靶向EGFR的RNA干扰诱导非小细胞肺癌细胞A549凋亡的研究  

Apoptosis of non-small cell lung cancer A549 cells by silencing EGFR with RNA interference

作　　者：游佳[1] 赵新宇[1] 陈县城[1] 徐宁[1] 邓洪新[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,四川成都610041

出　　处：《中华肿瘤防治杂志》2008年第16期1205-1209,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家重点基础研究发展计划(973计划)资助项目(2004CB518805)

摘　　要：目的:探讨短发夹状RNA(short hairpin RNA,shRNA)介导的靶向EGFR基因的RNA干扰(RNA interference,RNAi)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞A549的凋亡诱导作用。方法:构建靶向人EGFR基因的表达shRNA的质粒(pShEGFR),转染A549细胞,应用RT-PCR分析EGFR mRNA的表达情况,采用MTT法检测A549细胞的生长状况,通过流式细胞仪以及PI染色法进行细胞凋亡的定量检测和形态学分析。结果:pShEGFR转染组A549细胞中EGFR mRNA的表达水平,较转染同一载体连接无关序列的对照组(pShNEG组)、单纯脂质体组及PBS阴性对照组A549细胞均明显降低,F=425.640,P=0.000,其中较PBS阴性对照组细胞降低了74.5%。转染pShEGFR的A549细胞与3种不同对照组细胞相比,生长受到明显抑制,F=107.428,P=0.000,生长抑制率达54.9%,而转染pShNEG组和单纯脂质体组的A549细胞生长抑制率则分别为20%和3.1%。流式细胞仪检测结果显示,pShEGFR组细胞存在32.8%的凋亡率,PI荧光染色可见pShEGFR组细胞呈现出典型的凋亡形态学变化。结论:质粒介导的靶向人EGFR基因的pShEGFR能够有效地干扰NSCLC细胞系A549中EGFR基因的表达,进而抑制肿瘤细胞的生长,诱导肿瘤细胞的凋亡。OBJECTIVE：To investigate the therapeutic effect of plasmid-mediated shRNA targeting EGFR （pShEGFR） for NSCLC in vitro. METHODS： NSCLC cell line A549 was transfected with plasmid-mediated shRNA targeting pShEGFR as well as various controls. Reverse Transcription-PCR was used to detect the level of EGFR mRNA. MTT assay was used to assess the depressant effects of EGFR silencing on tumor cell growth. Flow cytometry（FCM）and PI staining were used to evaluate the apoptosis-inducing activity of pShEGFR. RESULTS： The expression of EGFR mRNA in the pShEGFR transfected A549 cells was lower than that of pShNEG, lipofectamine 2000 or PBS negative groups （F=425.640, P=0.000）, and transfection with pShEGFR resulted in specific silencing of EGFR with 74.5% decreases in EGFR mRNA transcription compared with PBS transfected ones. The decrease in EGFR expression inhibited tumor cell growth in vitro by 54.9%, F=107.428, P=0.000. The inhibiatory rates of pShNEG and lipofectamine 2000 group were 20% and 3.1%, respectively. Furthermore, in the pShEGFR-treated group, 32.8% of A549 cells were apoptosis confirmed by FCM and the characteristics of apoptosis were present in substantially greater numbers confirmed by PI staining. CONCLUSION： Plasmid-mediated EGFR RNA interference could efficiently down-regulate EGFR expression and suppress A549 cell growth in vitro, and moreover, induce apoptosis which may serve as the mechanism of antitumor effect of pShEGFR.

关 键 词：癌 非小细胞肺 受体 表皮生长因子 RNA干扰 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]