跨损伤DNA合成通路基因REV3L在宫颈癌细胞化疗增敏中的作用及其机制研究

发布时间：2018-05-11 21:30

本文选题：宫颈癌 + 跨损伤DNA合成　；参考：《复旦大学》2014年硕士论文

【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(P0.05)。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。
[Abstract]:Objective REV3L is a catalytic subunit of DNA polymerase 味 Pol 味, which plays a key role in transcriptional DNA synthesis pathway. The purpose of this study was to investigate the effect of REV3L gene on the chemosensitivity of cervical cancer cells and to explore the value of REV3L gene as a molecular target in cervical cancer. Methods Immunohistochemical method was used to detect the expression of Pol 味 protein in 123 cases of cervical carcinoma and 17 cases of normal cervical tissues. REV3L gene overexpression vector (pcDNA-REV3L) was constructed and introduced into cervical cancer cell line with low REV3L expression. The REV3L gene interference vector shRNA-REV3L was constructed and transfected into REV3L overexpression cervical cancer cell line. The proliferation of cervical carcinoma cell line was detected by CCK-8 assay and cell cycle change was detected by flow cytometry. The effect of REV3L gene on the tolerance of platinum drugs was detected by CCK-8 assay and apoptosis was detected by flow cytometry. The changes of Bcl-xl and Bax were detected by Western blot. The effect of REV3L gene on the formation of focal point of Y-H2AX was detected by immunofluorescence, and the changes of Y-H2AX and other proteins were detected by Western blot. Results the expression level of Pol 味 protein in cervical cancer tissue was higher than that in normal cervix tissue. The proliferation of cervical cancer cell line slowed down after the silencing of P0.05N. REV3L gene and the proliferation of cervical cancer cell line increased after overexpression of REV3L gene. The silencing of REV3L gene inhibited cell cycle progression through G 1 / S phase arrest. After overexpression of REV3L gene, the cell cycle progression could be promoted by crossing the checkpoint of G 1 / S phase cell cycle. After the silencing of REV3L gene, the cell flat clone formation rate was decreased, but the cell clone formation rate increased after overexpression. After down-regulation of REV3L gene expression in cervical cancer cell line, CCK-8 assay showed that the sensitivity of cervical cancer cell line to cisplatin was increased, the early apoptosis rate was increased by flow cytometry, and the pro-apoptotic protein was increased by Western blot, but the anti-apoptotic protein was decreased by flow cytometry. The increase of Y-H2AX focus formation in cells by immunofluorescence assay and the increase of Y-H2AX protein expression by Western blot. After overexpression of REV3L gene, CCK-8 showed increased resistance to cisplatin. Flow cytometry was used to detect the early apoptosis rate of cisplatin. Western blot was used to detect the decrease of pro-apoptotic protein while anti-apoptotic protein was increased. Immunofluorescence detection of Y-H2AX focus formation decreased and Western blot detected the decrease of Y-H2AX protein expression. Conclusion inhibiting the expression of REV3L gene in cervical cancer cells can increase the sensitivity of the cells to cisplatin, while overexpression of REV3L gene can increase its resistance to cisplatin. REV3L may be a molecular target for chemosensitization of cervical cancer.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

【参考文献】