MEX3A调节MMR通路介导MGMTneg型复发胶质瘤TMZ耐药的实验研究

MGMTneg recurrent gliomas are mostly associated with lower MMR proteins - PMS2 and MSH2 expression, and TMZ resistance. Yet, the mechanism is still obscure. Our preliminary data showed that TMZ treatment remarkably enhanced the expression of MEX3A. And up-regulation of MEX3A promoted TMZ resistance and decreased the expression of PMS2 and MSH2. Thus, we proposed the research hypothesis that MEX3A promotes TMZ resistance in MGMTneg recurrent gliomas through MMR signaling. In the current research program, we will study as follows: the relationship among MEX3A、PMS2 and MSH2 in gliomas tissue samples; the mechanism that MEX3A regulating the expression of MSH2 and PMS2; and whether MEX3A confers TMZ resistance thought deceasing the expression of MSH2 and PMS2. Expected research results will elucidate the mechanism that MEX3A promotes TMZ resistance in MGMTneg recurrent gliomas through MMR signaling and provide a novel treatment strategy targeting MEX3A.

MGMTneg型复发胶质瘤多伴有MMR蛋白-PMS2和MSH2的下调及对TMZ的耐药，但其中的机制不明。前期实验发现TMZ刺激可促进MEX3A的表达，MEX3A的表达又可影响胶质瘤对TMZ的敏感性及PMS2、MSH2的表达。据此，我们提出“MEX3A通过MMR通路调控MGMTneg型复发胶质瘤对TMZ的敏感性”的研究假说。本项目拟在胶质瘤标本中检测MEX3A、PMS2及MSH2的表达及三者间的相互关系；探讨MEX3A影响PMS2、MSH2表达的具体机制；在细胞系和动物模型中证明：MEX3A通过PMS2、MSH2调控MGMTneg型复发胶质瘤的TMZ耐药。预期结果将阐明MEX3A通过MMR通路影响MGMTneg型复发胶质瘤的耐药机制，建立以MMR通路为核心针对MEX3A治疗MGMTneg型复发胶质瘤的新策略。

研究背景：替莫唑胺（TMZ）治疗后复发的胶质瘤常常对替莫唑胺的敏感性降低，且多伴有DNA错配修复信号（MMR）分子异常表达，其中的机制尚不清楚。.研究内容：我们采用qRT-PCR和Western blot检测胶质瘤中MMR分子和MEX3A的表达，蛋白质免疫共沉淀和RNA免疫沉淀用于验证MEX3A与靶RNA和靶蛋白的相互作用，泛素化分析检测MEX3A对靶蛋白泛素化的作用，脱腺苷降解实验分析MEX3A对靶RNA的降解作用，流式细胞术和裸鼠原位模型用于检测MEX3A在胶质瘤替莫唑胺耐药性中的作用。.研究结果：我们的研究发现，替莫唑胺治疗后复发的胶质瘤中MSH2和PMS2的表达显著降低。体内外的实验发现，长期TMZ处理可显著提升MEX3A的水平，抑制PMS2和MSH2的表达。此外我们的体外实验发现，过表达MEX3A促进MSH2和PMS2 mRNA的脱腺苷降解。进一步的机制研究发现，MEX3A通过其KHs结构域靶向结合PMS2和MSH2 mRNA 3′-UTR的MRE（MEX-3识别元件）区域，进而招募CCR4-NOT复合体，MEX3A通过其RING 结构域调控CNOT6和CNOT7的泛素化激活，进而促进PMS2和MSH2 mRNA的脱腺苷降解。最后，我们的裸鼠原位模型证实，MEX3A可以降低胶质瘤细胞对替莫唑胺的敏感性。.科学意义：我们的研究结果显示，TMZ诱导胶质瘤耐药可能是通过激活MEX3A/CCR4-NOT信号通路从而抑制MMR分子表达实现的。MEX3A通过调节MSH2和PMS2脱腺苷降解从而降低胶质瘤细胞对替莫唑胺的敏感性，MEX3A有可能成为复发胶质瘤的预后指标或治疗靶点。