miR-134-5p对宫颈癌细胞增殖和凋亡的影响及其分子机制

Effect of miR-134-5p on proliferation and apoptosis of cervical carcinoma cell and its molecular mechanism

目的:观察微小核糖核酸-134-5p(mi R-134-5p)转染对宫颈癌细胞增殖和凋亡的影响,验证其可能的分子机制。方法:收集湖北医药学院附属人民医院肿瘤中心2016年5月至8月收治的8名宫颈癌患者肿瘤组织和相应癌旁组织。利用lipofectamine 2000将mi R-134-5p mimics转染至宫颈癌Hela和Si Ha细胞。采用MTT法和集落形成实验检测细胞增殖活性;流式细胞术(FCM)检测细胞周期和细胞凋亡;q RT-PCR检测宫颈癌组织和细胞mi R-134-5p m RNA表达以及宫颈癌细胞EGFR m RNA表达;Western blotting检测宫颈癌细胞EGFR信号通路相关蛋白的表达。结果:宫颈癌组织mi R-134-5p m RNA表达显著低于癌旁组织(P<0.01)。和转染mi R-NC的Hela和Si Ha细胞比较,转染mi R-134-5pmimics的宫颈癌Hela和Si Ha细胞mi R-134-5p m RNA表达显著升高;细胞增殖能力显著降低(转染第5天,Hela细胞:1.06±0.13 vs 1.32±0.07;Si Ha细胞:1.12±0.10 vs 1.42±0.12,均P<0.05);形成的集落数减少;G0/G1期细胞比例显著上升,S期和G2/M期细胞比例显著下降;细胞凋亡率显著增加[Hela细胞:(26.53±13.48)%vs(3.25±1.74)%;Si Ha细胞:(30.49±12.04)%vs(5.10±2.86)%,均P<0.05];EGFR m RNA和EGFR蛋白表达显著下调,其中EGFR m RNA,Hela细胞下调58%(P<0.01),Si Ha细胞下调41%(P<0.05);EGFR下游靶蛋白p-AKT、p-ERK1/2和Cyclin D1蛋白及p EGFR蛋白表达显著下调。结论:mi R-134-5p可显著抑制宫颈癌细胞增殖并促进细胞凋亡,其可能的分子机制是通过抑制EGFR基因的表达,抑制EGFR通路的活化。

Objective： To observe effect of miR-134-5p transfection on proliferation and apoptosis of cervical carcinoma cell and to verify its possible molecular mechanism. Methods： Eight pairs of cervical cancer and para-cancerous tissuesfrom the patients with cervical cancer who hospitalized in Center of Oncology, Renmin Hospital, Hubei University of Medicine during May to August 2016 were collected. miR-134-5p mimics were transfected into cervical carcinoma Hela and SiHa cells by lipofectomin 2000. MTT and colony formation assays were used to detect proliferation of cells. Flow cytometry （FCM）assay was used to test cell cycles and apoptosis of cells. Expressions of miR-134-5p mRNA in cervical carcinoma tissue and cell, and expression of EGFR mRNA in cervical carcinoma cell were detected by qRT-PCR assay. Expressions of EGFR pathway-related proteins in cervical carcinoma cell were examed by Western blotting assay. Results： Expression of miR-134-5p mRNA in cervical carcinoma tissue was significantly lower than that in paracarcinoma tissue （P〈0.01）. Comparing with the Hela and SiHa cells that transfected with miR-NC, expressions of miR-134-5p mRNA in the Hela and SiHa cells that transfected with miR-134-5p mimics were obviously increased, proliferation abilities of the cells significantly reduced （at the 5th day of the transfection, Hela cell：1.06 ± 0.13 vs 1.32 ± 0.07; SiHa cell： 1.12 ± 0.10 vs 1.42 ± 0.12, all P〈0.05）, apoptosis rates of the cells obviously increased （Hela cell： [26.53 ± 13.48]% vs [3.25 ± 1.74]%; SiHa cell： [30.49 ± 12.04]% vs [5.12 ±2.86]%, all P〈0.05）, number of formed colony decreased, ratio of G0/G1 phase cells increased, ratio of the cells in Sand G2/M phase decreased, apoptosis rate of the cells enhanced （all P〈0.05）, expressions of EGFR mRNA and EG-FR protein in the cells were remarkably down-regulated, among which EGFR mRNA in the the Hela cell down 58%（P〈0.01） and in the the SiHa cell down 41% （P〈0.05）, expressions of downstream target protein for EGFR, p-AKT,p-ERK/2 and Cyclin D1, as well as pEGFR proteins were evidently down-regulated. Conclusion： miR-134-5p couldsignificantly inhibit proliferation of the cervical carcinoma cells and promote their apoptosis, of which possible molecular mechanism might be inhibit activation of EGFR pathway through inhibiting expression of EGFR gene.