小分子干扰RNA沉默果蝇zeste基因增强子同源物2对宫颈癌Hela细胞顺铂敏感性的影响

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英文篇名：Effect of small interfering RNA silencing zeste gene enhancer homologue 2 gene on the sensitivity of Hela cells of cervical cancer to cisplatin 作者：秦海霞 ; 李少平 ; 朱利红 ; 张全华 ; 王世进 英文作者：QIN Hai-xia;LI Shao-ping;ZHU Li-hong;ZHANG Quan-hua;WANG Shi-jin;Department of Gynecology,the First Affiliated Hospital of Xinxiang Medical University,Henan Key Laboratory of Neural Rehabilitation; 关键词：zeste基因增强子同源物2 ; 宫颈癌 ; 小分子干扰RNA ; 顺铂 ; 药物敏感性 英文关键词：enhancer of zeste homolog 2;;cervical cancer;;small interfering RNA;;cisplatin;;drug sensitivity 中文刊名：XXYX 英文刊名：Journal of Xinxiang Medical University 机构：新乡医学院第一附属医院妇科河南省神经修复重点实验室; 出版日期：2019-02-05 出版单位：新乡医学院学报 年：2019 期：v.36;No.222 基金：河南省医学科技攻关计划项目(编号:201602157) 语种：中文; 页：XXYX201902004 页数：5 CN：02 ISSN：41-1186/R 分类号：21-25

摘要

目的探讨小分子干扰RNA(siRNA)沉默果蝇zeste基因增强子同源物2(EZH2)对宫颈癌Hela细胞顺铂敏感性的影响。方法培养宫颈癌Hela细胞,收集对数生长期Hela细胞继续培养,待贴壁细胞长满底部面积80%～90%时进行转染。将Hela细胞随机分为空白对照组、control siRNA组和EZH2 siRNA组,空白对照组Hela细胞不作处理,control siRNA组Hela细胞转染control siRNA,EZH2 siRNA组Hela细胞转染EZH2 siRNA。收集3组转染后Hela细胞,采用实时荧光定量聚合酶链反应检测Hela细胞中EZH2 mRNA表达,Western blot法检测Hela细胞中EZH2蛋白表达,流式细胞术检测Hela细胞的细胞周期。收集3组转染后Hela细胞,分别加入不同质量浓度(0. 0、12. 5、25. 0、50. 0、100. 0、200. 0 g·L~(-1))的顺铂,48 h后采用四甲基偶氮唑蓝法检测Hela细胞在顺铂作用下的体外存活率。结果 Control siRNA和EZH2 siRNA可高效转染Hela细胞,转染率均达90%以上。空白对照组、control siRNA组和EZH2 siRNA组Hela细胞中EZH2 mRNA相对表达量分别为396. 7±88. 4、389. 2±70. 6和98. 5±20. 3,EZH2 siRNA组Hela细胞中EZH2 mRNA相对表达量显著低于空白对照组和control siRNA组(t=2. 057、2. 015,P <0. 01),空白对照组与control siRNA组Hela细胞中EZH2 mRNA相对表达量比较差异无统计学意义(t=1. 476,P> 0. 05)。空白对照组、control siRNA组和EZH2 siRNA组Hela细胞中EZH2蛋白相对表达量分别为509. 4±110. 7、497. 5±80. 4和120. 4±31. 3,EZH2 siRNA组Hela细胞中EZH2蛋白相对表达量显著低于空白对照组和control siRNA组(t=2. 682、2. 597,P <0. 01),空白对照组与control siRNA组Hela细胞中EZH2蛋白相对表达量比较差异无统计学意义(t=1. 943,P> 0. 05)。EZH2 siRNA组G_0/G_1期细胞比例显著高于空白对照组和control siRNA组(t=2. 893、3. 087,P <0. 05),EZH2 siRNA组S期、G_2/M期细胞比例显著低于空白对照组和control siRNA组(EZH2 siRNA组与空白对照组比较:t=2. 526、5. 462,P <0. 05; EZH2 siRNA组与control siRNA组比较:t=2. 498、5. 417,P <0. 05),空白对照组与control siRNA组G_0/G_1期、S期及G_2/M期细胞比例比较差异均无统计学意义(t=0. 926、1. 017、0. 947,P> 0. 05)。不同质量浓度顺铂作用48 h后,同一质量浓度下EZH2 siRNA组Hela细胞的存活率明显低于空白对照组及control siRNA组(P <0. 05),且随着顺铂浓度的增加,3组Hela细胞的存活率均呈逐渐下降趋势。结论沉默EZH2表达可提高宫颈癌Hela细胞对顺铂的敏感性,而阻滞细胞周期可能是其主要作用机制之一。
        Objective To investigate the effect of small interference RNA( siRNA) silencing enhancer of zeste homolog 2( EZH2) on the sensitivity of Hela cells of cervical cancer to cisplatin. Methods The Hela cells of cervical cancer were cultured. The Hela cells in logarithmic growth phase were collected for further culture and transfection when the adherent cells reached 80%-90% of the basal area. The Hela cells were randomly divided into blank control group,control siRNA group and EZH2 siRNA group. The Hela cells in the blank control group were cultured normally with out treatment. The control siRNA and EZH2 siRNA were transfected into the Hela cells of the control siRNA group and the EZH2 siRNA group respectively. The Hela cells in the three groups were collected after transfection. The expression of EZH2 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction,the expression of EZH2 protein was detected by Western blot,and the cell cycle of Hela cells was detected by flow cytometry. The transfected Hela cells in the three groups were collected and added with cisplatin of different mass concentration( 0. 0,12. 5,25. 0,50. 0,100. 0,200. 0 g·L~(-1)). The survival rate of Hela cells was measured by thiazolyl blue method at 48 hours after treated with cisplatin. Results The control siRNA and EZH2 siRNA could efficiently transfect the Hela cells,and the transfection rate was more than 90%. The relative expression level of EZH2 mRNA in Hela cells of the blank control group,control siRNA group and EZH2 siRNA group was 396. 7 ±88. 4,389. 2 ± 70. 6 and 98. 5 ± 20. 3,respectively. The relative expression level of EZH2 mRNA in Hela cells of EZH2 siRNA group was significantly lower than that in the blank control group and control siRNA group( t = 2. 057,2. 015; P < 0. 01).However,there was no significant difference in the relative expression level of EZH2 mRNA in Hela cells between the blank control group and control siRNA group( t = 1. 476,P > 0. 05). The relative expression level of EZH2 protein in Hela cells of the blank control group,control siRNA group and EZH2 siRNA group was 509. 4 ± 110. 7,497. 5 ± 80. 4 and 120. 4 ± 31. 3,respectively. The relative expression level of EZH2 protein in Hela cells of the EZH2 siRNA group was significantly lower than that in the blank control group and control siRNA group( t = 2. 682,2. 597; P < 0. 01). There was no significant difference in the relative expression level of EZH2 protein in Hela cells between the blank control group and the control siRNA group( t =1. 943,P > 0. 05). The proportion of the cells in G_0/G_1 phase in the EZH2 siRNA group was significantly higher than that in the blank control group and control siRNA group( t = 2. 893,3. 087; P < 0. 05). The proportion of the cells in S phase and G_2/M phase in the EZH2 siRNA group was significantly lower than that in the blank control group and control siRNA group( EZH2 siRNA group compared with blank control group: t = 2. 526,5. 462; P < 0. 05. EZH2 siRNA group compared with control siRNA group: t = 2. 498,5. 417; P < 0. 05). There was no significant difference in the proportions of the cells in G_0/G_1,S and G_2/M phases between the blank control group and the control siRNA group( t = 0. 926,1. 017,0. 947; P > 0. 05). After 48 hours of cisplatin treatment with different mass concentration,the survival rate of Hela cells in the EZH2 siRNA group was significantly lower than that in the blank control group and control siRNA group at the same mass concentration( P < 0. 05). With the increase of cisplatin mass concentration,the survival rate of Hela cells in the three groups decreased gradually. Conclusion Silencing EZH2 expression can improve the sensitivity of Hela cells of cervical cancer to cisplatin,and blocking cell cycle may be the main mechanism.

引文

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