【作者】 杨莉；

【导师】 程玺；

【作者基本信息】 复旦大学 ， 肿瘤学（专业学位）， 2014， 硕士

【摘要】 目的REV3L是DNA聚合酶ζ(Polζ)的催化亚基,在跨损伤DNA合成通路(TLS)中起关键作用。本课题旨在研究REV3L基因对宫颈癌细胞化疗敏感性的影响,探讨REV3L基因在宫颈癌中作为分子靶点的价值。方法采用免疫组化法检测123例宫颈癌组织及17例正常宫颈组织中Polζ蛋白的表达水平。构建REV3L基因过表达载体pcDNA-REV3L后导入REV3L低表达的宫颈癌细胞系,构建REV3L基因干扰载体shRNA-REV3L并转染REV3L高表达宫颈癌细胞系后,通过CCK-8法检测对细胞增殖、流式细胞仪检测细胞周期变化、平板克隆形成实验检测克隆形成率的变化,进一步通过CCK-8法检测REV3L基因对铂类药物耐受性的影响,通过流式细胞术检测药物作用后细胞凋亡情况,并通过蛋白免疫印迹法检测凋亡相关蛋白Bcl-2、Bcl-xl及Bax等的变化情况。通过免疫荧光法检测顺铂处理后REV3L基因对Y-H2AX焦点形成情况的影响,蛋白免疫印迹法检测Y-H2AX等蛋白的变化。结果宫颈癌组织中Polζ蛋白表达水平较正常宫颈组织高(P<0.05)。REV3L基因沉默后宫颈癌细胞株增殖减慢,REV3L基因过表达后宫颈癌细胞株增殖加快：沉默REV3L基因通过G1/S期阻滞抑制细胞周期进展,而REV3L基因过表达后可以越过G1/S期细胞周期检查点促进细胞周期进展；REV3L基因沉默后细胞平板克隆形成率减少,而过表达后细胞克隆形成率增加。下调宫颈癌细胞系REV3L基因表达后CCK-8检测示宫颈癌细胞对顺铂的敏感性增加；流式细胞仪检测顺铂作用后早期凋亡率增加；Western blot检测促凋亡蛋白升高,而抗凋亡蛋白降低；免疫荧光检测细胞内Y-H2AX焦点形成增多,Western blot检测Y-H2AX蛋白表达升高。REV3L基因过表达后CCK-8示细胞对顺铂的抵抗性增加；流式细胞仪检测顺铂作用后早期凋亡率降低；Western blot检测促凋亡蛋白降低,而抗凋亡蛋白升高；免疫荧光检测细胞内Y-H2AX焦点形成减少,Western blot检测Y-H2AX蛋白表达降低。结论抑制REV3L基因在宫颈癌细胞中的表达,可提高细胞对顺铂的敏感性,而过表达REV3L基因增加其对顺铂的抵抗性。REV3L可能作为宫颈癌化疗增敏的分子靶点。更多还原

【Abstract】 Objectives. REV3L, the catalytic subunit of DNA Polymerase (?) (Po(?), plays a significant role in the DNA damage tolerance mechanism of translesion synthesis (TLS). The role of REV3L in chemosensitivity of cervical cancer and its mechanism needs exploration.Methods. We evaluated the expression of Po(?) protein in 123 paraffin-embedded cervical cancer tissues and 17 normal cervical tissues using immunohistochemistry. We transfected REV3L shRNA into cervical cancer cell lines with high expression of REV3L and REV3L cDNA were delivered into cervical cancer cell lines with low expression of REV3L, and we explored the effect of REV3L on cell proliferation, cell cycle and colony formation ability. Then we assessed the cytotoxicity of cisplatin by Cell Counting Kit-8 (CCK-8) assay, apoptosis rates by apoptosis analysis using flow cytometry after exposure to cisplatin, expression changes of anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia sequence 1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xl) and proapoptotic Bcl-2-associated x protein (Bax) by western blotting after cisplatin treatment. We used immunofluorescence to detect y-H2AX foci after cisplatin treatment.Results. The expression of Po(?) in cervical cancer is higher than that in normal tissue samples (P<0.05). Suppression of REV3L expression inhibits cell proliferation through G1/S arrest, and enhancement of REV3L expression promotes cell proliferation by inducing G1 phase to S phase transition. Suppression REV3L expression inhibits colony formation ability, and enhancement of REV3L expression promotes colony formation ability. Suppression of REV3L enhances the sensitivity of cervical cancer cells to cisplatin, and the overexpression of REV3L confers resistance to cisplatin as evidenced by the alteration of apoptosis rates, and significantly expression level changes of Bcl-2, Mcl-1, Bcl-xl, Bax and vice versa. Immunofluorescence shows cells deficient in REV3L expression exhibited intense y-H2AX staining in comparison with control cells after exposure to cisplatin, and cells with REV3L overexpression shows weaker y-H2AX staining compared with control cells.Conclusions. The results suggest that REV3L plays an important role in regulating cervical cancer cellular response to DNA damaging agents, which indicates the inhibition of REV3L as potential target of chemosensitizer of cervical cancer.更多还原