HPV18-E7介导的上皮间质转化与抑癌基因Rb的关系研究

Human papillomavirus type 18 E7 oncoprotein enhances invasion and migration by inducing Epithelial to Mesenchymal Transition through degradating Rb in HeLa cells

目的探究宫颈腺癌细胞中HPV18-E7介导的上皮间质化(EMT)与抑癌基因Rb之间的关系。方法针对HPV18-E7的siR N A转染入人宫颈腺癌HeL a细胞运用R ealtime PCR技术、免疫印迹实验在mRNA水平和蛋白水平检测E7与Rb的表达变化;Real-time PCR技术筛选特异性针对Rb基因的siR NA,并将其转染入HeL a-siE 7细胞运用免疫印记实验检测EMT相关的标记因子的表达变化,CCK-8细胞增殖实验和免疫荧光染色法检测细胞增殖能力及细胞骨架的变化;最后用染色质免疫沉淀和real-time PCR技术检测Rb是否通过作用于E-cadherin基因的启动子区来发挥调控其表达的作用。结果HeL a-siE 7细胞内R b表达较对照组细胞显著上调;HeL a-siE 7-siR b细胞较HeL a-siE 7细胞E-cadherin的表达明显下调,vimentin的表达却显著上调;HeL a-siE 7-siR b细胞增殖能力显著增强;沉默E7表达后细胞失去极性,呈卵圆形,而在沉默E7表达的基础上沉默Rb后,细胞又重新获取细胞极性变回梭形;染色质免疫沉淀实验结果表明抑癌基因Rb可直接作用于上皮性标记因子E-cadherin的启动子区。结论 HPV18-E7能够结合抑癌基因Rb使其失去活性从而致使其无法绑定在上皮标记因子E-cadherin启动子区来调控其表达,最终导致宫颈腺癌发生EMT,促进肿瘤发生侵袭转移。

Objective To confirm the role of Rb in HPV18-E7 regulated EMT. Methods HPV18-E7 siRNAs were transfected in human cervical cancer HeLa cells, real-time PCR, Western blot were performed to examine the expression of E7 and Rb. To further explore the role of Rb in HPV18-E7 induced EMT, siRb was transfected into HeLa/siE7 cells to determine the role of Rb in HPV18-E7 regulated EMT by CCK8 cell proliferation assay and Confocal fluorescent analysis, respectively. At last, ChIP assay was performed to probe the Rb binding site in the endogenous E-cadherin promoter. Results Decreasing of mRNA and protein level of E7 were significantly induced the expression of Rb. When siRb was transfected into HeLa/siE7 cells, depletion of Rb resulted in the down regulation of E-cadherin and upregulation of vimentin. In addition, in comparison with the HeLa/siE7 cells, HeLa/siE7/siRb cells showed a higher cell proliferation rate（ about 40%） in all five incubation periods examined. Furthermore, local polymerization of actin made the structures of HeLa/siE7/siRb cells to become fusiform more than HeLa/siE7 cells, and this structure was an important sign of cell migration. At last,ChIP assay was performed to probe the Rb binding site in the endogenous E-cadherin promoter, and then Rb could regulate the transcriptional activity through binding to the E-cadherin promoter. Conclusion In HeLa cells, HPV18-E7 can enhance invasion and migration by inducing EMT through combining with Rb, which binds to the E-cadherin promoter sequence and regulate its expression.