抑制miR-421表达增强宫颈癌细胞放疗敏感性

发布时间：2018-06-07 08:36

本文选题：宫颈癌 + miR-　；参考：《中国病理生理杂志》2017年05期

【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(P0.05)。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(P0.05)。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(P0.05)。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。
[Abstract]:Objective: to study the molecular mechanism of inhibiting miR-421 expression to enhance the radiosensitivity of cervical cancer cells. Methods: Liposome 2000 was used to transfect miR-421 inhibitor into four cervical cancer cell lines (HeLa La-SiHaHaC33A and CaSki). The expression of miR-421 in endometrial epithelial cells (ESC) and four cervical carcinoma cells was detected by real-time PCR with transfection negative nucleotides as control. The transfected cells were treated with different doses of ionizing radiation for 48 h. The cell viability, the leakage rate of lactate dehydrogenase (LDH) and apoptosis were measured by MTT Elisa and Annexin V-FITC/PI staining combined with flow cytometry. The levels of Bcl-2 and Bax protein in cleaved caspase-9, caspase-9 and caspase-9 were detected by Western blot. Results the expression of miR-421 in ESC cells was significantly lower than that in negative control group (P 0.05), but was highly expressed in 4 cervical cancer cell lines after transfection of miR-421 inhibitor. Under the same conditions, the viability of cervical cancer cells with low expression of miR-421 and the leakage rate of LDH after irradiation were significantly lower than those of the negative control group for 72 h. The results of P0.05 +. Western blot showed that the cell viability and LDH leakage rate after ionizing radiation were significantly higher than those in the negative control group. The protein levels of cleaved caspase-9 cleaved PARP and Bcl-2 were significantly increased in cervical cancer cells with low expression of miR-421, while the level of Bax protein was significantly decreased (P 0.05). Conclusion the low expression of ESC in normal endometrial epithelium and the high expression in cervical carcinoma cell line, the down-regulation of miR-421 expression can inhibit the viability of cervical cancer cells and significantly enhance the radiosensitivity of cervical cancer cells. The mechanism is realized at least in part by activating the apoptosis pathway of caspase-9 cells and thus promoting the expression of anti-apoptotic protein Bcl-2 and inhibiting the expression of pro-apoptotic protein Bax.
【作者单位】：济南市第五人民医院妇产科;中国计划生育研究所细胞生物学实验室;
【基金】：山东省医药卫生资助项目(No.2013WS0010)
【分类号】：R730.23

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