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目的 :研究沉默髓样细胞白血病1(myeloid cell leukemia-1,Mcl-1)基因表达对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)诱导人胃腺癌耐多柔比星(adriamycin,ADR)SGC7901/ADR细胞凋亡的影响,并探讨胃癌耐药细胞株耐T RAI L凋亡效应的可能机制。方法 :不同质量浓度的TRAIL(10、50和100 ng/m L)处理SGC7901/ADR细胞后,采用MTT法和FCM法分别检测细胞活力和细胞凋亡率,实时荧光定量PCR法和蛋白质印迹法分别检测SGC7901/ADR细胞中Mcl-1 m RNA和蛋白的表达水平。采用脂质体转染法将Mcl-1-si RNA转染至SGC7901/ADR细胞,实时荧光定量PCR和蛋白质印迹法检测Mcl-1-si RNA对Mcl-1 m RNA及蛋白表达水平的影响。采用TRAIL(50 ng/m L)处理Mcl-1基因沉默后的SGC7901/ADR细胞,MTT法和FCM法分别检测Mcl-1基因沉默后TRAIL对细胞增殖及凋亡率的影响,并进一步用蛋白质印迹法检测细胞色素C(cytochrome C,Cyt C)的表达水平以及凋亡相关蛋白caspase 3和caspase 9的激活情况。结果 :不同质量浓度的TRAIL(10、50和100 ng/m L)均能抑制人胃腺癌耐药株SGC7901/ADR细胞的增殖,并诱导其凋亡(P值均<0.05),但敏感性较低;TRAIL能明显提高SGC7901/ADR细胞中Mcl-1m RNA(P值均<0.001)和蛋白(P值均<0.05)的表达水平。Mcl-1-si RNA转染后,SGC7901/ADR细胞中Mcl-1 m RNA和蛋白表达均明显下调(P值均<0.001)。与阴性对照组(转染阴性对照-siR NA+TRAIL)相比,Mcl-1-siR NA转染后联合应用TRAIL组SGC7901/ADR细胞的增殖能力明显下降(P<0.001),细胞凋亡率明显增加(P<0.001),Cyt C、active-caspase 3和active-caspase 9蛋白的表达水平均明显增加(P值均<0.001)。结论 :TRAIL能上调胃癌耐药细胞株中抗凋亡蛋白Mcl-1表达,siR NA抑制Mcl-1表达可增强TRAIL对SGC7901/ADR细胞的致凋亡作用,提示胃癌耐药细胞株SGC7901/ADR耐TRAIL致凋亡效应可能与Mcl-1高表达有关。
Objective: To investigate the effect of silencing myeloid cell leukemia-1 (Mcl-1) gene on human gastric adenocarcinoma induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (ADR) SGC7901 / ADR cell apoptosis, and explore the possible mechanism of gastric cancer resistant T RAI L apoptosis effect. Methods: The viability and apoptosis rate of SGC7901 / ADR cells treated with various concentrations of TRAIL (10, 50 and 100 ng / m L) were determined by MTT and FCM respectively. Real-time PCR and Western blotting The expression of Mcl-1 m RNA and protein in SGC7901 / ADR cells were detected respectively. Mcl-1-si RNA was transfected into SGC7901 / ADR cells by lipofection method. The effect of Mcl-1-si RNA on Mcl-1 mRNA and protein expression was detected by real-time fluorescence quantitative PCR and Western blotting. The silencing of Mcl-1 gene by TRAIL (50 ng / m L) was used to treat SGC7901 / ADR cells. MTT and FCM were used to detect the effect of TRAIL on cell proliferation and apoptosis after Mcl-1 gene silencing. The expression of cytochrome C (Cyt C) and the activation of caspase 3 and caspase 9 were detected by Western blotting. Results: Different concentrations of TRAIL (10, 50 and 100 ng / m L) could inhibit the proliferation and induce the apoptosis of SGC7901 / ADR cells (all P <0.05), but the sensitivity TRAIL significantly increased the expression of Mcl-1mRNA (P <0.001) and protein (P <0.05) in SGC7901 / ADR cells. After Mcl-1-si RNA transfection, the expression of Mcl-1 mRNA and protein in SGC7901 / ADR cells were significantly down-regulated (all P <0.001). The proliferation of SGC7901 / ADR cells treated with Mcl-1-siR NA combined with TRAIL decreased significantly (P <0.001) compared with the negative control (transfected with negative control-siR NA + TRAIL) (P <0.001). The expressions of Cyt C, active-caspase 3 and active-caspase 9 were significantly increased (all P <0.001). CONCLUSION: TRAIL can up-regulate the expression of anti-apoptotic protein Mcl-1 in gastric cancer cell line SGC7901 / ADR induced by siR NA. The inhibitory effect of siR NA on the apoptosis of SGC7901 / ADR cell line SGC7901 / ADR TRAIL-induced apoptosis may be related to the high expression of Mcl-1.