肿瘤坏死因子相关凋亡诱导配体对Omi/HtrA2不同表达水平前列腺癌细胞的促凋亡作用

目的 观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)对前列腺癌细胞(PC-3M)不同丝氨酸蛋白酶Omi/HtrA2表达水平的促凋亡作用.方法 构建其小分子干扰RNA(siRNA)表达载体,辅助设计Omi/HtrA2特异性siRNA序列.合成后克隆入真核表达载体psiRNA-hH1neo.脂质体法转染psiRNA-Omi/HtrA2载体至PC-3M中,检测Omi/HtrA2在PC-3M细胞中的表达及psiRNA-Omi/HtrA2对Omi/HtrA2沉默效应后的转录和表达.用原位末端转移酶标记技术检测Omi/HtrA2基因沉默后,计算不同浓度TRAIL(50、100、200、500 μg/L)下PC-3M细胞凋亡指数(AI).结果 Omi/HtrA2在PC-3M细胞中高表达.酶切和DNA测序证实siRNA基因序列正确,且准确克隆入psiR-NA-hH1neo载体中.psiRNA-Omi/HtrA2载体可特异性抑制PC-3M细胞中Omi/HtrA2的表达.不同浓度TRAIL(50、100、200、500 μg/L)对转染psiRNA-Omi/HtrA2载体后PC-3M细胞AI分别为7.23、14.87、22.65、31.78.而未转染组分别为15.28、24.17、36.33、47.76.两组之间差异有统计学意义(P<0.05).结论 Omi/HtrA2在前列腺癌细胞凋亡过程中起重要作用,TRAIL促进前列腺癌细胞的凋亡,其效果与TRAIL浓度及Omi/HtrA2表达水平相关.

The apoptosis of human prostate cancel cell line (PC-3M) at different levels of Omi/HtrA2 when treated with TRAIL

Objective To study the apoptosis of human prostate cancel cell line (PC-3M) at dif-ferent levels of Omi/HtrA2 when treated with TNF-related apoptosis-inducing ligand (TRAIL). Methods siRNA expressing vector was constructed. The expression of omi/HtrA2 in PC-3M cells was detected by u-sing reverse transcription-polymerase chain reaction (RT-PCR) techniques. According to the software of Invivogen company, Omi/HtrA2 specific siRNA sequence was designed, synthesized in vitro and cloned in-to the expression vector psiRNA-hH1neo. The constructed psiRNA-Omi/HtrA2 was transiently transfected into PC-3M cells 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 blotting. The apoptesis index (AI) of PC-3M cells was determined by in situ end labeling techniques under different TRAIL concentrations (50, 100,200 and 500 μg/L). Results There was high level of the Omi/HtrA2 expression in PC 3M cells. Enzyme digestion a-nalysis and DNA sequencing confirmed the Omi/HtrA2 specific siRNA expression vector was constructed successfully. The expression of Omi/HtrA2 was down-regulated in PC-3M cells after the transfection of psiRNA-Omi/HtrA2. The AI of PC-3M cells in transfection group was 7. 23, 14. 87, 22. 65 and 31.78 re-spectively, and that in un-transfection group was 15.28, 24. 17, 36. 33 and 47. 76, respectively (P < 0. 05). Conclusion Omi/HtrA2 plays an important role in the apoptosis of PC-3M cells. TRAIL can in-duce the apoptosis of PC-3M cells concentration-dependently, and its effect was correlated with the expres-sion level of omi/HtrA2.