HPV-E6/E7通过上调AEG-1表达诱发宫颈癌的作用机制研究

Human papillomaviruses (HPV) of high-risk types are the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 is considered to contribute to disease initiation. However, experimental data indicate that the viral presence is not enough to induce cervical cancer and additional genetic and epigenetic events are presumably required, and the complete mechanism of which is still need to be elucidated. AEG-1 gene is newly identified oncogene and it's overexpression in malignant tumors has been related with the tumor development, and it's subcellular compartment localization also contributes to different functions, nevertheless, the role of AEG-1 in HPV induced cervical carcinoma has not been reported. In our previous studies, by using siRNA and full-lenghth gene transfection methods we found that AEG-1 expression level was closely coincided with the expression of E6/E7 in HPV positive or negative cervical carcinoma cell lines of Hela or C33A cells, which indicated that AEG-1 might be the key effector in the process of HPV-E6/E7 inducing cervical carcinoma formation. In the present study, we will use chromsome IP, EMSA, gene mutation and RNAi technique to explore the machnism of AEG-1 transcription activation regulated by HPV-E6/E7. Meanwhile, we will also use the anti-AEG-1 ScFv eukaryotic expression vectors which were constructed in our lab and could be expressed in the nucleus(with NLS peptid) or in the cytoplasmid(without NLS peptid) to inhibit AEG-1 function either within cytoplasmid or in nucleus, respectively to explore the effect of AEG-1 functions on cell malignant phenotype in E6/E7 positive cervical carcinoma cells. The aim of this study is to provide evidence and new theraputic target for the control, prevention and therapy of HPV positive cervical carcinomas.

高危型人乳头状瘤病毒(HPV)及其表达的E6/E7蛋白为公认的宫颈癌发病诱因，但需多种內源基因参与，具体机制及关键效应分子尚未完全阐明。AEG-1为新发现的与恶性肿瘤发生、发展密切相关的癌基因且其亚细胞定位与其功能发挥有关，但其在HPV所致宫颈癌中的作用及意义未见报道。课题组前期实验首次发现，宫颈癌组织中AEG-1与HPV表达成平行关系；阻断宫颈癌细胞中E6/E7表达或过表达E6/E7，可分别下调或上调AEG-1表达，提示AEG-1可能为E6/E7致癌的关键效应分子。本课题拟用启动子研究技术阐明E6/E7上调AEG-1表达的分子机制；同时以具有亚细胞定位功能的ScFv分别抑制胞浆或胞核AEG-1功能发挥，验证E6/E7高表达状态下AEG-1功能消失对逆转细胞恶性表型影响，确定AEG-1为HPV致癌的关键效应分子。最终为丰富E6/E7致癌机制的理论、发掘宫颈癌诊治新靶点提供理论及实验依据。

高危型人乳头状瘤病毒(HPV)及其表达的E6/E7蛋白为公认的宫颈癌发病诱因，但需多种內源基因参与，具体机制及关键效应分子尚未完全阐明。AEG-1为新发现的与恶性肿瘤发生、发展密切相关的癌基因且其亚细胞定位与其功能发挥有关，但其在HPV所致宫颈癌中的作用及意义未见报道。课题组前期实验首次发现，宫颈癌组织中AEG-1与HPV表达成平行关系；阻断宫颈癌细胞中E6/E7表达或过表达E6/E7，可分别下调或上调AEG-1表达，提示AEG-1可能为E6/E7致癌的关键效应分子。本课题采用不同亚细胞定位的抗AEG-1链可变区抗体（ScFv）真核表达载体转染细胞，MTT细胞增殖实验、克隆形成实验、划痕实验、western blot检测转染细胞中与细胞恶性表型相关的信号通路中主要信号分子PI3K, Akt1, Erk1/2等蛋白水平的变化，结果均证实HPV-E6/E7高表达条件下，抑制胞核内或胞浆内AEG-1功能可有效逆转HPV-E6/E7诱发的肿瘤细胞恶性表型并影响其下游信号通路中效应分子的表达。进一步通过荧光报告载体、CHIP及位点突变等启动子研究手段证实，E6/E7可通过与转录调控因子SP1等相互作用，影响AEG-1启动子活性，进而最终影响AEG-1表达水平。