白藜芦醇在调控二硫键氧化还原酶类似蛋白基因表达中的作用

Role of resveratrol in regulation of disulfide-bond A oxidoreductase-like protein gene transcription

目的探讨白藜芦醇对二硫键氧化还原酶类似蛋白(DsbA-L)基因转录的影响。方法将25μmol/L白藜芦醇加入HepG2细胞和3T3-L1细胞,干预24h后收获细胞,用于后续实验提取RNA和蛋白;以二甲基亚砜(DMSO)处理组作为对照。采用实时荧光定量聚合酶链反应(PCR)和Western印迹法检测3T3-L1细胞和HepG2细胞DsbA-L mRNA和蛋白表达水平。以荧光素酶报告基因实验分析白藜芦醇对DsbA-L基因启动子活性的影响。采用电泳迁移率变动分析法(EMSA)检测白藜芦醇对转录因子Sp1与DsbA-L基因内含子1区域Sp1结合位点的结合能力的影响。采用Western印迹法检测Sp1蛋白表达水平。结果在3T3-L1细胞和HepG2细胞中,白藜芦醇处理组DsbA-L蛋白和DsbA-L mRNA的表达水平均显著高于DMSO对照组(P值均<0.05)。白藜芦醇处理组的DsbA-L基因启动子报告基因质粒p(-186to+432)Luc的相对荧光素酶活性显著高于DMSO对照组(P<0.05);与包含该Sp1结合位点的DsbA-L基因启动子报告基因质粒p(-186to+432)Luc相比,不包含该Sp1结合位点的两个报告基因质粒,Sp1位点缺失突变的p(-186to+432)Luc mut和3’端部分缺失的p(-186to+391)Luc在白藜芦醇干预后的相对荧光素酶活性与DMSO对照组的差异均无统计学意义(P值均>0.05)。与DMSO对照组相比,白藜芦醇处理后转录因子Sp1与DsbA-L基因内含子区DNA特异结合形成的DNA-Sp1复合物的条带灰度减弱。白藜芦醇处理后3T3-L1细胞和HepG2细胞中Sp1的蛋白表达均显著低于DMSO对照组(P值均<0.05)。结论白藜芦醇通过抑制Sp1表达,上调DsbA-L基因的转录,从而促进DsbA-L蛋白的表达。

Objective To explore the potential mechanism of resveratrol-induced up-regulation of the disulfide-bond A oxidoreductase-like protein （DsbA-L） gene. Methods 3T3-L1 cells and HepG2 cells were cultured with resveratrol （25μmol/L） and harvested 24 h later. The cells treated with dimethyl sulfoxide （DMSO） were taken as controls. The DsbA-L mRNA and protein levels were detected by real-time quantitative polymerase chain reaction （PCR） and Western blotting analysis, respectively. The promoter activity of DsbA-L gene was detected by dual luciferase reporter assay system. The effect of resveratrol on the binding activity of transcription factor Spl to the DsbA-L intronic sequence was analyzed by electrophoretic mobility shift assay （EMSA）. The expression of Spl protein was determined by Western blotting analysis. Results The mRNA and protein levels of DsbA-L in resveratrol group were significantly higher than those in DMSO group （all P〈0. 05）; so was the luciferase activity of p〈- 186 to ＋ 432） Luc （ P〈0.05）. The luciferase activity of the plasmid which contained DsbA-L promoter and intron region encompassing a Spl-binding site was significantly increased after treated with resveratrol, whereas resveratrol had no effect on the luciferase activity of the reporter plasmids, which did not contain the Sp1 site. Furthermore, compared with the DMSO control, resveratrol reduced the intensity of the DNA- Spl complex which represented the binding of transcription factor Sp1 to the Spl-binding site of DsbA-L, as well as the protein level of Spl （both P〈0.05）. Conclusion Resveratrol may increase the expression of DsbA-L gene via inhibiting the expression of Spl.