论文部分内容阅读
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。
OBJECTIVE: To verify the site of cholesterol-sensitive activity of the promoter of prohibitin (PHB) gene and provide an experimental basis for the application of PHB in targeted gene therapy of prostate cancer. Methods: PHB promoter was amplified by PCR and cloned into pGL3-Basic luciferase reporter vector. The constructed recombinant plasmids were transfected into human prostate cancer PC-3 cells by liposome, and then cultured in low cholesterol medium and normal cholesterol medium respectively to detect the luciferase activity of each segmented recombinant plasmid of PHB promoter Variety. The promoter region of PHB, which is about 200 bp, was defined. The sterol regulatory element (SRE) homologous sites were identified and mutated. The results showed that the PHB gene was involved in the cholesterol-sensitive regulatory region. Results: The PHB promoter and each fragment product were constructed into plasmids. PC-3 cells transfected with pPHB-179 (-35 / +138) had significantly lower luciferase activity (P <0.05) compared to PC-3 cells transfected with the complete PHB promoter (pPHB-1192) PC-3 cells transfected with SRE point mutation had significantly higher luciferase activity compared to PC-3 cells transfected with pPHB-1192 after point mutations were located on the PHB promoter at a possible SRE of -117 to -108 bp Decreased (P <0.05). Conclusion: The activity of PHB promoter in human prostate cancer PC-3 cells is regulated by cholesterol. The cholesterol-sensitive site of action in the PHB promoter is located at the-117 to -108 bp SRE homology sites. PHB may be an effective tool for targeted gene therapy of prostate cancer.