shRNA干扰沉默ERCC1对A549/DDP细胞增殖与凋亡的影响

ERCC1-shRNA inhibits the proliferation and promotes the apoptosis of lung cancer A549/DDP cells

目的前期研究发现ERCC1基因可能参与顺铂继发耐药,文中旨在探讨短发夹RNA(shRNA)靶向沉默切除修复交叉互补基因组1(ERCC1)对不同浓度顺铂处理的肺腺癌顺铂耐药细胞株A549/DDP增殖和凋亡的影响。方法实验分为阴性对照组(shRNA)、空白对照组(lipofectamine 2000)、ERCC1-shRNA1组(ERCC1-shRNA1)、ERCC1-shRNA2组(ERCC1-shRNA2)。设计并合成靶向人的ERCC1基因shRNA1、shRNA2;采用脂质体lipofectamine 2000转染入A549/DDP细胞,通过Real-time PCR和Western blot分别检测转染前后ERCC1 mRNA和蛋白表达;MTT法检测转染后各组细胞增殖抑制率的变化;流式细胞术检测细胞周期和凋亡。结果成功构建ERCC1-shRNA并转入A549/DDP,ERCC1-shRNA1组、ERCC1-shRNA2组mRNA表达量[(0.20±0.04)、(0.47±0.28)]明显低于阴性对照组和空白对照组[(0.96±0.12)、(0.84±0.07)],差异均有统计学意义(P<0.01)。ERCC1-shRNA1组、ERCC1-shRNA2组蛋白表达量[(0.24±0.10)、(0.37±0.11)]明显低于阴性对照组和空白对照组[(1.32±0.13)、(1.45±0.23)],差异均有统计学意义(P<0.01)。ERCC1-shRNA1组IC50值[(7.78±0.54)]明显低于阴性对照组和空白对照组[(16.71±2.33)、(16.69±1.69)],差异均有统计学意义(P<0.01)。ERCC1-shRNA1组处于G0/G1期细胞比例[(82.99±4.23)%]较阴性对照组、空白对照组[(72.87±3.23)%、(71.75±4.56)%]明显增高,细胞周期阻滞于G0/G1期,差异有统计学意义(P<0.05)。6.25、12.5μg/mL顺铂作用后ERCC1-shRNA1组细胞凋亡率[(11.91±1.41)%、(29.97±3.14)%]较0μg/mL顺铂作用下[(8.17±0.67)%]明显升高(P<0.05)。结论 ERCC1-shRNA能有效沉默A549/DDP细胞中ERCC1基因的表达,细胞增殖显著受抑而凋亡明显增强。

Objective Studies show that the ERCC1 gene may be involved in secondary cisplatin resistance. This article aims to investigate the effects of shRNA targeting silencing excision repair cross-complementation group 1 （ERCCI-shRNA） on the pro- liferation and apoptosis of lung cancer A549/DDP cells treated with different concentrations of cisplatin. Methods Lung cancerA549/DDP cells were divided into a negative control, a blank control, an ERCCI-shRNA1, and an ERCCI-shRNA2 group. Human interfering RNA （RNAi） targeting the human ERCC1 gene was con- structed and transfected into the A549/DDP cells using Lipofectamine 2000. The mRNA and protein expressions of ERCC1 in the A549/ DDP cells were detected by real-time PCR and Western blot respec- tively, the proliferation-inhibition rate was assessed by MTF, andtheir cell cycle and apoptosis were determined by flow cytometry. Results ERCCI-shRNA was successfully constructed and trans- fected into the A549/DDP cells. Both the mRNA and protein expressions of ERCC1 were significantly lower in the ERCCI-shRNA1 （0.20±0.04 and 0.24±0.10） and ERCCI-shRNA2 （0.47±0.28 and 0.37±0.11 ） than in the negative control （0.96±0.12 and 1.32± 0.13） and blank control groups （0.84±0.07 and 1.45±0.23） （P〈0.01）. Compared with the negative and blank control groups, the ERCCI-shRNAI group showed a significantly decreased IC50 value （16.71±2.33 and 16.69±1.69 vs 7.78±0.54, P〈0.01 ） and an in- creased proportion of G0/G1 phase cells （ [72.87±3.23] and [71.75±4.56] vs [ 82.99±4.23] %, P〈0.05）, with the cell cycle arres- ted in the G0/G1 phase. The apoptosis rate of the cells in the ERCCI-shRNA1 group was remarkably lower after treated with cisplatin at the concentrations of 6.25 and 12.5 μg/mL than at 0 μg/mL （[8.17±0.65] and [ 11.91±1.41] vs [29.97±3.141%, P〈0.05）. Conclusion ERCCI-shRNA can inhibit the proliferation and enhance the apoptosis of A549/DDP cells by silencing the expression of the ERCC1 gene.