Prohibitin启动子中胆固醇活性作用位点研究

 目的：验证抑制素（prohibitin，PHB）基因启动子胆固醇敏感活性作用位点，为PHB在前列腺癌靶向基因治疗中的应用提供实验依据。方法：采用PCR分段扩增PHB启动子，克隆入pGL3-Basic萤光素酶报告基因载体。将构建的重组质粒用脂质体转染至人前列腺癌PC-3细胞，再分别培养于低胆固醇培养基和普通胆固醇培养基中，检测PHB启动子各分段重组质粒萤光素酶活性的变化。限定PHB启动子约200 bp的胆固醇敏感启动区域，找出其中的固醇调节元件（sterol regulatory element，SRE）同源位点并进行点突变，证实PHB基因的胆固醇敏感调控位点。结果：PHB启动子和各分段产物构建质粒完整。与转染完整PHB启动子（pPHB-1192）的PC-3细胞比较，转染pPHB-179 (-35/+138)的PC-3细胞萤光素酶活性显著降低（P<0.05）。点突变位于PHB启动子上的-117到-108 bp的SRE可能位点后，与转染pPHB-1192的PC-3细胞比较，转染SRE点突变的PC-3细胞萤光素酶活性显著降低（P<0.05）。结论：PHB启动子在人前列腺癌PC-3细胞中的活性受胆固醇调节。PHB启动子中对胆固醇敏感的作用位点位于-117到-108 bp的SRE同源位点。PHB可能成为前列腺癌靶向基因治疗的有效工具。

Cholesterol-sensitive action sites in promoter of prohibitin gene

AIM: To verify the cholesterol-sensitive sites in the promoter of prohibitin (PHB) gene. METHODS: A series of successive PHB promoter truncated segments were amplified by PCR and cloned into pGL3-Basic luciferase reporter vector. Human prostate cancer PC-3 cells were transfected with the recombinant plasmids and then cultured in normal medium (NM) or cholesterol-depleted medium (CDM). The activity of PHB promoter was detected by luciferase activity analysis to find the cholesterol-sensitive region of approximately 200 bp in PHB promoter. Point mutations in sterol regulatory element (SRE) homologous loci of this 200　bp region were made to confirm the cholesterol-sensitive role of this SRE site. RESULTS: Plasmids with PHB promoter segments were successfully constructed. Luciferase activity in PC-3 cells transfected with PHB promoter segment pPHB-179 (-35/+138) plasmid was significantly decreased compared with the cells transfected with the whole PHB promoter pPHB-1192 plasmid (P<0.05). After point mutation was made in the SRE homologous locus located at -117 to -108 bp and this plasmid was transfected into PC-3 cells, the luciferase activity in these cells was significant decreased compared with the cells transfected with pPHB-1192 plasmid (P<0.05). CONCLUSION: Cholesterol regulates the activity of PHB promoter in human prostate cancer PC-3 cells. The cholesterol-sensitive site in PHB promoter is located at -117 to -108 bp of SRE homologous locus. PHB may become a new target of gene therapy for prostate cancer.