宫颈癌中HPV16 E6蛋白诱导内质网应激-自噬反应的研究

摘要

Objective:To investigate the effects of HPV16 E6 on endoplasmic reticulum (ER) stress-autophagic response in the cervical cancer C33A cells. Methods:The eukaryotic expression vector of HPV16 E6 was constructed and transfected via lipofectamine into C33A cells. Experimental cells was classified into 3 groups: pcDNA3.1 (-)-HPV16 E6 group,pcDNA3.1 (-) group and C33A group. Fluorescent quantitation-ploymerase chain reaction was used for detecting expression of mRNA of HPV16 E6, and Western blot was explored to measure expression of protein of HPV16 E6,Beclin 1,LC3Ⅱ and GRP78 in transfected cells. Flow cytometry(FCM) was employed to observe the effect of transfection on the autophagic of C33A, and proliferation was analyzed by MTT assay. Results:Eukaryotic expression vector pcDNA3.1 (-)-HPV16 E6 was constructed successfully. The eukaryotic expression vector pcDNA3.1 (-)-HPV16 E6 significantly improved the expression of mRNA and protein of HPV16 E6 in C33A cells. The cell proliferations of HeLa cells increased , and FCM investigation showed the autophagic rate of pcDNA3.1 (-)-HPV16 E6 group was higher than that in pcDNA3.1 (-) group and C33A group (P<0.05). The expression of Beclin 1, LC3Ⅱ and GRP78 were significantly improved after transfection with vector pcDNA3.1(+)-HPV16 E6(P<0.05). Furthermore, GRP78 protein level was reduced and the autophagic rate was decreased by treatment with ER stress inhibitor. Conclusions:ER stress-autophagic response plays an important role in the process of HPV16 E6 inducing cervical cancer. So it might be one of the new strategies for gene therapy of cervical carcinoma.%目的：研究宫颈癌C33A细胞中HPV16 E6蛋白过表达对内质网应激－自噬反应的影响。方法：构建HPV16 E6的真核表达载体pcDNA3．1（－）－HPV16 E6，转染C33A细胞[pcDNA3．1（－）－HPV16 E6组]，同时设空载体转染组[pcDNA3．1（－）组]和空白对照组（C33A组），荧光定量聚合酶链反应（PCR）和蛋白质印迹法（Western blot）检测其C33A细胞中HPV16 E6 mRNA及蛋白表达的变化，及其对自噬蛋白Beclin 1、LC3Ⅱ、内质网应激标记蛋白葡萄糖调节蛋白78（GRP78）表达的影响，四甲基偶氮唑盐（MTT）法检测细胞生长的变化；流式细胞仪检测细胞自噬的变化。结果：成功构建了HPV16 E6的真核表达载体pcDNA3．1（－）－HPV16 E6，其可以使C33A细胞中HPV16 E6 mRNA及蛋白表达增加；HPV16 E6在C33A中过表达可促进肿瘤细胞的生长，pcDNA3．1（－）－HPV16 E6转染后C33A的自噬率较pcDNA3．1（－）组和C33A组增高，差异有统计学意义（P＜0．05）；HPV16 E6过表达使Beclin 1、LC3Ⅱ和GRP78蛋白表达增加，阻断内质网应激通路后GRP78表达减少，细胞自噬率降低。结论：内质网应激－自噬反应在HPV16感染导致宫颈癌的过程中具有重要作用，这为宫颈癌的基因治疗提供了新靶点。