摘要
背景与目的:已证实非小细胞肺癌中表皮生长因子受体(epidermal growth factor receptor,EGFR)的过度表达和活化与肿瘤分期、放化疗敏感度下降密切相关。许多阻滞和下调EGFR的方法被用来抑制肿瘤增殖以改善预后,EGFR目前成为公认的肿瘤基因治疗的重要靶点。本研究采用靶向EGFR的短发夹RNA(short hairpinRNA,shRNA)表达重组质粒,观察能否在人肺腺癌细胞株A549细胞中引发RNA干扰(RNAi)效应及其对A549细胞生长及药物敏感性的影响。方法:构建靶向EGFR的shRNA重组质粒(pShEGFR)和非特异性的shRNA重组质粒(pShNEG),阳离子脂质体将其转染至肺腺癌A549细胞分别命名为A549/pShEGFR、A549/pShNEG和对照组A549,免疫荧光和Western blot法检测转染后细胞中EGFR表达变化;克隆形成实验观察细胞生长变化;流式细胞仪检测细胞周期和凋亡;MTT法检测细胞对顺铂、多柔比星、紫杉醇的敏感性。结果:与对照组A549细胞和A549/pShNEG相比,A549/pShEGFR可显著抑制A549细胞中EGFR表达,转染后第6天抑制率达74.1%(P<0.01);A549/pShEGFR组细胞克隆形成抑制率为62.8%。与对照组A549细胞和A549/pShNEG相比,A549/pShEGFR组细胞阻滞在G0/G1期(63.2±1.1,64.5±1.4vs.74.2±0.8;P<0.01),凋亡细胞比例显著增加(5.8±1.4,7.7±1.2vs.25.6±1.2;P<0.01);与A549组相比,A549/pShEGFR可将A549细胞对顺铂、多柔比星、紫杉醇的敏感性分别提高6.7、5.5、6.6倍。结论:瞬时转染靶向EGFR基因的shRNA表达重组质粒能够通过下调EGFR蛋白水平抑制肺腺癌A549细胞生长,并提高细胞对不同化疗药物的敏感性。
Background and purpose：Up-regulation of expression and activation
of epidermal growth factor receptor（EGFR）in non-small cell lung carcinoma（NSCLC）is associated with advanced tumor stage,and resistance to radiochemotherapy.EGFR has been identified as an important target in anticancer therapy by blocking EGFR function with the detailed understanding of the biology of EGFR protein and
its downstream signal pathway.In this study,we investigated the effects of short hairpin RNA（shRNA）targeting EGFR on cell growth and its sensitivity to cytotoxic drugs in lung adenocarcinoma A549 cell line.Methods：Recombined plasmids of short hairpin RNA targeting EGFR（pShEGFR）and of negative control shRNA（pShNEG）were respectively transfected into A549 cells,and named A549/pShEGFR,A549/pShNEG,and control cell group A549.The protein level of EGFR was observed by immunofluorescence,and then further quantified by Western blot.The effect of shRNA targeting EGFR on tumor cell growth was assessed by colony formation assay,cell cycle and apoptosis by flow cytometry,and the sensitivity of A549 to cytotoxic drugs by 3-4,5-dimethylthiozol-2yl-2,5-diphenyltetrazolium bromide MTT assay.Results：A549/pShEGFR significantly down-regulated EGFR expression by 74.1% on day 6 after transfection and the colony number by 62.8% in A549（P〈0.01）.Compared with the other two groups,A549/pShEGFR caused G1 arrest（63.2±1.1,64.5±1.4 vs.74.2±0.8;P〈0.01）,induced apoptosis（5.8±1.4,7.7±1.2vs.25.6±1.2;P〈0.01）,and subsequently increased the sensitivity to cisplatin,doxorubicin and paclitaxel by 6.7,5.5,6.6 fold when compared with recombined plasmid A549 respectively（P〈0.01）.Conclusions：Our data suggested that shRNA could effectively inhibit the growth and enhance the sensitivity of A549 cells to different cytotoxic drugs by downregulating the expression of EGFR gene.
出处
《中国癌症杂志》
CAS
CSCD
2007年第11期833-837,共5页
China Oncology
基金
中国博士后科学基金资助项目(2005037488)
上海市科委重大科技攻关项目(04DZ19208).
