RNAi沉默ERCC1基因对肺腺癌细胞顺铂敏感性的影响  

The effect of RNAi silencing ERCC1 gene on the cisplatinum sensitivity of lung adenocarcinoma cell line

作　　者：王巍炜[1] 巫正伟[1] 王德光[1] 张勇[1] 

机构地区：[1]昆明医科大学第三附属医院胸外科,650118

出　　处：《重庆医学》2016年第28期3904-3906,共3页Chongqing medicine

基　　金：云南省科技厅-昆明医科大学2013年联合基金资助项目(2013FB164);昆明医科大学第三附属医院博士基金资助项目(BS55201510)

摘　　要：目的目的用包含切除修复交叉互补(ERCC1)基因特异RNA序列的慢病毒,将其转染入宣威肺腺癌细胞XWCL05中,沉默ERCC1基因表达,初步探讨其基因沉默效果及对顺铂敏感性的影响,为顺铂耐药逆转提供思路和依据。方法经293T细胞包装后构建ERCC1基因RNAi的慢病毒载体及阴性对照,分别使用XWCL05空白组(空白对照)、XWCL05-neg组(转染空载体)、XWCL05-RNAi组(转染ERCC1-RNAi慢病毒)。Western blot法检测转染前后XWCL05细胞中ERCC1蛋白的表达。12.5μg/mL顺铂作用48h后,用四甲基偶氮唑蓝(MTT)法检测顺铂对各组XWCL05细胞增殖抑制率的影响;流式细胞术检测各组中XWCL05细胞的凋亡率。结果 (1)Western blot结果显示,XWCL05-RNAi组中ERCC1蛋白表达水平显著低于XWCL05-neg组(P<0.05),而XWCL05-neg和XWCL05空白组差异无统计学意义(P>0.05)。(2)MTT法检测显示相同浓度顺铂作用下,XWCL05-RNAi组的增殖抑制率显著高于XWCL05-neg组和XWCL05空白组(P<0.05),而XWCL05-neg组。(3)流式细胞术检测结果显示,XWCL05-RNAi组凋亡率显著高于XWCL05-neg组和XWCL05空白组(P<0.05)。而顺铂作用后,各组凋亡率较前均显著升高(P<0.05),且XWCL05-RNAi组凋亡率显著高于其余两组。结论 ERCC1基因RNAi的慢病毒能够有效地沉默宣威肺腺癌细胞XWCL05中ERCC1基因的表达,并增强XWCL05对顺铂的敏感性。Objective RNAi targeted ERCC1 were used to transfected Xuanwei lung adenocarcinoma cell line XWCL05 by lentivirus in order to further investigate ERCC1 gene silencing effect on the cisplatinum sensitivity of XWCL05. We wish this could be reference for cisplatinum-resistance reversion. Methods Lentivirus contained ERCC1 targeted RNAi were cultured in 293T cell line and XWCL05 wild t ype（control）, XWCL05-neg （blank vector） and XWCL05-RNAi（ERCC1-RNAi） were cultured for further a- nalysis. Western blot was applied to test ERCC1 expression in XWCL05 before and after transfeetion. After co-cultured with eisplatinum（concentration 12.5 μg/mL）for 48 hours,MTT was used to test cell proliferation and flow cytometry（FCM）was used to test cell necrosis. Results （1）Western blot showed ERCC1 expression was significantly lower in XWCLO5-RNAi（P〈0. 05） and its expression between XWCL05 wild type and XWCL05-neg showed no significantly difference（P〈0.05）. （2） In MTT test, there was significant proliferation inhibiting effect in XWCL05-RNAi group compared with XWCLOS-neg group. （3）In FCM, the necrosis rate of XWCL05-RNAi was significantly higher（P〈0.05）than that of other two groups and there was also no significant difference between XWCL05 wild type and XWCL05-neg. However after treated with cisplatinum,the necrosis rate of these 3 groups increased significantly when compared with their counterpart before the treatment of cisplatinum（P〈0.05）and necrosis rate of XWCL05- RNAi decreased the most. Conclusion ERCC1 RNAi lentivirus can effectively silence ERCC1 gene expression in XWCL05 and enhance XWCL05 sensitivity to cispaltinum.

关 键 词：遗传载体 RNA干扰 ERCC1 肺腺癌细胞 

分 类 号：R734.2[医药卫生—肿瘤]