p53-miR-138-AGO2通路调控NSCLC细胞辐射敏感性的机制研究

In our study, we found the abundance of miR-138 was correlated with the epression level of p53 in non-small cell lung cancer (NSCLC) cells. In H460 cell which expressed wildtype p53, the expression level of miR-138 was higher than that in H1299, which is p53 deficient. The expression level of miR-138 increased when p53 over-expressed in H1299. There was an up-regulation of miR-138 and p53 in H460 cell after 10Gy radiation, while no alteration of them in H1299. We had demonstrated that AGO2 is a target gene of miR-138, over-expressed miR-138 as well as knock-down AGO2 could increase the sensitivity to radiation in H460 cell. The microarray result showed that miR-138 could down-regulate dozens of miRNAs which had been reported relating to radiation. On the basis of the results, we speculated that miR-138 is up-regulated by p53 and enhances the sensitivity to radiation in NSCLC cells by targeting AGO2. This project will validate the speculation above.

根据现有工作基础，我们发现在非小细胞肺癌（NSCLC）中miR-138丰度与p53表达水平存在相关性，p53野生型细胞（H460）中miR-138表达水平显著高于p53缺失型细胞（H1299）。在H1299中外源表达p53后，miR-138表达水平升高。10Gy剂量照射两种细胞后，H460中miR-138水平显著上升，H1299中无明显变化。此外，我们已证实AGO2是miR-138的靶基因，且在H460细胞中过表达miR-138或敲低AGO2均能使细胞辐射敏感性增加。芯片结果显示miR-138能够下调其他miRNA，据报道这些miRNA多与辐射相关。综合上述工作基础及国内外文献报道，我们推测存在一条影响NSCLC细胞辐射敏感性的新途径：p53上调miR-138，miR-138靶向AGO2蛋白调控下游miRNA，并通过这些miRNA及其靶基因调控细胞的辐射敏感性，本立项将对上述推测进行验证。

本研究中，我们鉴定了在人非小细胞肺癌细胞中，p53通过与启动子区域相关序列的结合作用正向调控miR-138的转录水平。 更有趣的是，结合已发表相关文献，我们证实p53与miR-138的相互作用具有单向性和种属特异性：在大鼠和小鼠细胞中，miR-138能够靶向p53，但miR-138却未受到p53的转录调控；在人类细胞中，p53能够激活miR-138转录，但p53 mRNA 3’UTR却缺失了miR-138的靶位点，miR-138不能靶向p53。我们继续深入研究发现在人非小细胞肺癌中，miR-138能够特异靶向AGO2。而AGO2作为microRNA加工成熟的关键蛋白进一步调控了miR-130b的含量，相应的我们检测到miR-130b在人肺细胞肺癌中的特异靶标GADD45a发生变化，进而使细胞周期产生G2/M期阻滞，改变了细胞的辐射敏感性。