摘要
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 observe the inhibition of prostate cancer cell PC-3 by RNA interference on Aurora-A oncogene. Methods The expression of Aurora-A in prostate cancer cell was detected by immunohis- tochemical staining. The small interference RNAs（siRNAs） for Aurora-A oncogene was designed and synthesized in vitro, and was transfected into prostate cancer cell PC-3 by liposome. The transfection efficiency was detected by fluorescence microscope and flow cytometer. The cell inhibition rate was measured by MTT assay and apoptosis rate was measured by flow cytometer. The expression of Aurora-A protein and the expressions of Aurora-A mRNA were detected separately by the western blot and RT-PCR. Results Most Aurora-A proteins were found in the PC-3 cytoplasm. The transfection efficiency was more than90%. The 24th-hour, 48th-hour and 72th inhibition rates in siRNA1 group were （ -hour cell 0. 377 ± 0.035） %. （0. 597 ± 0. 065） % and（0. 749 ± 0. 061 ） % respectively. When it was compared with others groups, there were obvious difference. The RT-PCR detection showed that the relative value of Aurora-A mRNA was （1. 354±0. 087）, there were also obviously difference between the siRNA1 group and other groups. The expression of Aurora-A protein was obviously lower than in the control group. Conclusion RNA interference for Aurora-A oncogene can reduce the expression of Aurora-A mRNA and protein, inhibit cell prolif- eration and induce cell apoptosis.
出处
《山东医学高等专科学校学报》
2012年第1期1-4,F0003,共5页
Journal of Shandong Medical College
基金
山东省中青年科学家科研奖励基金项目(No.2006BS03036)
