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

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

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—hH1 neo。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、500μg／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，且准确克隆人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斗g／L）对转染psiRNA-Omi／HtrA2载体后PC．3M细胞A1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。

Objective To study the apoptosis of human prostate cancel cell line （PC-3M） at different 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 using 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 into the expression vector psiRNA-hH1 neo. 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 apoptosis index （AI） of PC-3M cells was determined by in situ end labeling techniques under different TRAIL concentrations （50, 100,200 and 500μg/L）. Results There was high level of the Omi/HtrA2 expression in PC 3M cells. Enzyme digestion analysis and DNA sequencing confirmed the 0mi/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 respectively, 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 induce the apoptosis of PC-3M cells concentration-dependently, and its effect was correlated with the expression level of omi/HtrA2.