表达Ki67-siRNA的溶瘤腺病毒抑制人肾癌ACHN细胞Ki67基因表达及增殖研究

Inhibition of Ki67 expression in human renal carcinoma ACHN cells by oncolytic adenovirus harboring specific Ki67-siRNA leads to suppression of proliferation and induction of apoptosis

目的:研究表达Ki67-siRNA的溶瘤腺病毒(ZD55-Ki67)对肾癌ACHN细胞Ki67基因表达及增殖抑制作用。方法:ZD55-Ki67、溶瘤腺病毒ZD55、表达Ki67-siRNA的增殖缺陷腺病毒Ad-Ki67感染人肾癌ACHN细胞。Western印迹法检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、Western印迹、免疫组织细胞化学法检测Ki67表达;原位末端标记法(TUNEL)检测细胞凋亡;MTT法检测细胞存活;结晶紫染色法检测细胞毒作用。结果:感染ZD55-Ki67、ZD55的ACHN细胞表达E1A;抑制Ki67表达作用依次为ZD55-Ki67>Ad-Ki67>ZD55。ZD55-Ki67、ZD55、Ad-Ki67感染的ACHN细胞凋亡率(%)分别为(56.3±3.1)、(25.3±2.5)、(37.0±3.0),均显著高于对照组(5.5±2.1)(P<0.01),每种病毒之间均有显著差异(P<0.01);存活率(%)分别为(40.9±2.2)、(71.3±6.6)、(86.8±3.1),每种病毒之间均有显著差异(P<0.01);对ACHN细胞的细胞毒作用ZD55-Ki67>ZD55>Ad-Ki67。结论:表达Ki67-siRNA的溶瘤腺病毒具有显著的抑制肾癌ACHN细胞Ki67基因表达、诱导凋亡、杀伤肾癌细胞作用。

Objective： To evaluate the effects of suppressing Ki67 gene expression on the cell proliferation and apoptosis of human renal carcinoma ACHN cells with oncolytic adenovirus ZD55, an E1B-55KD gene-deleted adenovirus, and ZD55-Ki67, a ZD55 armed with small in- terference RNA targeting Ki67 gene（Ki67-siRNA）.Methods： Renal carcinoma ACHN cells were infected with ZD55-Ki67,ZD55 and Ad-Ki67, the replication-deficient adenovirus expressing Ki67-siRNA, respectively. Ki67 expressing levels of ACHN cells were detected by RT-PCR, Western blot and immunocytocbemistry analysis. Cell apoptosis was measured by TUNEL assay. Cell proliferation was assayed by MTF method. ACHN cells were stained with crystal violet to assay tumor-selective cytotoxicity. Results： Western blot assay of E1A expression indicated ACHN cells infected with ZD55-Ki67 and ZD55 expressed E1A, but the cells infected with Ad-Ki67 did not. Significant reduction of Ki67 mRNA and protein content was observed in the lysates from ACHN cells infected with ZD55-Ki67. Similar significant reduction in the proportion of ACHN cells manifesting immunoreactivity for Ki67 was seen after infected with ZD55-Ki67.ZD55-Ki67 treatment of ACHN cells resulted in increased apoptotic cell death than ZD55 and Ad-Ki67 treatment. Results of crystal violet stain and MTr assay demonstrated that the antitumor effect of ZD55-KJ67 was more potent than both ZD55 and Ad-Ki67. Condusion：ZD55-Ki67 and ZD55 can replicate selectively in ACHN cells and result in cancer-specific cytotoxicity. The significant knockdown of Ki67 expression by ZD55-Ki67 treatment of ACHN cells is closely associated with apoptotic changes. The antitumor effect of ZD55-Ki67 is better than those of ZD55 and Ad-Ki67. Oncolytic adenovirus armed with siRNA against Ki67 gene may prove to be a useful novel tool for renal cancer therapy.