miR-154靶向CUL2调控HPV16 E7-pRb途径在宫颈癌发生中的作用

Cervical cancer is one of the most common malignant tumors that seriously threatens women's lives. E3 ubiquitin ligase Cullin2 (CUL2) is essential in the HPV16 E7 oncoprotein-mediated pRb degradation process. Our early work found that miR-154 expression was significantly down-regulated in cervical cancer, and overexpression of miR-154 can inhibit the proliferation and migration of cervical cancer cells in vitro, but the mechanism is unclear. Bioinformatics prediction and preliminary experiments results show that miR-154 combines the 3’UTR of CUL2 mRNA. So we speculate that miR-154 can inhibit the occurance of cervical cancer through silencing the expression of CUL2. In this study, we will adopt luciferase reporter assay to clear whether miR-154 can directly combine with the CUL2; establish the CIN cell line by transfetion of the HPV16 E7 overexpression lentiviral vector into human immortalized epidermal cell (HaCaT) to simulate the process of cervical cancer, then establish and screen the cervical lesion cells with stable high and low expression of miR-154 by lentivirus transfection technology further; finally confirm the effect of miR-154 targeting CUL2 on HPV16E7-mediated cervical cancer in vitro and in vivo. We expect to improve the pathogenesis of cervical cancer and find a new target for preventing the occurrence of cervical cancer.

宫颈癌是严重威胁妇女生命的最常见恶性肿瘤之一。E3泛素连接酶Cullin2(CUL2)在HPV16E7癌蛋白介导的pRb降解过程中至关重要。本课题组前期发现宫颈癌中miR-154表达显著下调，上调miR-154表达可显著抑制宫颈癌细胞的增殖和迁移，但机制不清。生物信息学预测和预实验结果显示，miR-154高度保守，可与CUL2结合。故我们推测：miR-154可靶向沉默CUL2，抑制宫颈癌的发生。本项目拟通过荧光素酶报告基因实验验证miR-154对CUL2的直接作用；采用人永生化表皮细胞，构建HPV16E7过表达慢病毒载体，建立具有癌前病变特性的细胞系，模拟宫颈癌变进程，进一步利用慢病毒转染技术，建立并筛选稳定高、低表达miR-154的宫颈病变细胞株，通过细胞、动物实验证实miR-154靶向CUL2在HPV16E7介导宫颈癌发生中的作用。旨在完善宫颈癌发病机制，为阻断宫颈癌的发生提供新靶点。

宫颈癌是严重威胁妇女生命的最常见恶性肿瘤之一。HPV16E7癌蛋白介导的pRb降解是宫颈癌发生的经典途径，E3泛素连接酶Cullin2(CUL2)在HPV16E7癌蛋白介导的pRb降解过程中至关重要，因此miR-154可调控CUL2成为阻断宫颈癌发生的重要靶点。然而，miR-154在宫颈癌中的作用尚未见报道。本项目首先通过荧光素酶报告基因实验验证miR-154对CUL2的直接作用；然后采用人永生化表皮细胞，构建HPV16E7过表达慢病毒载体，建立具有癌前病变特性的细胞系，模拟宫颈癌变进程，进一步利用慢病毒转染技术，建立并筛选稳定高、低表达miR-154的宫颈病变细胞株，通过细胞、动物实验双重证实了miR-154靶向CUL2调控HPV16E7-pRb途径在宫颈癌发生中的作用。研究结果为进行宫颈癌病因学和发病机制研究提供了新的理论依据，为宫颈癌前病变的早期发现与诊断提供可靠的生物标志，为宫颈癌及癌前病变预防和治疗策略的制定开拓了新的思路。