靶向沉默异黏蛋白基因对膀胱癌细胞凋亡的影响及其作用机制

Effect of siRNA-mediated silencing metadherin on apoptosis of bladder cancer cells and its mechanisms

目的探讨siRNA干扰技术靶向沉默异黏蛋白(metadherin,MTDH)基因对膀胱癌细胞凋亡的影响及其作用机制。方法采用RT-PCR和Western blot分别检测膀胱上皮永生化SV-HUC-1细胞和人膀胱癌BIU-87细胞中MTDH mRNA及其蛋白表达;利用脂质体转染MTDH-siRNA靶向沉默MTDH基因,以RT-PCR和Western blot检测MTDH的沉默效果及B细胞淋巴瘤/白血病-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、p53和磷酸化Akt(p-Akt)的表达;流式细胞仪检测MTDH基因沉默对BIU-87细胞凋亡的影响。结果 MTDH基因在人膀胱癌BIU-87细胞中m RNA表达(0.82±0.05 vs 0.25±0.03)和蛋白表达(0.42±0.06 vs 0.18±0.05)明显高于膀胱上皮永生化SV-HUC-1细胞(P均<0.05);与NC组相比,siRNA MTDH2组细胞中MTDH2 mRNA(0.31±0.03 vs 1.02±0.03)和蛋白(0.29±0.02 vs 0.48±0.05)的表达、siRNA MTDH3组中MTDH3mRNA(0.22±0.05 vs 1.02±0.03)和蛋白(0.18±0.08 vs 0.48±0.05)的表达显著降低(P均<0.05),且siRNA MTDH3组的降低幅度大于siRNA MTDH2组;siRNA NC组及siRNA MTDH1组差异无统计学意义(P>0.05);靶向沉默MTDH基因后,与NC组相比,siRNA MTDH组BIU-87细胞的凋亡率(38.86%±2.52%vs 9.95%±1.26%)、p53(1.58±0.07 vs 0.61±0.04)及Bax蛋白(0.86±0.06 vs 0.43±0.03)的表达显著升高,p-Akt(0.31±0.03 vs 0.75±0.08)和Bcl-2蛋白(0.35±0.08 vs 1.26±0.12)的表达显著下降,差异有统计学意义(P均<0.05),NC组与siRNA NC差异无统计学意义(P>0.05)。结论 MTDH基因沉默可能通过上调p53、Bax和下调p-Akt、Bcl-2蛋白的表达诱导BIU-87细胞凋亡。

Objective To investigate the effect of siRNA-mediated silencing metadherin （MTDH） gene on apoptosis of bladder cancer cells and its mechanisms. Methods RT-PCR and Western blot were used to detect the expression of MTDH mRNA and protein in bladder epithelial immortalized SV-HUC-1 cells and human bladder cancer BIU-87 cells. MTDH gene was targeted silenced by liposomal transfection of MTDH-siRNA, and the silencing effect of MTDH and expression of B cell lymphoma/leukemia -2 gene （Bcl-2）, Bcl-2 related X protein （Bax）, p53 and phosphorylated Akt （p-Akt） were detected by RT-PCR and Western blot. The effect of MTDH gene silencing on apoptosis of BIU-87 cells was detected by flow cytometry. Results The mRNA and protein expressions of MTDH gene in human bladder cancer BIU-87 cells were significantly higher than those in bladder epithelial immortalized SV- HUC-1 cells [（0.82 ± 0.05） vs （0.25 ± 0.03）, （0.42 ± 0.06） vs （0.18 ± 0.05）; P 〈 0.05, respectively]. Compared with NC group, the mRNA and protein expressions of MTDH2 in cells of siRNA MTDH2 group and their expressions of MTDH3 in cells of siRNA MTDH3 group decreased significantly [MTDH2：（0.31 ± 0.03） vs （1.02 ± 0.03）, （0.29 ± 0.02） vs （0.48 ± 0.05）; MTDH3：（0.22 ± 0.05） vs （1.02 ± 0.03）, （0.18 ± 0.08） vs （0.48 ± 0.05）; all P 〈 0.05], and the decrease in siRNA MTDH3 group was greater than that in siRNA MTDH2 group; While, there was no significant difference between siRNA NC group and siRNA MTDH1 group （P 〉 0.05）. After silencing of MTDH, compared with NC group, the apoptosis rate of BIU-87 cells [（38.86% ± 2.52%） vs （9.95% ± 1.26%）], p53 [（1.58 ± 0.07） vs （0.61 ± 0.04）] and Bax protein [（0.86 ± 0.06） vs （0.43 ± 0.03）] in siRNA MTDH group were significantly higher, while p-Akt protein [（0.31 ± 0.03） vs （0.75 ± 0.08）] and Bcl-2 protein [（0.35 ± 0.08） vs （1.26 ± 0.12）] decreased significantly, and the differences were all statistically significant （all P 〈 0.05）, but the difference between NC group and siRNA NC was not significant （P 〉 0.05）. Conclusion MTDH gene silencing may induce apoptosis of BIU-87 cells by up regulating the expression of p53, Bax protein and down regulating the expression of p-Akt, Bcl-2 protein.