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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 investigate the effect of Bcl-2 associated athanogene-1 (BAG-1) on the drug resistance of cisplatin (DDP) in lung cancer A549 cells by vector-mediated shRNA knockdown. Methods: shRNA targeting vector BAG-1 was constructed and transfected into A549 cells. In the experimental group, the cell line stably transfected with pGCsi-BAG-1 (BAG-1-shRNA) was used, the negative control group was transfected with the SC-shRNA without any relevant sequence, and the untransfected parental A549 cells Control. The effect of pGCsi-BAG-1 transfection on the expression of BAG-1 and Bcl-2 in A549 cells was detected by Western blotting. MTT assay and flow cytometry were used to detect the effect of pGCsi-BAG-1 transfection on the proliferation and apoptosis of A549 cells after DDP treatment. Results: The BAG-1 and Bcl-2 protein expression in BAG-1-shRNA group was significantly lower than that in SC-shRNA group and control group (all P <0.05). With the increase of DDP concentration (2.5 ~ 40μg / ml), the proliferation inhibition rate of each group also increased. When the DDP concentration was 2.5μg / ml, the proliferation inhibition rate of A549 cells in BAG-1-shRNA group was significantly higher than that in SC -shRNA group and control group [(22.26 ± 4.89)% vs (10.07 ± 3.82)%, (8.12 ± 4.09)%, all P <0.05]. Compared with SC-shRNA group and control group, the apoptosis rate of BAG-1-shRNA group was significantly increased after treated with DDP (2.5μg / ml) for 24 h [(37.8 4 ± 3.62)% vs (16.80 ± 2.81)% , (17.10 ± 3.11)%, P <0.05]. Conclusion: Down-regulation of BAG-1 can inhibit the proliferation of A549 cells induced by DDP and promote its apoptosis.