Zhan Mei, Zhou Yuling, Han Zhong Chao
State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China.
Int J Hematol. 2004 May;79(4):400-4. doi: 10.1532/ijh97.a10310.
Plasminogen activator inhibitor type 1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease, myocardial infarction, and cerebrovascular events. Previous studies on variations in plasma PAI-1 levels and associations between PAI-1 levels and PAI-1 genotypes have suggested that PAI-1 expression maybe regulated in a genotype-specific manner by insulin, hypertriglyceridemic very low-density lipoprotein, and lipoprotein. We investigated whether basal transcription of the PAI-1 gene also is regulated in a genotype-specific manner. Allele-specific polymerase chain reaction-amplified fragments containing a 4G/5G polymorphism of the PAI-1 gene promoter were ligated into the chloramphenicol acetyltransferase (CAT) reporter gene. The constructs of p4G-CAT or pSG-CAT and pSV-beta-galactosidase as an internal control were transiently cotransfected into human HepG2 hepatoma cells. Electrophoresis mobility shift assays (EMSA) employed a fragment from positions -687 to -664 (4G allele) or from -688 to -664 (5G allele) labeled with adenosine triphosphate tagged with phosphorous 32 in the gamma position and used nuclear extracts of HepG2 cells. Analysis of CAT produced by constructs containing the PAI-1 4G or 5G allele showed similar 3-fold increases in CAT activity in the PAI-1 4G/4G and PAI-1 5G/5G constructs, compared with the CAT activity in the pCAT3-Basic construct. Analyses using the probes containing the 4G or 5G allele site in the EMSA assay revealed no difference in the binding of nuclear protein. Our in vitro assay of basal transcription suggests no difference in the transcriptional activities of the alleles of the PAI-1 4G/5G polymorphism.
1型纤溶酶原激活物抑制剂（PAI-1）已被证明是冠状动脉疾病、心肌梗死和脑血管事件的独立危险因素。先前关于血浆PAI-1水平变化以及PAI-1水平与PAI-1基因型之间关联的研究表明，PAI-1的表达可能受胰岛素、高甘油三酯血症极低密度脂蛋白和脂蛋白以基因型特异性方式调节。我们研究了PAI-1基因的基础转录是否也以基因型特异性方式受到调节。将含有PAI-1基因启动子4G/5G多态性的等位基因特异性聚合酶链反应扩增片段连接到氯霉素乙酰转移酶（CAT）报告基因中。将p4G-CAT或pSG-CAT以及作为内对照的pSV-β-半乳糖苷酶构建体瞬时共转染到人HepG2肝癌细胞中。电泳迁移率变动分析（EMSA）使用来自-687至-664位（4G等位基因）或-688至-664位（5G等位基因）的片段，该片段用γ位标记有32P的三磷酸腺苷标记，并使用HepG2细胞的核提取物。对含有PAI-1 4G或5G等位基因的构建体产生的CAT分析表明，与pCAT3-Basic构建体中的CAT活性相比，PAI-1 4G/4G和PAI-1 5G/5G构建体中的CAT活性有相似的3倍增加。在EMSA分析中使用含有4G或5G等位基因位点的探针进行分析，结果显示核蛋白的结合没有差异。我们对基础转录的体外分析表明，PAI-1 4G/5G多态性等位基因的转录活性没有差异。
