Screening And Identification Of MYOD Downstream Long Non-coding RNA And The Function Analysis Of Two LncRNAs During Myoblast Differentiation

Skeletal muscle differentiation and development is an exquisitely orchestrated network which involve in many kinds of functional molecules.Long non-coding RNA(lncRNAs)is a class of non-coding RNA regulates various of biological functions,and it is widely studied in cancer and tumor tissues.So far,some functional lncRNAs have been reported in muscle differentiation,but there are still a lot of unlocked lncRNA remained elusive.In this study,we utilised lncRNA and mRNA microarray analysis to identify potential lncRNAs regulated by MyoD in muscle cells.And two lnc RNAs were found to play an important role in controlling muscle cell differentiation.The main results were as follows:1.Small interfering RNA(siRNA)were utilised to suppress Myo D protein and mRNA expression.Knockdown of MyoD caused a decrease in expression of the downstream marker genes,MyoG and MyHC.In contrast,no significant changes were found in other upstream genes.Microarray analysis were used to screen differentially expressed lncRNAs and mRNAs.335 up-regulated and 662 down-regulated lnc RNA were identified after MyoD knockdown.By selecting lncRNAs and mRNAs with Pearson's correlation analysis,we found 1,396 mRNAs in the network.GO and KEGG pathway analyses were carried out with respect to the co-expressed mRNAs to predict the function of the differentially expressed lncRNAs.We found the majority of genes were involved in muscle formation and muscular movement.2.Thirty-seven intergenic lncRNAs were selected due to the higher expression and larger fold changes in microarray.Quantitiative real-time PCR was used to identify the distribution pattern in mouse tissues.The results showed that nine lncRNAs(MM9LINCRNAEXON11961-,AK141672,AK031663,ENSMUST00000150337,AK017263,ENSMUST00000154720,AK160312,AK163925,ENSMUST00000104935)displayed high expression patterns in muscle;eight lnc RNAs,including AV570737,displayed high expression patterns in testis;four lnc RNAs,including AK139402,displayed high expression patterns in brain;seven lncRNAs,including AK052777,displayed high expression patterns in liver and kiney;nine lncRNAs,including AK143003,exhibited ubiquitous expression.3.MyoD eukaryotic expression vector was used to transfect C2C12 cells to identify nine candidated lnc RNAs.The results showed that AK143003,AK017263 and ENSMUST00000150337 were significant changed,in accordance with MyoD overexpression.Through the analysis of time course expression,we found all three lncRNAs were promptly activated at the early stage of differentiation.However,AK143003 gradually increased until terminal differentiation,while AK017263 and ENSMUST00000150337 decreased slightly during differentiation4.Through sequence analysis,we found AK017263 was an intergenic lncRNA,which were predicted to bind Myo D and Myo G at the start site of transcription.The full lengh of AK017263 were obtained by RACE.Further analyses showed its noncoding ability and nuclear localisation.Using q-PCR and western blotting analysis the expression of MyoG and MyHC after knockdown of AK017263.The results showed the mRNA and protein expression of Myo G and MyHC were significantly decreased,indicating AK017263 could positively regulated muscle differentiation.5.Through sequence analysis,we found AK143003 was an intergenic lncRNA,which were located next to a coding gene,Mxd4.Using Northern blot,RACE and in vitro translation system,we demonstrated AK143003 was a non-coding RNA with single copy.Real-time PCR analysis showed that the AK143003 transcripts were localised nearly equal amounts in the cytoplasm and nucleus during the proliferative stage,while all transcripts were transferred to the cytoplasm during cell differentiation.Silencing of AK143003 stimulated the accumulation of myogenic marker genes,whereas AK143003 overexpression led to their decreased synthesis.In addition,knockdown of AK143003 could cause the increased expression of Mxd4 and vice versa,indicating AK143003 could negative control cell differentiation through inhibiting the Mxd4 expression.But the function of Mxd4 in cell differentiation was not reported.So,Real-time PCR and Western Blotting were used to detect the expression of Myo G and MyHC after Mxd4 knockdown and overexpression.The results showed Mxd4 promoted muscle cell differentiation,in accordance with the previous assumption,indicating Mxd4 could be one of the factor in AK143003-mediated cell differentiation.This study identified 37 lncRNAs which related to Myo D,demonstrated two novel lncRNA,AK017263 and AK143003,which could regulate muscle cell differentiation.Our research provides a new evidence for the function of lnc RNA in muscle differentiation network,and laid the theory basis for lncRNA application in livestock breeding.