Sirt1 AS LncRNA Promotes C2C12 Proliferation By Inhibiting MIR-34A

Sirt1 is a NAD-dependent deacetylase, which regulates various physiological processes through deacetylase activity. Precise level of Sirt1 is important for many normal physiological processes including energy metabolism, development and aging. The transcriptional and post-transcriptional regulations were involved in Sirt1 precise expression. MiR-34 a is a important miRNA to Sirt1,which inhibites Sirt1 expression by targeting Sirt1 3′ UTR. Natural antisense transcripts(NATs) exist ubiquitously as pivotal molecules to regulate coding gene expression at epigenetic, transcriptional and post-transcriptional level.Bioinformation analysis demonstrated that Sirt1 exists natural antisense transcription. Therefore, we speculated that Sirt1 AS lncRNA regulates C2C12 myoblasts proliferation by regulating Sirt1 expression.In this study, we identified Sirt1 AS lncRNA by bioinformatics analysis and detected it expressed in various mouse tissues, as well as in C2C12 myogenesis differentiation. Moreover, the half-life of the Sirt1 mRNA and Sirt1 AS lncRNA were detected. To verify the function of Sirt1 AS lncRNA and miR-34 a, we constructed the epression plasmids and transfected C2C12 cell. We detected the expression of Sirt1 by real-time PCR and Western Blot Furthermore, the proliferation of C2C12 were assessed through CCK8, EDU assay and flow cytometry. Then, we proved the interaction of Sirt1 AS lncRNA and miR-34 a to Sirt1 by dual luciferase reporter and RNA stabilities assay.The main results are as follows: 1. Identification of Sirt1 antisense long non-coding RNABioinformatic analysis prediction of Sirt1 NATs,which was classified as AS lncRNA according to its coding potential calculation. The results of PCR production gel extraction and sequencing indicated that Sirt1 AS lncRNA existed in mouse genome transcripts. Sirt1 AS lncRNA expressed in all detected mouse tissues, and the level of Sirt1 AS lncRNA expression was greater in spleen, but was less in muscle.The levels of Sirt1 mRNA decreased faster than Sirt1 AS lncRNA during C2C12 differentiation, but the levels of miR-34 a gradually elevated. The half-life of Sirt1 mRNA was about 6 h, whereas that of the Sirt1 AS lncRNA was approximate 10 h. Therefore, Sirt1 AS lncRNA was more stable than Sirt1 mRNA. 2. Sirt1 AS lncRNA promotes Sirt1 expression by inhibiting miR-34aOverexpression of miR-34 a downregulated the levels of Sirt1 mRNA and protein(P < 0.05). In contrary, overexpression of Sirt1 AS lncRNA increased the level of Sirt1 protein. Moreover, overexpression of Sirt1 AS lncRNA resisted downregulation of Sirt1 level by miR-34 a. Sirt1 AS lncRNA competed with miR-34 a bonding Sirt1 3′ UTR to improve Sirt1 expression. 3. Sirt1 AS lncRNA promotes C2C12 cells proliferationIn consist with Sirt1, Sirt1 AS lncRNA was elevated in the C2C12 cells proliferation. Overexpression of Sirt1 AS lncRNA increased the levels of cyclinB, cyclinD and CyclinE(P < 0.05), rescued the depression of them by miR-34 a. Sirt1 AS lncRNA promoted C2C12 cells proliferation(P < 0.05) and resisted the inhibition of miR-34 a.In summary, we first identified Sirt1 AS lncRNA, which increased Sirt1 expression by inhibiting miR-34 a and further promoted C2C12 cells proliferation.