抑制miR-421表达增强宫颈癌细胞放疗敏感性  

Inhibition of miR-421 expression enhances radiosensitivity of cervical cancer cells

作　　者：潘雨露[1] 吴淑霞[1] 石翠格 任兴业[1] 

机构地区：[1]济南市第五人民医院妇产科,山东济南250022 [2]中国计划生育研究所细胞生物学实验室,北京100081

出　　处：《中国病理生理杂志》2017年第5期798-804,共7页Chinese Journal of Pathophysiology

基　　金：山东省医药卫生资助项目(No.2013WS0010)

摘　　要：目的:研究抑制miR-421表达增强宫颈癌细胞放疗敏感性的分子机制。方法:运用脂质体2000将miR-421 inhibitor转染4株宫颈癌细胞系HeLa、SiHa、C33A和CaSki,以转染阴性核苷酸为对照,real-time PCR检测子宫内膜上皮细胞ESC细胞和4株宫颈癌细胞中miR-421的表达水平;转染的细胞经不同剂量电离辐射(0、2、4、6和8 Gy)处理48 h后运用MTT法、ELISA和Annexin V-FITC/PI染色法联合流式细胞术分别测定细胞活力、乳酸脱氢酶(LDH)漏出率和细胞凋亡率;运用Western blot检测细胞中cleaved caspase-9、caspase-9、cleaved PARP、PARP、Bcl-2和Bax蛋白的水平。结果:miR421在ESC细胞中低表达,而在4株宫颈癌细胞中高表达,转染miR-421 inhibitor后miR-421显著低于阴性对照组(P<0.05)。相同条件下,辐照后低表达miR-421的宫颈癌细胞活力和LDH漏出率显著低于阴性对照组,72 h的细胞凋亡率明显增加(P<0.05)。Western blot结果显示,电离辐射后与阴性对照组比较,低表达miR-421的宫颈癌细胞中cleaved caspase-9、cleaved PARP和Bcl-2的蛋白水平明显增高,而Bax蛋白水平显著降低(P<0.05)。结论:miR-421在正常子宫内膜上皮ESC细胞中低表达,而在宫颈癌细胞系中高表达;下调miR-421表达能抑制宫颈癌细胞的活力,显著增强宫颈癌细胞的辐射敏感性,其机制至少部分通过激活caspase-9细胞凋亡通路进而促进抗凋亡蛋白Bcl-2表达和抑制促凋亡蛋白Bax表达而实现。AIM：To investigate the molecular mechanism of inhibition of miR-421 expression promoting radiosensitivity in the cervical cancer cells.METHODS： Cervical cancer lines HeLa, SiHa, C33A and CaSki were transfected with miR-421 inhibitor or negative control nucleotide using Lipofectamine 2000 kit, and the levels of miR-421 expression in the cervical cancer lines and endometrial epithelium cell line ESC were detected by real-time PCR. These cells with transfection were exposed to various doses of X-ray （0, 2, 4, 6 and 8 Gy）. After 48 h, the cell viability, LDH leakage rate and apoptotic rate were measured respectively by MTT assay, ELISA and flow cytometry with Annexin V-FITC/PI staining. The protein levels of cleaved caspase-9, caspase-9, cleaved PARP, PARP, Bcl-2 and Bax were monitored by Western blot.RESULTS： Low miR-421 levels was found in the ESC cells, while high miR-421 levels were observed in the HeLa, SiHa, C33A and CaSki cells. The level of miR-421 in the cells transfected with miR-421 inhibitor was significantly lower than that in negative control group （P〈0.05）. The viability and LDH leakage rate of the cervical cancer cells with low miR-421 expression were notablely lower than those in negative control group, and the apoptotic rate at 72 h was remarkablely increased （P〈0.05） under the same conditions. The results of Western blot indicated that, after exposure to ionizing radiation, the protein levels of cleaved caspase-9, cleaved PARP and Bcl-2 were significantly increased, while the protein level of Bax was significantly decreased in the cervical cancer cells with low miR-421 expression compared with negative control group （P〈0.05）.CONCLUSION： miR-421 is lowly expressed in the normal endometrial epithelial cells, but highly expressed in the cervical cancer cells. Down-regulation of miR-421 expression significantly inhibits the growth and enhances the radiosensitivity of cervical cancer cells at least partly via activating caspase-9 apoptosis pathway, thus promoting Bcl-2 and

关 键 词：宫颈癌 miR-421 电离辐射 放疗敏感性 细胞凋亡 

分 类 号：R730.23[医药卫生—肿瘤]