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目的:探讨NOTCH1基因沉默对人源性膀胱癌细胞增殖及分化的影响。方法:用pGenesil质粒构建靶向NOTCH1的shRNA真核表达载体psiRNA1并将其转染到膀胱癌细胞系BIU87中,四甲基偶氮唑盐微量酶反应比色法(MTT)、流式细胞术(FCM)检测NOTCH1沉默后膀胱癌细胞生长变化、细胞周期和凋亡情况。WesternBlot和RT-PCR法检测NOTCH1基因的蛋白和mRNA在转染前后表达变化。结果:转染psiRNA1质粒后BIU87细胞中NOTCH1基因在蛋白和mRNA表达水平相对于对照组明显下调(P<0.05),BIU87的生长受到明显抑制,呈现一定的时间依赖性(<60h),凋亡率增加,G_0/G_1期细胞明显增多,与对照组相比差异有统计学意义(P<0.05)。结论:沉默NOTCH1基因能够抑制膀胱癌细胞的增殖和分化,NOTCH1在膀胱癌中可能具有癌基因的作用。
Objective: To investigate the effect of NOTCH1 gene silencing on the proliferation and differentiation of human bladder cancer cells. Methods: The shRNA expression vector psiRNA1 targeting NOTCH1 was constructed by pGenesil plasmid and transfected into bladder cancer cell line BIU87. Methyl thiazolyl tetrazolium assay (MTT), flow cytometry (FCM) to detect the growth of bladder cancer cells after NOTCH1 silencing, cell cycle and apoptosis. The expression of NOTCH1 mRNA and protein was detected by Western Blot and RT-PCR before and after transfection. Results: The expression of NOTCH1 mRNA and protein in BIU87 cells was significantly downregulated (P <0.05), and the growth of BIU87 was significantly inhibited (P <0.05) The rate of G0 / G1 phase cells increased significantly compared with the control group (P <0.05). Conclusion: Silencing NOTCH1 gene can inhibit the proliferation and differentiation of bladder cancer cells. NOTCH1 may play an oncogene role in bladder cancer.