人乳头瘤病毒16型E6、E7反义RNA对人宫颈癌SiHa细胞的恶性逆转作用

Reversal Effect of Antisense RNA Targeting Human Papillomavirus 16(HPV16) E6E7 on Malignancy of Human Cervical Cancer Cell Line SiHa

背景与目的:人乳头瘤病毒16型(humanpapillomavirus16,HPV16)的早期基因E6、E7可分别诱导p53的泛素化降解和pRb的高磷酸化失活,与宫颈癌的发生发展关系密切。本研究通过真核表达载体介导HPV16型E6、E7基因反义RNA在SiHa细胞中的表达,探讨反义RNA能否降低E6、E7基因的表达和诱导细胞凋亡。方法:利用pEGFP构建HPV16型E6、E7基因反义RNA的真核表达载体并转染SiHa细胞,利用RT-PCR和Westernblot技术检测转染后E6、E7基因的mRNA和蛋白的变化,利用MTT法检测SiHa细胞转染后细胞增殖活性,利用流式细胞仪和激光共聚焦显微镜检测转染后细胞的凋亡情况。结果:转染携带HPV16型E6、E7反义基因的质粒后,SiHa细胞的E6、E7基因的mRNA和蛋白表达均明显下调;MTT结果显示转染后细胞的增殖活性(0.50±0.05)与转染空载体细胞(1.01±0.06)和未转染细胞(1.28±0.06)比较明显降低(P<0.05);流式细胞仪检测结果显示转染后细胞凋亡率(59.3±11.3)%与转染空载体细胞(9.4±1.8)%和未转染细胞(2.1±0.4)%比较有显著性差异(P<0.05)。激光共聚焦显微镜结果示转染后细胞凋亡明显增多。结论:HPV16型E6、E7基因反义RNA可降低宫颈癌细胞中E6、E7癌基因的表达,诱导宫颈癌细胞凋亡。

BACKGROUND ＆ OBJECTIVE： The ubiquitinated degradation of p53 can be induced by human papillomavirus type 16 （HPV16） early gene 6 （E6）; the phosphorylated inactivation of pRb can be induced by HPV16 E7. They are closely associated with the carcinogenesis and progression of cervical cancer. This study was to investigate the effects of HPV16 E6E7 antisense RNA on the expression of oncogene E6 and E7 and apoptosis of human cervical cancer cell line SiHa harboring HPV16 genome. METHODS：The antisense sequence of HPV16 E6E7 was cloned into eukaryotic expression vector pEGFP to prepare recombinant plasmid containing E6E7AS, which was transfected into SiHa cells. The mRNA and protein levels of E6 and E7 gene were detected by Western blot and reverse transcfiption-polymerase chain reaction （RT-PCR）. Cell proliferation after transfection was evaluated by MTT assay. The apoptosis of transfected SiHa cells was assessed by flow cytometry （FCM） and confocal laser microscopy. RESULTS： After transfection of HPV16 E6E7 antisense RNA, the mRNA and protein levels of HPV16 E6 and E7 in SiHa cells were obviously decreased. The proliferation activity of SiHa/E6E7AS cells was significantly lower than that of SiHa/EGFP and SiHa cells （0.50±0.05 vs. 1.01±0.06, 1.28±0.06, P〈 0.05）. The apoptosis rate was significantly higher in SiHa/E6E7AS cells than in SiHa/EGFP and SiHa cells E（59.3±11.3）% vs. （9.4±1.8）%, （2.1±0.4）%, P 〈0.05]. The apoptotic cells increased noticeably after transfection. CONCLUSION： Down-regulating HPV16 E6 and E7 with antisense RNA induces apoptosis in SiHa cells, and may be useful for HPV-associated malignancy gene therapy.