靶向表皮生长因子受体基因的短发夹RNA对肺腺癌A549细胞生长及药物敏感性的影响

Effects of transient transfection of short hairpin RNA targeting EGFR into lung adenocarcinoma A549 cell line on cell growth and sensitivity to cytotoxic drugs

目的探讨瞬时转染靶向表皮生长因子受体(EGFR)基因的短发夹RNA(shRNA)表达载体对肺腺癌A549细胞生长及药物敏感性的影响。方法构建靶向EGFR的shRNA载体(pShEGFR)和非特异性的shRNA载体(pShNEG),阳离子脂质体将其转染至肺腺癌A549细胞,免疫荧光和Western blot法检测转染后细胞中EGFR蛋白表达变化;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;MTI法检测细胞对顺铂、阿霉素、泰素的敏感性。结果与未转染组和pShNEG转染组细胞相比,pShEGFR可显著抑制A549细胞中EGFR蛋白表达,转染后第6天抑制率达74.1%(P<0.01);pShEGFR转染组细胞克隆形成抑制率为62.8%。与未转染组和pShNEG组相比,pShEGFR组细胞阻滞在G_0/G_1期(P<0.01),凋亡细胞比例显著增加(P<0.01);与未转染组相比,pShEGFR可将A549细胞对顺铂、阿霉素、泰素的敏感性分别提高6.7、5.5、6.6倍。结论瞬时转染靶向EGFR基因的shRNA表达载体能够通过下调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。

Objective To determine the effects of vector-based short hairpin RNA （shRNA） targe-ting EGFR on cell growth and sensitivity to cytotoxic drugs in lung adenocarcinoma A549 cell line. Methods Vector-based short hairpin RNA targeting EGFR （pShEGFR） and negative control shRNA （pShNEG） were respectively transfected into A549 cells. The protein level of EGFR was observed by immunofluores-cence, 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 cytotoxie drugs by 3- [ 4,5-dimethylthiozol-2 yl ] -2,5-diphenyhetrazolium bromide [ MTT] assay. Results Vectors expressing shRNA against EGFR significantly down-regulated the receptor expression by 74.1% on day 6 after transfection and the colony number by 62.8% in A549 （ P 〈 0.01 ）. Vector-based shRNA against EGFR caused G1 arrest, induced apoptosis, and subsequently increased the sensitivity to eisplatin, doxorubicin and paclitaxel by 6.7, 5.5, 6.6 fold in A549, respectively （ P 〈 0. 01 ）. Conclusion Vector-based shRNA may effectively inhibit the growth and enhance the sensitivity of A549 cells to cytotoxic drugs through downregulation of EGFR gene expression.