miR-351靶向TRAF6调控失神经肌萎缩的机制研究

Prevention of the denervated skeletal muscle atrophy is a major challenge in the field of neuroscience. TRAF6 plays an important role in the denervated muscle atrophy process, and can be used as drug development potential targets to prevent muscle atrophy. miRNA targeted drugs is more effective than the drugs for single gene. The previous studies, from our project through proteomics analysis, Western blot, miRNA chip analysis, TargetScan prediction and luciferase reporter gene assay, indicated that the expression of TRAF6 was up-regulated in the muscle after peripheral nerve injury, and a negative correlation was found between the expression of miR-351 and the expression of TRAF6 in denervated skeletal muscle. Combined with the results predicted by the online software-TargetScan and preliminary experimental validation, we inferred that miR-351 could participate in the regulation of the expression of TRAF6. But the regulatory relationship between miR-351 and TRAF6 and the regulatory role on denervated skeletal muscle atrophy is not clear. Therefore, we hypothesized that: miR-351 expression decreased after the target muscle denervation, promoting the up-regulation of TRAF6, causing muscle atrophy through the activation of protein degradation system (ubiquitin-proteasome and autophagy systems). So, whether the miR-351 can be used as a molecular target for inhibiting denervated muscle atrophy? The aim of this study is to validate the presence of the direct interaction and to elucidate the interaction site between miR-351 and TRAF6 by dual-luciferase reporter gene assay, to elucidate the effects of miR-351 on the biological behavior of myoblast in vitro, to investigate the regulatory effect of miR-351 on myotube atrophy process in vitro, to elucidate the inhibitory effects of miR-351 on denervated muscle atrophy and the mechanism. This study will be to enrich the molecular regulation mechanism of denervated muscle atrophy, and to provide new targets for clinical treatment of denervated muscle atrophy.

失神经肌萎缩的防治是神经科学领域的一大难题。TRAF6在失神经肌萎缩过程中起重要作用，并可作为防治肌萎缩的潜在药物开发靶点。本项目组通过miRNA芯片筛选、软件预测及初步实验首次验证了TRAF6为miR-351调控的靶基因。因此我们推测：在失神经肌萎缩过程中miR-351通过对其靶基因TRAF6的上调作为促发因素，诱导泛素化蛋白酶体水解系统和自噬过程的过度激活，引起肌萎缩。那么是否miR-351可以作为抑制失神经肌萎缩的分子靶点？本项目拟用荧光素酶报告基因系统确定miR-351与靶基因之间相互作用关系并明确其相互作用位点；体外在细胞水平探讨miR-351对成肌细胞生物学行为的影响，在肌管水平探讨miR-351对肌管萎缩过程的调控作用；体内观察miR-351对失神经肌萎缩的抑制作用及机制。本研究将进一步丰富失神经肌萎缩的分子调控机制，为失神经肌萎缩的临床治疗提供新的分子干预靶点。

失神经肌萎缩的防治是神经科学领域的一大难题。本研究发现肿瘤坏死相关因子6（TRAF6）在失神经肌萎缩过程中以及地塞米松诱导的C2C12肌管萎缩过程中表达上调。体外研究发现抑制TRAF6的表达可以减轻地塞米松诱导的C2C12肌管萎缩；体内研究显示在失神经支配胫前肌中注射TRAF6 siRNA，结果TRAF6 siRNA注射组的胫前肌湿重比以及肌纤维截面积均明显大于对照组，TRAF6 siRNA注射组胫前肌中的TRAF6表达受到明显抑制，TRAF6下游靶基因MuRF1和MAFBx也受到显著抑制。由此可见，TRAF6在控制肌萎缩过程中起非常重要作用。那么寻找到TRAF6的miRNA调控方式，一方面可以更好地理解肌萎缩的分子调节机制与进程。另一方面将寻找到一个更有利于控制肌萎缩的药物靶点。我们的研究发现miR-351在失神经肌萎缩过程中的表达逐渐下降，miR-351和TRAF6在失神经肌萎缩过程中的表达呈负相关；荧光素酶报告基因系统分析发现，miR-351可以显著抑制含有野生型TRAF6 3'-UTR质粒的荧光素酶活性，对含有突变型TRAF6 3'-UTR质粒的荧光素酶活性没有影响，这些结果提示miR-351与TRAF6 3'-UTR之间存在相互作用。那么在失神经肌萎缩过程调控miR-351的表达是否可以调控肌萎缩，我们在失神经支配胫前肌中注射miR-351 agamir，结果发现实验组胫前肌湿重比以及肌纤维截面积均明显大于对照组；miR-351 agamir注射组胫前肌中的TRAF6表达受到明显抑制，TRAF6下游靶基因MuRF1和MAFBx也受到明显抑制。综上所述，miR-351可通过靶向抑制TRAF6的而延缓失神经支配骨骼肌萎缩。