人乳头瘤病毒E6蛋白通过上调氯离子蛋白通道5的表达促进宫颈癌细胞迁移

目的: 探讨人乳头瘤病毒（human papillomavirus，HPV）E6蛋白与氯离子通道蛋白5（chloride voltage gated channel 5，CLCN5）之间的关系以及CLCN5对宫颈癌细胞迁移的影响。方法: 采用RNA干扰技术敲低HPV E6基因，实时荧光定量PCR（RT qPCR）和蛋白质印迹法检测敲低HPV E6后宫颈癌HeLa细胞中CLCN5 mRNA及蛋白的表达水平。通过PCR扩增CLCN5基因片段，并将其克隆至表达载体pCDNA3.1 3×Flag（简称pCDNA3.1）质粒中，构建重组质粒pCDNA3.1 CLCN5。将目的质粒pCDNA3.1 CLCN5及其对照pCDNA3.1质粒分别转染宫颈癌HeLa细胞，蛋白质印迹法检测CLCN5蛋白的表达。Transwell迁移实验检测过表达CLCN5对宫颈癌HeLa细胞迁移的影响。同时，将CLCN5的小干扰RNA及其阴性对照分别转染宫颈癌HeLa细胞，Transwell迁移实验检测敲低CLCN5对宫颈癌HeLa细胞迁移的影响。结果： RT qPCR和蛋白质印迹检测结果显示，敲低HPV E6可显著降低CLCN5的mRNA和蛋白表达水平。质粒双酶切鉴定以及核酸测序比对结果证实重组质粒pCDNA3.1 CLCN5构建成功。蛋白质印迹检测结果显示CLCN5蛋白在宫颈癌HeLa细胞中被有效过表达或敲低。Transwell迁移实验结果表明，过表达CLCN5可促进宫颈癌细胞的迁移，敲低CLCN5可抑制宫颈癌细胞的迁移。 结论： HPV E6蛋白通过上调CLCN5的表达促进宫颈癌细胞迁移。

Human papillomavirus E6 protein promotes the migration of cervical cancer cells by up-regulating the expression of chloride voltage-gated channel 5

Objective: To investigate the relationship between human papillomavirus(HPV) E6 protein and chloride voltage gated channel 5(CLCN5), and to explore the effect of CLCN5 on the migration of cervical cancer cells. Methods: RNA interference technique was used to knock down the E6 gene of HPV, real time quantitative PCR(RT qPCR) and Western blotting were used to detect the expression of CLCN5 mRNA and protein in cervical cancer HeLa cells, after knocking down HPV E6 gene. The CLCN5 gene fragment was amplified by PCR and then cloned into pCDNA3.1 to construct the recombinant expression plasmid pCDNA3.1 CLCN5. The control vector pCDNA3.1 and the target plasmid pCDNA3.1 CLCN5 were transfected into cervical cancer HeLa cells respectively, after which Western blotting was used to detect the expression of CLCN5. Transwell migration assay was used to detect the effect of overexpression of CLCN5 on the migration of cervical cancer HeLa cells. Meanwhile, negative control and CLCN5 small interfering RNA were transfected into cervical cancer HeLa cells.Transwell migration assay was used to detect the effect of knockdown of CLCN5 on the migration of cervical cancer HeLa cells. Results: The results of RT qPCR and Western blotting showed that both the mRNA and protein level of CLCN5 was significantly decreased after HPV E6 was knocked down. The result of transwell migration showed that over expression of CLCN5 could promote cell migration, while knockdown of CLCN5 could inhibit cell migration. Conclusion: HPV E6 protein promotes the migration of cervical cancer cells by up regulating the expression of CLCN5.