The Functional Study Of Lnc RNA-AK003290 In Myoblast Proliferation And Differentiation

Long non-coding RNAs(LncRNAs)are widely found in mammals,and RNAs that are defined as more than 200 nucleotides in length and with no protein-coding capacity.The present study found that lncRNAs play an important role in many life activities,such as body immunity,muscle differentiation,growth and development and so on.Based on the previous results,we identified a lncRNA-AK003290 which was highly expressed in muscle.In this study,the effect of lncRNA-AK003290 on the proliferation and differentiation were dectected by using RNAi and overexpression experiments in C2C12 myoblasts.The results are as follows:1.The full length cDNA sequence of AK00390 was obtained by RACE and Northern blot experiments.The protein-coding capacity of the gene was predicted by CPC software,we found AK00390 can not encode protein;Meanwhile,AK003290 was highly expressed in muscle tissues and continued to increase during myoblast differentiation.AK003290 was located in nucleus during myoblast proliferation,and distributed both in nucleus and cytoplasm at 3days after myoblast differentiation.2.The mRNA and protein levels of the two marker genes ki67 and N-Ras were detected by interfering and overexpression experiments.The expression of these two genes were not changed,and the RTCA xCELLence system and EDU staining were used to identify the effect of AK003290 on myoblast differentiation,the results showed that AK003290 had no significant effect on myoblast proliferation.3.The expression of myoblasts differentiation marker genes were detected by QRT-PCR,Western Blot and immunofluorescence experiments after knockdown and overexpression of AK003290.After the AK003290 konckdown,the mRNA levels of MyHC MyoG,MyoD and Trdn were significantly decreased,MyHC and MyoG protein levels were also significantly reduced.Immunofluorescence results showed that the cells fusion rate decreased,blocking myoblasts to myotubes.Overexpressing results were the opposite of knockdown.comfirming AK003290 can positively regulate myoblast differentiation.In addition,it was found that AK003290 could promote the migration of myoblasts by cell scratch assay.4.Microarray was used to detect differentially expressed genes which were regulated by AK003290,the up-regulated genes were mainly involved in the metabolic pathway,and the down-regulated genes were mainly involeved in myocardial deseases and autophagy pathways.At the same time,29 differentially expressed genes were selected and verified by qRT-PCR,25 genes of which were consistent with the results of microarray,and this proved the reliability of the microarray results.The results of this study showed that AK003290 was highly expressed in muscle tissues and had a positive effect on myogenic differentiation.The results established the foundation for further elaborating how AK003290 regulates the expression of muscle differentiation-related genes,and laid a theoretical foundation for further study of its mechanism in muscle cells.