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目的采用RNA干扰技术特异性阻断前列腺癌细胞PC-3中Aurora-A的表达,观察其对肿瘤细胞生长的抑制作用。方法采用免疫组化方法检测Aurora-A在前列腺癌细胞中的表达。体外化学合成特异针对Aurora-A基因的小干扰RNA(small interfering RNA,siRNA),以脂质体转染前列腺癌细胞系PC-3,荧光显微镜及流式细胞仪确定最佳转染效率。MTT法检测细胞增殖抑制率,流式细胞仪检测细胞凋亡率。RT-PCR和Western-blot检测Aurora-A mRNA和蛋白的表达。结果 Aurora-A蛋白主要表达于PC-3细胞胞浆中;脂质体的最佳转染效率高于90%;siR-NA1组24h、48h、72h细胞增殖抑制率分别为(0.377±0.035)%、(0.597±0.065)%、(0.749±0.061)%,与各组比较均有显著性差异;siRNA1组细胞凋亡率为(50.593±1.208)%,与各组比较均有显著性差异;RT-PCR检测siRNA1组Aurora-A mRNA相对值为(1.354±0.087),与各组比较均有显著性差异;Western-blot检测显示siRNA 1组蛋白表达量明显低于对照组。结论应用RNAi技术靶向沉默Aurora-A基因可以下调Aurora-A mRNA及蛋白的表达,抑制细胞增殖,促进细胞凋亡。
Objective To inhibit the expression of Aurora-A in PC-3 prostate cancer cells by RNAi technique and observe its inhibitory effect on tumor cell growth. Methods Immunohistochemistry was used to detect the expression of Aurora-A in prostate cancer cells. In vitro, small interfering RNA (siRNA) specific to Aurora-A gene was chemically synthesized, and the optimal transfection efficiency was determined by lipofection of prostate cancer cell line PC-3, fluorescence microscope and flow cytometry. The inhibitory rate of cell proliferation was detected by MTT assay and the apoptosis rate was detected by flow cytometry. The expression of Aurora-A mRNA and protein was detected by RT-PCR and Western-blot. Results Aurora-A protein was mainly expressed in the cytoplasm of PC-3 cells. The optimal transfection efficiency of liposomes was higher than 90%. The cell proliferation inhibition rate of siR-NA1 group was (0.377 ± 0.035) %, (0.597 ± 0.065)%, (0.749 ± 0.061)%, respectively. There was a significant difference between the two groups (P <0.05). The apoptosis rate of siRNA1 group was (50.593 ± 1.208)%, The relative value of Aurora-A mRNA in siRNA1 group was (1.354 ± 0.087) by RT-PCR, which showed significant difference compared with each group. Western-blot showed that the expression level of siRNA1 protein in group siRNA1 was significantly lower than that in control group. Conclusion Targeting silencing of Aurora-A by RNAi can downregulate Aurora-A mRNA and protein expression, inhibit cell proliferation and promote apoptosis.