncRNAs have been identified that have important effects on muscle fiber growth,but the functions and mechanisms of many lncRNAs are still unclear.In our previous study,we found differentially expressed lncRNA AK017263 which was significantly down-regulated after knockdown of MyoD in C2C12 cells based on lncRNA and mRNA microarray analysis.In this study,C2C12 cells were used to study the effects of AK017263 on the proliferation and differentiation by using RNAi and overexpression experiments.The main results are as follows:1.Quantitative real-time PCR results showed that the expression of AK017263 was significantly up-regulated during myoblast differentiation.AK017263 was highly expressed in leg muscle,longissimus dorsi and tongue.2.Quantitative real-time PCR and Western Blot were used to confirm the change of the mRNA and protein levels of the proliferative marker genes Ki67 and N-RAS by interference and overexpression experiments.Overexpression of AK017263 could promote the expression of proliferation marker genes,while AK017263 knockdown significantly decreased the expression of proliferation marker genes.EdU and flow cytometry results also showed that AK017263 could promote the proliferation of C2C12 cells.3.Quantitative real-time quantitative PCR,Western Blot and immunofluorescence experiments were used to verify the change of the mRNA and protein levels of differentiation marker genes MyoD,MyoG and MyHC by interference and overexpression experiments.The results showed that the expression of the differentiation marker genes was up-regulated after overexpression of AK017263,and AK017263 knockdown significantly decreased the expression of differentiation marker genes,indicating that AK017263 could promote the myoblasts differentiation.In addition,AK017263 was found to promote the expression of MyHC1/slow and inhibit the expression of MyHC2a?MyHC2b genes.4.Online software prediction showed that AK017263 may interact with Mef2 D which is a member of MEF2 family,and the expression of Mef2 D gene mRMA level was verified after AK017263 interference or overexpression.The results showed that the expression of Mef2 D was decreased after AK017263 interference,and the expression of Mef2 D was up-regulated after AK017263 overexpression.The above test results indicated that there is a positive correlation between the expression of Mef2 D and AK017263.The results of these experiments showed that lncRNA-AK017263,which is highly expressed in mouse muscle,can promote the proliferation and differentiation of C2C12 cells,and it can exert different functions on different muscle fiber types.We also preliminarily predicted the mechanism of AK017263 in promoting C2C12 cells differentiation.The results of this study laid the foundation for further understanding of the AK017263 regulation mechanism,and also provided new evidence for the development of important lncRNAs during skeletal muscle growth.