增殖及非增殖腺病毒载体介导的RNA干扰对肾癌细胞杀伤作用

Comparison of the anti-tumor effects of RNA interference mediated by replication-competent adenovirus and replication-deficient adenovirus expressing small interference RNA against Ki67 gene in renal carcinoma cells

目的比较荷载Ki67基因小于扰RNA(Ki67-siRNA)的增殖腺病毒ZD55-Ki67及非增殖腺病毒Ad-Ki67对肾癌细胞的杀伤作用。方法MOI=10的ZD55-Ki67、Ad-Ki67感染肾癌786-0细胞,2d后收集细胞,蛋白印迹法检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、蛋白印迹法、免疫细胞化学法检测Ki67表达;原位末端标记法(TUNEL)检测凋亡。4 d后噻唑蓝(MTT)法检测细胞存活率,7 d后结晶紫染色法检测细胞毒性作用。结果感染ZD55-Ki67的786-0细胞表达E1A,感染Ad-Ki67的细胞不表达E1A。ZD55-Ki67、Ad-Ki67感染的786-0细胞Ki67 mRNA表达率分别为(37.9±2.3)%、(64.1±1.9)%;Ki67蛋白表达率分别为(42.5±2.4)%、(60.1±2.2)%;Ki67染色阳性率分别为(20.8±2.8)%、(32.3±2.5)%;786-0细胞存活率分别为(22.2±3.0)%、(60.4±3.4)%;凋亡率分别为(53.0±3.7)%、(35.3±2.5)%,两种病毒处理之间差异均有统计学意义(P<0.01)。结论ZD55-Ki67抑制786-0细胞Ki67表达及增殖、诱导凋亡及细胞毒性作用均显著优于Ad-Ki67。增殖腺病毒介导的RNA干扰杀伤肾癌细胞作用优于非增殖腺病毒。

Objective To compare the anti-tumor effects of RNA interference mediated by replication-competent adenovirus （ ZD55-Ki67 ） and replication-deficient adenovirus （ Ad-Ki67 ） expressing small interference RNA targeting K/67 gene in renal carcinoma cells （RCC）. Methods Renal carcinoma 786-0 cells were infected with ZD55-Ki67 and Ad-Ki67 （ MOI = 10） respectively. The E1A expression of 786-0 cells was detected by Western blot. The Ki67 expression levels of 786-0 cells were detected by RTPCR and Western blot and immunocytochemistry respectively. Cell apoptosis was measured by TUNEL assay. Cell proliferation was assayed by MTT method. 786-0 cells were stained with crystal violet to assay tumor-selective cytotoxicity. Results Western blot assay of E1A expression indicated 786-0 cells infected with ZD55-Ki67 expressed El A, but the cells infected with Ad-Ki67 did not. In comparison with 786-0 cells infected with Ad-Ki67 ,the significant reductions of Ki67 mRNA （ 37.9 ± 2.3 vs 64.1 ± 1.9） and protein content （42.5 ± 2.4 vs 60. 1 ± 2.2） and the proportion of cells with immunoreactivity of Ki67 （20.8 ± 2.8 vs 32.3 ± 2.5 ） were observed in 786-0 cells infected with ZD55-Ki67. ZD55-Ki67 treatment resulted in the increased apoptotic cell death of 786-0 cells as compared with Ad-Ki67 treatment （53.0 ± 3.7 vs 35.3 ± 2.5 ）. Results of crystal violet stain and MTT assay revealed that the anti-tumor effect of ZD55-Ki67 was more potent than that of Ad-Ki67 （ 22.2 ± 3.0 vs 60.4 ± 3.4）. Conclusion ZD55- Ki67 can replicate selectively and mediate RNA interference in RCC. The anti-tumor effects of replication- competent adenovirus expressing Ki67-siRNA was better than that mediated by replication-deficient adenovirus expressing Ki67-siRNA.