Explore The Biological Function Of LncRNA Xist/miR-486-5p During Myoblast Proliferation And Differentiation

Purpose:This study aimed to explore the Lnc RNA-mi RNA axis that plays an important role in myoblast proliferation and differentiation using multi-omics data integration analysis and cellular experimental models to lay the foundation for the mechanism study of the muscle atrophy-regeneration process.Materials and Methods: In this study,the Lnc RNAs and mi RNAs that were significantly changed in the proliferation and differentiation of myoblasts,were firstly mined by bioinformatics methods,and studied the regulatory relationships between the above RNAs using association analysis to obtain the Lnc RNA-mi RNA axis with potentially important roles in the proliferation and differentiation of adult muscle cells.Specifically,a hind limb muscle wasting atrophy model was established by tail suspension in C57BL/6 mice,and then lowered the mice from the suspension system after the onset of muscle atrophy(day 14 of tail suspension)to build a muscle regeneration model.Muscles were collected at 0,3,7 and14 days of suspension and1 and 7 days of de-suspension,and mi RNA microarrays were used to analyze mi RNAs that play important functions in muscle atrophy and regeneration.In addition,the RNA sequencing data of myoblasts under microgravity stimulation from public database and Lnc RNA microarray data of myoblast proliferation and differentiation were used to explore the Lnc RNAs that changed significantly in response to microgravity stimulation and during proliferation and differentiation of adult myoblasts Subsequently,the Lnc RNA-mi RNA axis,which plays a potentially important role in the proliferation and differentiation of myoblasts,was obtained by combing the above data and the targeting relationship network analysis of Lnc RNA-mi RNA.On the basis of the above analysis,we further used molecular biology and cell biology methods to verify the regulatory effect of the above Lnc RNA-mi RNA axis on the proliferation and differentiation of myoblasts.On the one hand,a dual luciferase reporter system was used to verify the Lnc RNA-mi RNA interactions obtained during the above analysis in HEK-293 T cell line.On the other hand,relevant lnc RNA and mi RNA overexpression and knockdown models were established in C2C12 cell lines,and the effects of overexpression and knockdown on cell proliferation and differentiation trends,respectively,were analyzed at the cellular level using Edu staining analysis and Dio staining analysis.At the molecular level,RT-q PCR and Western Blot were used to analyze the expression changes of molecular markers of proliferation and differentiation such as Pcna,Myod,Myog and Hsp90 proteins.Based on the above experiments,this study further verified the importance of the aforementioned Lnc RNA-mi RNA axis in the proliferation and differentiation process of myoblasts using rescue experiments.Finally,the mi RNA database was used to predict mi RNA potential target m RNAs.The RNA-sequencing data of myoblasts under microgravity stimulation and the RNA-sequencing data of myoblast during proliferation and differentiation from public databases were used to mine the m RNAs that significantly changed in myoblast in response to microgravity stimulation and during proliferation and differentiation.Combined with the above data,it was obtained that Lnc RNA-mi RNA has potential target m RNAs during the process of myoblast proliferation and differentiation,and simple experimental validation was performed.Results:(1)Firstly,the mi RNA array found 65 mi RNAs significantly upregulated during muscle atrophy and significantly downregulated during muscle regeneration,86 mi RNAs significantly downregulated during muscle atrophy and significantly upregulated during muscle regeneration.GSE108040 data analysis showed that366 Lnc RNAs(log FC≧1,p<0.05)were significantly up-regulated and 509 Lnc RNAs(log FC≦-1,p<0.05)were remarkably down-regulated during myoblast proliferation,603 Lnc RNAs(log FC≧1,p<0.05)were notably up-regulated and 492 Lnc RNAs(log FC≦-1,p<0.05)were obviously down-regulated during myoblast differentiation.We noted that Lnc RNA Xist was significantly changed both during proliferation and differentiation of myoblasts and after being stimulated by gravity,suggesting that Lnc RNA Xist might be involved in the process of myoblast proliferation and differentiation.Among the mi RNAs that Lnc RNA Xist might bind to,151 were significantly changed during muscle atrophy and regeneration.Among them,mi R-486-5p was significantly down-regulated during muscle atrophy and significantly up-regulated during muscle regeneration.The above integration analysis suggests that Lnc RNA Xist may regulate mi R-486-5p expression,and has a potential biological function in myoblast proliferation and differentiation.(2)We found that overexpression of Lnc RNA Xist significantly reduced the proliferation and differentiation ability of C2C12 cells,and knockdown of lnc RNA Xist significantly improved the proliferation and differentiation ability of C2C12 cells.Overexpression of mi R-486-5p significantly enhanced the proliferation and differentiation of C2C12 cells,and knockdown of mi R-486-5p significantly inhibited the proliferation and differentiation ability of C2C12 cells.The above results suggest that Lnc RNA Xist and mi R-486-5p may be involved in the regulation of myoblast proliferation and differentiation.(3)We found that overexpression of Lnc RNA Xist significantly down-regulated mi R-486-5p levels in C2C12 cells,and knockdown of Lnc RNA Xist significantly up-regulated mi R-486-5p levels in C2C12 cells.Back-complementation of mi R-486-5p in Lnc RNA Xist-overexpressing cells can largely eliminate the inhibitory effect of Lnc RNA Xist overexpression on the proliferation and differentiation of C2C12 cells.Using an inhibitor to simultaneously reduce mi R-486-5p levels in cells knocking down Lnc RNA Xist also eliminated the promoting effect of Lnc RNA Xist knockdown on the proliferation and differentiation of C2C12 cell.Meanwhile,dual-luciferase reporter gene experiments indicated that mi R-486-5p may directly bind to Lnc RNA Xist.These results suggest that Lnc RNA Xist may regulate mi R-486-5p content through adsorption of mi R-486-5p,and then participate in the regulation of myoblast proliferation and differentiation.(4)Five databases including Target Scan obtained 1039 potential targets of mi RNA-486-5p.GSE108040 data analysis found that 3383 m RNAs(log FC≧1,p<0.05)were significantly up-regulated and 3070 m RNAs(log FC ≦-1,p<0.05)were significantly down-regulated during myoblast proliferation;3849 m RNAs(log FC≧1,p<0.05)were significantly up-regulated,and 3723 m RNAs(log FC≦-1,p<0.05)were significantly down-regulated during myoblast differentiation.Under gravity,analysis of GSE165565 data revealed that 1744 m RNAs(log FC≧1,p<0.05)were up-regulated and22 m RNAs(log FC ≦-1,p<0.05)were down-regulated in myoblasts.The above integration analysis suggests that Lnc RNA Xist/mi R-486-5p may regulate myoblast proliferation through the targets Foxo1,Pten,Akt,and Stk4,and myoblast differentiation through Dock3,Unc5 c and Atxn7.We found that overexpression of mi R-486-5p significantly downregulated Stk4 expression in C2C12 cells,and knockdown of mi R-486-5p significantly upregulated Stk4 levels.Dual-luciferase reporter experiments suggested that mi R-486-5p could directly bind to the 3’untranslated region of Stk4 gene,indicating that Stk4 is a potential downstream target gene of mi R-486-5p.Meanwhile,we also found that overexpression of Lnc RNA Xist significantly upregulated Stk4 levels in C2C12 cells,and knockdown of Lnc RNA Xist significantly downregulated Stk4 levels in C2C12 cells.The above results suggest that Stk4 may be one of the potential downstream targets of Lnc RNA Xist/mi R-486-5p axis.Taken together,based on the above experimental evidences,it is suggested that the Lnc RNA Xist/mi R-486-5p axis may be an important pathway regulating the proliferation and differentiation of myoblasts.(5)It was newly found that overexpression of Stk4 significantly reduced the proliferation ability of C2C12 cells,and knockdown of Stk4 significantly improved the proliferation ability of C2C12 cells;overexpression of mi R-486-5p significantly downregulated Stk4 expression in C2C12 cells,and knockdown of mi R-486-5p significantly upregulated Stk4 levels.Dual-luciferase reporter experiments suggested that mi R-486-5p could directly bind to the 3’ untranslated region of Stk4 gene,indicating that Stk4 ia a potential downstream target gene of mi R-486-5p.Complementing of Stk4 expression in cells overexpressing mi R-486-5p could partially eliminate the enhancement of mi R-486-5p overexpression on the proliferation of C2C12cells;Using si RNA to reduce Stk4 expression in mi R-486-5p knockdown cells also abolished the inhibitory effect of mi R-486-5p on C2C12 cell proliferation.The above results indicated that Stk4 mainly mediated the regulation of mi R-486-5p on myoblast proliferation.Additionally,overexpression of Lnc RNA Xist significantly upregulated Stk4 levels in C2C12 cells,and knockdown of Lnc RNA Xist significantly downregulated Stk4 levels in C2C12 cells.Concurrent overexpression of mi R-486-5p in Lnc RNA Xist-overexpressing cells blocked Stk4 upregulation caused by lnc RNA Xist overexpression,and concurrent inhibition of mi R-486-5p in Lnc RNA Xist-knockdown cells eliminated the downregulation of Stk4 caused by Lnc RNA Xist knockdown.It indicated that mi R-486-5p might mediate the regulation of the Stk4 level by Lnc RNA Xist and thus participate in the process of myoblast proliferation.Taken together,the above experimental evidence suggested that the Lnc RNA Xist/mi R-486-5p/Stk4 axis may be an important pathway for regulating the proliferation of myoblasts.Conclusions: This study combined bioinformatics analysis and experimental methods to identify and validate the Lnc RNA Xist/mi R-486-5p axis as an important pathway in the regulation of myoblast proliferation and differentiation.It was also demonstrated that Stk4 mediates Lnc RNA Xist/mi R-486-5p to regulate myoblast proliferation.This study provides a new explanation for the mechanism related to wasting muscle atrophy and muscle regeneration,and provides a new target for the prevention and treatment of wasting muscle atrophy.