摘要
背景与目的:促、抑凋亡因子间的相互作用与肿瘤的发生、发展密切相关。Omi/HtrA2是新近发现的一种凋亡调节因子,PED/PEA-15是一种广泛表达的抗凋亡蛋白。本研究旨在探讨Omi/HtrA2对PED/PEA-15表达和前列腺癌细胞PC-3凋亡的影响。方法:构建Omi/HtrA2的表达载体和siRNA载体,并用脂质体法分别将两载体转染至PC-3细胞中,Westernblot和ELISA法检测Omi/HtrA2对PED/PEA-15表达和细胞凋亡的影响;Caspase-8检测试剂盒检测PED/PEA-15对Caspase-8活性的影响;Westernblot、RT-PCR法检测Omi/HtrA2特异siRNA序列对其转录、翻译的影响,流式细胞仪检测siRNA导致Omi/HtrA2基因沉默后PC-3细胞凋亡的变化。结果:酶切和DNA测序证实Omi/HtrA2的表达载体和siRNA载体构建成功。通过转染Omi/HtrA2表达载体高表达Omi/HtrA2可抑制PED/PEA-15表达,并增加肿瘤细胞的凋亡率;抑制PED/PEA-15的表达可提高Caspase-8活性。siRNA沉默Omi/HtrA2基因后PC-3细胞对顺铂的敏感性降低。结论:Omi/HtrA2可通过抑制抗凋亡蛋白PED/PEA-15表达而在PC-3细胞凋亡中发挥重要作用。
BACKGROUND ＆ OBJECTIVE： The interaction between pro- apoptotic factors and anti-apoptotic factors is closely related to the genesis and development of tumors. Omi/HtrA2 is a novel gene involved in the regulation of apoptosis. PED/PEA-15 is a widely expressed anti-apoptotic protein. This study was to explore the effects of Omi/HtrA2 on PED/PEA-15 expression and apoptosis of prostate cancer cell line PC-3. METHODS： Omi/ HtrA2 expression and specific siRNA vectors were constructed and transiently transfected into PC-3 cells. The effect of Omi/HtrA2 on PED/PEA-15 expression was assayed by Western blot, and its effect on apoptosis of PC-3 cells was analyzed by ELISA. Caspase-8 activity was assayed using Caspase-8 colorimetric assay kit. The effects of Omi/HtrA2-specific siRNA sequence on its transcription and translation were analyzed by reverse transcription- polymerase chain reaction （RT-PCR） and Western blot. The sensitivity of PC-3 cells to cisplatin （DDP） after Omi/HtrA2 gene silencing was determined by flow cytometry. RESULTS： Enzyme digestion analysis and DNA sequencing confirmed Omi/HtrA2 expression, and specific siRNA vectors were successfully constructed. After transfection of Omi/HtrA2 expression vector, PED/PEA-15 expression was inhibited, Caspse-8 activity was promoted, and the apoptosis of PC-3 cells was enhanced. The sensitivity of PC-3 cells to DDP was suppressed after Omi/HtrA2 gene silencing. CONCLUSION： Omi/HtrA2 can promote the apoptosis of PC-3 cells through inhibiting PED/PEA-15 expression.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2006年第6期677-682,共6页
Chinese Journal of Cancer
基金
国家自然科学基金资助项目(No.30070756)~~
