siRNA沉默肺癌细胞中RECQL4基因表达诱导癌细胞凋亡的机制研究

siRNA-induces lung cancer cell apoptosis by silencing RECQL4 gene expression:a mechanistic study

目的探讨小干扰RNA(siRNA)沉默RECQL4基因对人肺癌A549细胞凋亡的影响及分子机制。方法采用qRT-PCR检测人支气管上皮HBE细胞及不同肺癌细胞A549、NCI-H1975和NCI-H1299细胞中RECQL4基因的表达情况。将RECQL4两条特异性siRNA分别转染至A549细胞中(分别记为si-RECQL4-1组和si-RECQL4-2组),转染无功能siRNA的A549细胞为阴性对照(NC组),未转染的A549细胞为空白对照(Blank组)。转染48 h后以qRT-PCR检测各组A549细胞中RECQL4 mRNA表达量,免疫印迹检测RECQL4、Bax、Bcl-2和Cleaved Caspase-3蛋白表达水平。AnnexinⅤ-FITC/PI双染色法检测细胞凋亡情况。DCFH-DA探针技术检测细胞中活性氧(ROS)含量,比色法检测细胞内丙二醛(MDA)还原型谷胱甘肽(GSH)的含量。结果与HBE细胞相比,A549、NCI-H1975和NCI-H1299细胞中RECQL4的表达量明显升高(P<0.05),A549细胞中RECQL4的表达量明显高于NCI-H1975和NCI-H1299细胞(P<0.05)。转染两种RECQL4 siRNA均能明显抑制A549细胞中RECQL4 mRNA和蛋白的表达(P<0.05),其中si-RECQL4-1组沉默效果更好。与Blank组相比,si-RECQL4-1组A549细胞中Bax和Cleaved Caspase-3蛋白表达及ROS和MDA含量明显升高(P<0.05),Bcl-2蛋白表达水平和GSH含量明显降低(P<0.05)。结论siRNA沉默肺癌A549细胞中RECQL4基因表达能够诱导细胞凋亡,其作用机制与调控凋亡相关蛋白表达及氧化应激有关。

Objective To investigate the effect of siRNA silencing RECQL4 gene on apoptosis of human lung cancer A549 cells and its molecular mechanism.Methods qRT-PCR was used to detect the expression of RECQL4 gene in human bronchial epithelial(HBE)cells and various lung cancer cell lines A549,NCI-H1975 and NCI-H1299.A549 cells were transfected with two specific siRNAs of RECQL4(si-RECQL4-1 and si-RECQL4-2 groups),and a non-functional siRNA(as negative control,NC group).Untransfected A549 cells were used as blank controls(blank group).At 48 h after transfection,the expression of RECQL4 mRNA in all groups of A549 cells was detected by qRT-PCR,the protein expression of RECQL4,Bax,Bcl-2 and cleaved caspase-3 by Western blotting,cell apoptosis by Annexin V-FITC/PI double staining,the content of reactive oxygen species(ROS)in cells by DCFH-DA probe technique,and the contents of malondialdehyde(MDA)and reduced glutathione(GSH)in cells by colorimetry.Results Compared with HBE cells,the expression of RECQL4 in A549,NCI-H1975 and NCI-H1299 cells was significantly increased(P<0.05).The expression of RECQL4 in A549 cells was significantly higher than that in NCI-H1975 and NCI-H1299 cells(P<0.05).Transfection of the two RECQL4 siRNAs significantly inhibited the mRNA and protein expression of RECQL4 in A549 cells(P<0.05),but the silencing effect was more prominent in the si-RECQL4-1 group.Compared with the blank group,the protein expression of Bax and cleaved caspase-3,the ROS and MDA contents in A549 cells of si-RECQL4-1 group were significantly increased(P<0.05),and the protein expression of Bcl-2 and GSH content were significantly decreased(P<0.05).Conclusion siRNA silencing of RECQL4 gene expression in lung cancer A549 cells may induce cell induce apoptosis.The underlying mechanism is related to the regulation of apoptosis-related protein expression and oxidative stress.