hTERT介导NRF2降低结肠癌细胞内活性氧水平促进结肠癌侵袭转移的机制研究

The expression of human telomerase reverse transcriptase (hTERT) is closely related to the invasion and metastasis of colon cancer. We advance experiment found expression of hTERT can significantly reduce the active oxygen levels in colon cancer cells. Immunofluorescence assay showed that hTERT is mainly expressed in the nucleus and hTERT may regulated intracellular gene expression to decrease the levels of intracellular reactive oxygen species (ROS). Nf-E2 related factor 2 (NRF2) is a key molecule in the regulation of cellular reactive oxygen species. Pre-experimental results showed that NRF2 was highly expressed in colon cancer tissues, and overexpression of hTERT could up regulate the expression of NRF2, suggesting that NRF2 was regulated by hTERT. Two luciferase experiments showed that hTERT could up regulate the NRF2 promoter activity. The literature and our previous work showed that hTERT could be used as an assistant transcription factor to regulate gene expression. Accordingly, this paper puts forward hypothesis that "hTERT through the recruitment of certain nuclear transcription factor promotes the expression of NRF2, reduce the level of reactive oxygen species in colon cancer cells, promote the invasion and metastasis of colon carcinoma". In this paper, we will use CO-IP, ChIP, animal model and other experimental methods to confirm the above hypothesis, the hypothesis will be able to provide a new molecular target for the prevention and treatment of colon cancer.

文献报道人端粒酶逆转录酶(hTERT)的表达与结肠癌侵袭转移密切相关。我们前期预实验发现过表达hTERT可以显著降低结肠癌细胞活性氧水平,免疫荧光实验表明hTERT主要表达在细胞核内,提示hTERT可能通过调控细胞内基因表达降低细胞内活性氧水平。文献报道Nf-E2相关因子2(NRF2)是调控细胞活性氧关键分子。预实验结果发现NRF2在结肠癌组织中高表达，过表达hTERT可上调NRF2表达水平,提示NRF2受hTERT调控。双荧光素酶实验表明hTERT可上调NRF2启动子活性。文献及我们前期工作表明hTERT可作为辅助转录因子调控基因的表达。据此,本课题提出“hTERT通过募集某些核转录因子促进NRF2表达，降低结肠癌细胞内活性氧水平，促进结肠癌侵袭转移”的假说。本课题拟采用CO-IP、ChIP、动物模型等实验方法对上述假说进行证实,该假说的证实将可以为结肠癌的防治提供新的分子靶标。

结肠癌是我国常见的消化系统肿瘤之一，其发病率及死亡率呈逐年增高趋势。结肠癌细胞的侵袭转移是导致患者死亡的主要原因。化疗是结肠癌患者术后的主要治疗手段，通过提高肿瘤细胞内活性氧水平杀伤肿瘤细胞，但目前相关研究表明肿瘤细胞可以通过降低细胞内活性氧水平从而在化疗药物作用下存活。因此研究结肠癌细胞内活性氧调控机制将可以为结肠癌治疗提供潜在靶点。本项目发现端粒酶逆转录酶hTERT通过招募核转录因子YBX1，促进结肠癌细胞内NRF2的表达，从而下调结肠癌细胞内活性氧水平，主要研究内容如下：.（1）hTERT与NRF2在结肠癌组织中高表达且与结肠癌不良预后相关：课题组在不同人群中均发现，NRF2与hTERT在结肠癌组织中高表达，并且二者表达呈正相关；高表达hTERT或NRF2的患者生存时间显著低于低表达患者，且同时高表达NRF2及hTERT的患者生存时间最低；.(2)hTERT与NRF2均可促进结肠癌增殖、转移：课题组发现过表达NRF2及hTERT均可以促进结肠癌增殖、转移；.(3)NRF2发挥促进结肠癌增殖、转移依赖于hTERT的表达：课题组发现干扰hTERT可以抑制NRF2促进结肠癌增殖、转移带来的效应，提示NRF2发挥功能依赖于hTERT的表达。.(4)hTERT通过招募YBX1促进NRF2的表达：课题组首先发现，干扰hTERT后，NRF2的mRNA水平及蛋白水平均显著降低，进一步通过双荧光素酶实验发现，干扰hTERT表达后，结肠癌细胞内NRF2的启动子水平较前明显降低，提示hTERT通过转录水平影响NRF2的表达；其次课题组通过CO-IP实验探寻hTERT上结合的蛋白，并通过DNA pull-down方法探寻NRF2启动子区域上可能结合的核转录因子，将二者取交集后筛选出11个备选蛋白，进一步通过WB实验验证发现YBX1在其中发挥着关键作用；最后课题组发现抑制YBX1表达后，即使过表达hTERT，依旧不能回复NRF2的启动子活性，提示hTERT的确可能通过结合YBX1发挥促进NRF2表达的功能。.本项目通过上述实验，初步证实了“hTERT招募YBX1促进NRF2启动子活性，从而在转录水平提高NRF2的表达，继而促进了结肠癌增殖及转移”这一科学假说，为结肠癌增殖、转移提供了新的理论依据，并为结肠癌治疗提供了潜在的治疗靶点。