hTERT非逆转录酶活性促胃癌转移的分子机制研究

Literatures and our previous studies have showed that the human telomerase reverse transcriptase (hTERT) is related with tumor invasion and metastasis, but the mechanism is unclear. Scholars reported that TERT can be combined with Brg1 (brahma-related gene 1) and activate Wnt/β-catenin signal pathway and further participate in the development of nervous system independent of its reverse transcriptase. Our previous preliminary experiments demonstrated that hTERT transfection significantly increased heparanase expression in tumors. Bioinformatics predicted that there were binding sites of c-myc, a target molecular in the downstream of Wntβ-catenin signal pathway, in the promoter of heparanase. The dual luciferase experiments suggested that c-myc could enhance the promoter transcriptional activity of heparanase promoter. Based on the above analysis, we presume that hTERT could activate Wnt/β-catenin signal pathway through combining with Brg1 to promote the expression of c-myc, the latter would enhance the transcription of heparanase. This result will ultimately contribute to the invasion and metastasis of gastric cancer. Based on the above hypothesis, this study will first confirm that c-myc can be binded to the promoter of heparanase and increase the transcription of heparanase promoter. And then we will construct expression vector of hTERT independent of its reverse transcriptase and transfected with gastric cancer. We will further study the role and mechanism of hTERT in the activavtion of Wnt/β-catenin signal pathway, c-myc expression, heparanase transcription, and invasion and metastasis of gastric cancer by in vitro and in vivo experiments. This study will clarify the mechanism of hTERT in the advance of gastric cancer from another point of view. The results will provide a new theoretical basis for intervention to hTERT as a target for gastric cancer.

文献及我们的研究表明,人端粒酶逆转录酶(hTERT)与肿瘤转移密切相关,但机制不清.文献报道,TERT非逆转录酶活性可通过与Brg1作用,活化Wnt信号,参与神经系统发育.我们前期预实验表明hTERT转染可以明显增加肿瘤细胞肝素酶表达,生物信息学预测到Wnt下游靶分子c-myc在肝素酶启动子上有结合位点,双荧光素酶实验提示c-myc可增强肝素酶启动子转录活性,提示hTERT可通过Brg1活化Wnt信号,促进c-myc表达,增强肝素酶转录,进而促进胃癌转移.基于以上假说,本课题拟采用分子生物学技术,首先证实c-myc可与肝素酶启动子结合,然后构建hTERT非逆转录酶表达载体,从体内、外二方面证明hTERT非逆转录酶活性在Wnt信号活化、c-myc表达、肝素酶转录以及胃癌转移中的作用及其机制.本课题将从另一个角度阐明hTERT在胃癌进程中的作用,为以hTERT为靶点的胃癌干预提供新的理论依据.

人端粒酶逆转录酶（hTERT）的活化是端粒酶活化的前提，既往研究一直锚定于端粒酶与肿瘤细胞无限增殖方面。而文献及我们的研究表明，hTERT与肿瘤转移亦密切相关，但机制不清。我们拟通过体内外侵袭转移实验及临床分析阐明hTERT促胃癌侵袭及与胃癌预后的关系，进一步采用双荧光素酶实验、ChIP及免疫共沉淀等实验揭示hTERT通过与c-Myc交互作用共同调控肝素酶转录表达的作用机制。结果发现hTERT高表达可促进胃癌体内外侵袭转移能力，并且与肝素酶的表达呈明显正相关；hTERT可通过激活肝素酶启动子活性而上调肝素酶mRNA及蛋白的表达水平；转录因子c-Myc可直接结合肝素酶启动子而上调肝素酶转录活性，而Co-IP实验证实hTERT与c-Myc存在交互作用而形成转录复合体，hTERT可募集c-Myc结合于肝素酶启动子而发挥促肝素酶表达的作用；进一步研究发现，hTERT与c-Myc存在相互调控的正反馈机制，共同促进和维持肝素酶的高表达及胃癌的不断侵袭转移；通过临床病理分析，发现hTERT、c-Myc及肝素酶表达存在明显正相关，并且与胃癌的不良预后密切相关。因此，我们的研究揭示了hTERT在胃癌侵袭转移中的重要作用，靶向hTERT的肿瘤治疗可作为一潜在应用价值的新治疗手段。