摘要
目的检测人前列腺癌细胞(PC-3M)中Omi／HtrA2的表达情况,并构建其小干扰 RNA(siRNA)表达载体,探讨Omi／HtrA2对PC-3M凋亡的影响。方法以细胞免疫组织化学和逆转录-聚合酶链反应(RT-PCR)法检测Omi／HtrA2在前列腺癌细胞系(PC-3M)和正常前列腺细胞中的表达。利用软件辅助设计Omi／HtrA2特异性siRNA序列,体外合成后将其克隆入真核表达载体 psiRNA-hHlneo。以脂质体法转染psiRNA-Omi／HtrA2载体至PC-3M中,以RT-PCR和Western blot法检测psiRNA-Omi／HtrA2对Omi／HtrA2转录和表达的沉默效应。用噻唑蓝(MTT)比色法和流式细胞法检测siRNA导致Omi／HtrA2基因沉默后,PC-3M细胞凋亡的变化。结果细胞免疫组织化学和RT-PCR均显示Omi／HtrA2在PC-3M细胞中高表达。酶切和DNA测序证实合成的siR- NA基因序列正确,并已被准确克隆入psiRNA-hHlneo载体中。psiRNA-Omi／HtrA2载体可特异性抑制PC-3M细胞中Omi／HtrA2的表达。转染psiRNA-Omi／HtrA2载体的PC-3M细胞凋亡下调。结论促凋亡因子Omi／HtrA2可导致前列腺癌细胞凋亡,Omi／HtrA2的表达对前列腺癌的发展有重要作用。
Objective To study the expression of Omi/HtrA2 in human prostate cancer cell line （PC-3M）, construct siRNA expressing vector and investigate the effect of Omi/HtrA2 on PC-3M apoptosis. Methods The expression of Omi/HtrA2 in human prostate cancer cell line （PC-3M） was detected by means of RT-PCR and immunohistochemical techniques.According to the software of Invivogen company, Omi/HtrA2 specific siRNA sequence was designed, synthesized in vitro and cloned into the expression vector psiRNA-hHlneo. The constructed psiRNA-Omi/HtrA2 was transiently transfected into human prostate cancer cell line （PC-3M） and the inhibitory effect of psiRNA-Omi/HtrA2 on the expression of Omi/HtrA2 in PC-3M cells was detected using RT-PCR and Western Blot. The apoptosis of cancer cells was determined by flow cytometry and MTT techniques. Results The expression levd of Omi/ HtrA2 was up-regnlated in PC-3M cells. Enzyme digestion analysis and DNA sequencing confirmed that the Omi/HtrA2 specific siRNA expression vector was constructed successfully. The apoptosis of PC-3M and the expression of Omi/HtrA2 were down-regulated in prostate cancer ceils after the transfection of psiRNA-Omi/HtrA2. Conclusion Omi/HtrA2 can result in the apoptosis of prostate cancer ceils. Omi/ HtrA2 expression might be involved in prostate cancer development.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第3期344-347,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30070756)
