hTERT促进p53泛素化挽救氧化应激条件下肿瘤细胞铁死亡的分子机制研究

Literature and our study showed that human telomerase reverse transcriptase (hTERT) is associated with tumor metabolism. Scholars reported that the p53 can induce tumor cell iron death under oxidative stress. Our previous preliminary experiments demonstrated that hTERT transfection significantly reduce p53 expression in tumors. Bioinformatics predicted that there were protein - protein interactions between hTERT and the E3 ligase such as MDM2, beta -TRCP. Co immunoprecipitation experiments confirmed that hTERT-MDM2 binding, known MDM2 is the E3 ubiquitin ligase of p53 that hTERT may promote p53 ubiquitination through binding to MDM2 in order to reduce the mortality of cancer cells under oxidative stress. Based on the above hypothesis, we presume that hTERT and p53 influence tumor cell iron death in oxidative stress conditions together, and then confirmed that hTERT and MDM2 binding to each other. We will further study the role and mechanism of hTERT in promoting MDM2 mediated p53 ubiquitination and reduce the binding of p53 and core transcription factor Sp1 of hTERT by in vivo and in vitro experiments. For further activation of hTERT and promote tumor occurrence, development and save the iron death in oxidative stress.

文献及我们的研究表明，人端粒酶逆转录酶（hTERT）与肿瘤代谢相关。文献报道p53在氧化应激条件下可以诱导肿瘤细胞铁死亡，我们前期预实验表明hTER转染可以明显减少肿瘤细胞p53的表达，生物信息学预测到hTERT与MDM2、β-TRCP 等E3连接酶之间均可能存在蛋白-蛋白相互作用。免疫共沉淀实验证实hTERT与MDM2能结合，已知MDM2是p53的E3泛素化连接酶，提示hTERT可能通过结合MDM2，促进p53泛素化，降低肿瘤细胞在氧化应激条件下的铁死亡。基于以上假说，本课题拟采用分子生物学技术，首先证实hTERT与p53在氧化应激条件下共同作用影响肿瘤细胞铁死亡，然后证实hTERT与MDM2结合，再次从体内、外二方面证明hTERT在促进MDM2介导p53泛素化，减少p53与hTERT核心转录因子Sp1的结合，进一步活化hTERT,促进肿瘤的发生发展及挽救氧化应激下的铁死亡。

胃癌是严重威胁人类健康的恶性肿瘤；我国胃癌的发病率已跃居肿瘤发病率第二位，其肿瘤死亡率排第三位，每年有29万患者死于胃癌。研究人端粒酶逆转录酶 hTERT 在胃癌细胞中调控铁死亡的作用及分子机制，为胃癌的治疗提供新的方向。利用cck-8、MDA及GSH试剂盒检测发现在erastin处理后，胃癌细胞生长抑制率、MDA升高，GSH含量降低，进而发现胃癌细胞可发生铁死亡，并通过透射电镜成像观察到细胞线粒体发生皱缩、变小。通过数据库分析，发现hTERT与铁死亡存在一定相关性，故采用构建过表达及干扰hTERT的稳定胃癌细胞株，进一步功能实验分析发现hTERT的表达可抑制胃癌细胞发生铁死亡；同时利用CO-IP、荧光共定位及泛素化检测等试验，结果显示hTERT可通过与MDM2相结合促进p53的泛素化降解，抵抗胃癌细胞发生铁死亡。在研究结果中示铁死亡的发生与糖代谢相关，采用代谢组学、Seahorse试验、葡萄糖摄取、乳酸生成等发现hTERT可影响胃癌细胞发生代谢重编程，通过检测细胞内G6PD活性、GSSG/GSH、NADPH/NADP，发现hTERT通过促进细胞内PPP 通路，进而使NADPH生成增加，从而抑制Erastin诱导的铁死亡。总之，hTERT通过与MDM2结合促进p53的泛素化降解，同时诱导胃癌细胞发生代谢重编程，促进ppp通路的发生，进而挽救胃癌细胞在erastin诱导下发生铁死亡。