shRNA下调BAG-1表达降低肺癌A549细胞对顺铂的耐药性

Reduction of cisplatin resistance of lung cancer A549 cells through down-regula- ting the expression of BAG-1 mediated by shRNA

目的:通过载体介导的shRNA下调BAG-1基因(Bcl-2 associated athanogene-1)表达,探讨其对肺癌A549细胞顺铂(cisplatin,DDP)耐药性的影响。方法:构建靶向BAG-1的shRNA干扰载体pGCsi-BAG-1,稳定转染A549细胞。实验组使用稳定转染pGCsi-BAG-1的细胞株(BAG-1-shRNA),阴性对照组使用无关序列质粒转染的细胞株(SC-shRNA),对照组使用未转染的亲本A549细胞株(Control)。Western blotting检测pGCsi-BAG-1转染对A549细胞BAG-1、Bcl-2表达的影响。MTT法、流式细胞术分别检测pGCsi-BAG-1转染对DDP处理后A549细胞的增殖和凋亡的影响。结果:成功构建稳定干扰BAG-1表达的A549细胞株,BAG-1-shRNA组细胞中BAG-1和Bcl-2蛋白表达显著低于SC-shRNA组和对照组(均P<0.05)。随DDP(2.5~40μg/ml)浓度增加,各组细胞增殖抑制率也随之升高,DDP浓度为2.5μg/ml时,BAG-1-shRNA组A549细胞的增殖抑制率即显著高于SC-shRNA组和对照组[(22.26±4.89)%vs(10.07±3.82)%,(8.12±4.09)%,均P<0.05]。与SC-shRNA组和对照组相比,DDP(2.5μg/ml)处理24 h后,BAG-1-shRNA组凋亡率显著升高[(37.8 4±3.62)%vs(16.80±2.81)%、(17.10±3.11)%,P<0.05]。结论:下调BAG-1表达可抑制DDP作用下的A549细胞的增殖并促进其凋亡。

Objective：To detect down regulation of the expression of BAG-1 gene in lung cancer A549 by transfected a vector contained shRNA, and to investigate its effects on the cisplatin （DDP） resistance of A549 cells. Methods： Inter ference vector pGCsi-BAG-1 target BAG-1 was constructed and stable transferred into A549 cells. Cells stable transferred by pGCsi-BAG-1 were used as an experimental group （BAG-I-shRNA） , cells transferred by non-sense vector were used as a negative control group （SC-shRNA） , and the parent A549 cells were used as a control group. Western blotting was performed to detect the effect of pGCsi-BAG-1 transfection on the BCL-2 and BAG-1 expression of A549 cells. MTF meth od and flow cytometry was used to detect the influence on proliferation and apoptosis of A549 cells after DDP treatment. Results： An A549 cell line where BAG-1 was stably interfered was successfully constructed. And the expression of BCL 2 protein in cells of the BAG-l- shRNA group was significantly lower than that of SC-shRNA group and the control group （ all P 〈 0.05 ）. With increasing of the concentration of DDP, the proliferation inhibition rate of each cell was increased. When DDP concentration was 2.5 p^g,/ml, the cell proliferation inhibition rate of the BAG-I-shRNA group was significant ly higher than that of SC-shRNA group and the control group （ [ 8.12 ± 4.09 ] % vs [ 10.07± 3.82 ] %, [ 22.26 ± 4. 89 ] % ; all P 〈 0.05）. Compared with the SC-shRNA group and the control group, the apoptosis rate of the BAG-1- shRNA group was significantly increased after 24 h treatment with DDP （ [ 37.84 ±3.62 ] % vs [ 16.80 ±2.81 ] %, [ 17.10 ±3.11 ] % , P 〈 0.05 ）. Conclusion ： Down-regulation of BAG-1 expression can inhibit the proliferation of A549 cells after DDP treatment and promote its apoptosis.