DNA甲基化损伤后错配修复蛋白MLH1诱导胶质瘤细胞自噬的机制

After DNA methylated by temozolomide (TMZ), DNA base mismatches and activates Mismatch repair (MMR). MMR subsequently involves in the induction of autophagy which is an important way of tumor cells to repair damages. It remains unclear that how MMR induces autophagy. MLH1 is an important component of the MMR. In previous experiments, we found that after knockdown of MLH1 or inhibition of ATM phosphorylation, TMZ lost the ability to induce autophagy of U251 cells. Besides, knockdown of MLH1 led to significant depression of ATM phosphorylation. Therefore, we hypothesized that MLH1 induced autophagy through ATM phosphorylation after DNA methylation damages. In order to confirm this hypothesis and further elucidate the mechanism for MLH1 induced autophagy, we intend to make use of RNA interference, co-immunoprecipitation, confocal laser scanning microscopy and other technologies to research the mode of action of MLH1 with ATM, to explore the mechanism of that how phosphorylated ATM induces autophagy, to look for possible signal factors, to evaluate the effect of these signal factors on TMZ anti-glioma activity, to find out valuable therapeutic targets, to determine the expression of these signal factors in clinical glioma specimens and finally figure out the relationship between these factors with chemoresistance and relapse of glioma, which could provide experimental support to improve therapeutic effect of chemotherapies on glioma.

替莫唑胺（TMZ）甲基化DNA后，DNA出现碱基错配并激活错配修复系统（MMR）。MMR随后参与诱导细胞自噬，而细胞自噬是肿瘤细胞修复损伤的重要方式。MMR如何诱导细胞自噬仍不清楚。MLH1是MMR的重要组成蛋白。我们在前期实验中发现，沉默MLH1的表达或抑制ATM磷酸化后，TMZ均不能使U251细胞出现自噬，而且沉默MLH1的表达能使ATM磷酸化被明显抑制。因此我们提出假设：DNA甲基化损伤后MLH1通过ATM磷酸化诱导细胞自噬。为进一步证实这一假设并阐明MLH1诱导细胞自噬的机制，我们拟利用RNA干扰、免疫共沉淀、激光共聚焦等技术研究MLH1与ATM的作用方式，探索磷酸化ATM诱导细胞自噬的机制，寻找可能的信号因子，并观察这些信号因子对TMZ抗胶质瘤活性的影响，遴选有价值的作用靶点，最后研究临床胶质瘤标本中相关信号因子的表达及与胶质瘤耐药复发的关系，为提高胶质瘤的化疗疗效提供实验支持。

错配修复（MMR）与自噬均是肿瘤修复损伤的重要方式。替莫唑胺（TMZ）甲基化DNA 后，MMR被激活并诱导自噬发生，但机制不明。本课题即关注MMR的重要因子MLH1在自噬发生中的作用，以下为主要研究内容及发现：.1. MLH1对自噬的影响.用siRNA沉默MLH1的表达后，TMZ诱导的自噬小体比例明显下降（由34.02 ± 1.77%降至10.85 ± 1.36 %）。而HCT116细胞转染表达MLH1后，TMZ可使自噬标记LC3BⅡ明显升高。表明MLH1是自噬的关键调控因子。.2. MLH1对ATM磷酸化的作用.siRNA沉默MLH1的表达后，TMZ促进ATM、AMPK、ULK1磷酸化的能力被明显抑制。这表明TMZ诱导ATM、AMPK、ULK1的磷酸化受MLH1调控。.3. MLH1调控ATM磷酸化的机制.激光共聚焦显示MLH1与pATM均在细胞核内高表达。免疫共沉淀结果显示MLH1与ATM能相互共沉淀。当沉默NBS1表达后，MLH1与ATM的相互作用被明显抑制。这表明MLH1与ATM通过NBS1蛋白相互作用。.4. ATM对自噬的作用.ATM磷酸化被抑制后TMZ诱导的LC3BⅡ表达下降，自噬小体比例由29.90 ± 2.14 %降至9.24 ± 0.38 %。这表明ATM参与调控细胞自噬。.5. ATM调控自噬的机制.AMPK磷酸化被抑制后，LC3BⅡ及自噬激酶ULK1磷酸化明显下降。ATM磷酸化被抑制后TMZ诱导的AMPK、ULK1磷酸化及自噬小体比例均明显下降，表明ATM通过AMPK-ULK1调控自噬。.6. 干预MLH1-ATM-自噬对TMZ细胞毒性的影响.MLH1-NBS1-ATM-AMPK-ULK1不同节点干预对TMZ细胞毒性有不同影响。沉默MLH1或NBS1后，TMZ诱导的细胞凋亡数均不同程度下降。而抑制ATM、AMPK磷酸化则增强TMZ的毒性。.7. 相关因子在胶质瘤复发前后的表达差异.初步结果表明，NBS1，及NBS1相关的MRE11、RAD50在胶质瘤复发前后表达无明显差异。.综上，MMR系统通过MLH1-NBS1-ATM诱导ATM磷酸化，再通过ATM-AMPK-ULK1信号通路诱导细胞自噬，减轻TMZ的细胞毒性。.这是首次系统阐明MMR诱导细胞自噬的分子机制，为胶质瘤的治疗提供了新的思路。