Identification And Functional Analysis Of MicroRNAs Involving In The Process Of Autophagy Induced By Leucine Deprivation In C2C12 Cell

Branched-chain amino acids(BCAAs) are a series of essential amino acids having aliphatic side-chains with a branch, containing leucine, isoleucine and valine which cannot be synthesised in animal bodies. It has been reported that BCAAs, especially leucine, are not only nutrient elements supplying for animal requirement, but also can be served as regulatory molecules for protein degradation, protein synthesis and protein turnover in the skeletal muscle of mammals. Some reports demonstrated that leucine can regulate autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. Furthermore, mTOR phosphorylation ULK1 may be the bridge between leucine and autophagy.microRNAs (miRNAs) are short non-coding RNA molecules, on average only 22-23 nucleotides long and are found in all eukaryotic cells. microRNAs bind to three prime untranslated regions (3’-UTR) of the target mRNA and then inhibit translation or promote degradation of the target mRNA. Increasing reports illustrated that microRNA serves as an important role in cell proliferation, cell death, cell differentiation and so on. However, the role of microRNA in autophagy induced by leucine deprivation is still obscure.Based on the results of microRNA chip and quantitative reai-time PCR, this project filtered the differentially expressed microRNAs between C2C12 myoblasts in full medium and C2C12 myoblasts induced autophagic activity by cultured in medium without leucine. The target gene of candidate microRNA was predicted by TargetScan software and veryfied by dual luciferase report gene test and western blot. Furthermore, the role of the microRNA serving in the process of autophagy induced by leucine deprivation in C2C12 myoblasts was validated through microRNA transfection. The main results were listed as follows:1. As other cell lines, leucine deprivation can induce autophagic activity in C2C12 myoblasts. The results of confocal microscope for colocalization of LC3 and LAMP1, as well as western blot for LC3-II and p62 in C2C12 myoblasts cultured in medium without leucine demonstrated that in C2C12 myoblasts,4 h to 8 h of leucine deprivation can induce autophagic activity(P<0.05), with the deprivation time prolonging, the autophagic activity decresed.2. Leucine can serve as an important regulator for C2C12 myoblasts cell cycle. When leucine deprivation in C2C12 myoblasts, the percent of G1 cells significantly increased and S cells significantly decreased, which indicated that lecine deprivation induced cells arresting in G1 phase. 3.86 differentially expressed microRNAs were filtered between C2C12 myoblasts in full medium and C2C12 myoblasts induced autophagic activity by leucine deprivation through microRNA chip and quantitative PCR.4. The result of microRNA chip showed that in C2C12 myoblasts cultured in medium without leucine, all 8 members in miR-17 microRNA family were dowm-expressed, the result of quantitative PCR illustrated that miR-20a and miR-106b were most significant. Furthermore, the same phenomenon happened in C2C12 myotubes.5. It was reported that c-myc is the transcriptional factor of miR-20a and miR-106b, based on this conclusion we found that c-myc got dowmexpression in C2C12 myoblasts and myotubes while leucine deprivation.6. It was demonstrated that ULK1, an essential gene in autophagy, was one target of miR-20a and miR-106b through the bioinformatics analysis and dual luciferase report gene test as well as western blot.miR-20a and miR-106b binded to the 485-491bp in 3’-UTR of ULK1 mRNA and inhibited the translation of its mRNA.7. Through the confocal microscope for colocalization of LC3 and LAMP1, as well as western blot for LC3-Ⅱand p62, we found taht miR-20a/miR-106b overexpressing C2C12 myoblasts got lower sensitivity to the autophagic activity induced by leucine deprivation. miR-20a and miR-106b can inhibit the autophagic activity induced by leucine deprivation in C2C12 myoblasts.Colligated with all of the results, a conclusion came up that leucine deprivation induces autophagic activity in C2C12 myoblasts via miR-20a/miR-106b, which could target and inhibit the expression of ULK1. c-myc, the transcription factor of these two microRNAs, is inhibited during leucine deprivation in C2C12 myoblasts and C2C12 myotubes.