小干扰RNA对肾癌细胞Ki67基因表达及其增殖的抑制作用

Effects of siRNA against Ki67 gene on the proliferation and apoptosis of human renal carcinoma cells

目的探讨小干扰RNA(siRNA)对人肾癌细胞增殖基因Ki67表达及其增殖、凋亡的影响。方法将Ki67 siRNA(100 nmol/L)转染人肾癌786-0细胞。采用RT-PCR、免疫印迹、免疫组化技术检测Ki67 mRNA及蛋白表达,MTT法检测细胞增殖,免疫组化TUNEL法检测细胞凋亡。结果Ki67 siRNA处理组786-0细胞Ki67 mRNA表达(37．6±1．9)%、Ki67蛋白表达(46．4±0．9)%、免疫组化Ki67表达吸光度(A)值52．5±2．3,阴性siRNA对照组分别为(97．3±0．9)%、(95．3±0．9)%、114．5±4．9,2组比较差异均有统计学意义(P＜0．01)。Ki67 siRNA处理组细胞增殖抑制率(63．6±1．6)%、凋亡细胞阳性率(41．7±0．6)%,阴性siRNA对照组分别为(2．8±0．2)%、(10．3±1．4)%,2组比较差异有统计学意义(P＜0．01)。结论肿瘤增殖基因Ki67 siRNA能抑制人肾癌786-0细胞Ki67基因表达,进而抑制其增殖,促进其凋亡,有望成为肾癌基因治疗的有效工具。

Objective To evaluate the effects of small interfering RNA（siRNA）against Ki67 gene on the proliferation and apoptosis of human renal carcinoma cell line 786-0 cells. Methods The human renal carcinoma 786-0 cells were treated with Ki67-siRNA （ 100 nmol/L）. The mRNA expression of Ki67 was detected by RT-PCR. The protein expression of Ki67 was detected by Western blot and immunohistochemical technique, respectively. The proliferation of 786-0 cells was detected by MTT assay. The apoptosis of 786-0 cells was detected by TUNEL assay. Results RT-PCR and Western blot analysis showed that the Ki67 mRNA and Ki67 protein expression levels of the 786-0 cells treated with Ki67-siRNA were （ 37.6 ± 1.9） % and （46.4 ± 0.9 ） %, respectively, which were significantly lower than those of controls [ （ 97.3 ± 0.9） % and （95. 3 ± 0.9） %, P 〈 0.01 1. The Ki67 positive expression rate of 786-0 cells treated with Ki67-siRNA by immunohistochemical technique was 52.5 ± 2.3, which was significantly lower than that of controls （ 114.5 ± 4.9 ,P 〈 0.01 ）. The proliferation-inhibiting rate and apoptosis rate of the 786-0 cells trea- ted with Ki67-siRNA were （ 63.6 ± 1.6） % and （ 41.7 ± 0.6 ） %, respectively, which were significantly higher than those of controls [（2.8±0.2）% and （10.3±1.4）%,P〈0.011. Conclusions siRNA against Ki67 gene can inhibit the proliferation and induce the apoptosis by blocking Ki67 expression of human renal carcinoma 786-0 cells. The inhibition of Ki67 expression by siRNA may be a promising approach in gene therapy for renal cancer.