Department of Haematology, Oslo University Hospital, Oslo, Norway.
Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway.
J Cell Biochem. 2019 Aug;120(8):12924-12936. doi: 10.1002/jcb.28563. Epub 2019 Mar 12.
Single nucleotide polymorphisms (SNPs) may play an important role in the risk of certain diseases. We have previously shown that the -287T/C SNP of the tissue factor pathway inhibitor (TFPI) gene promoter region exerts differential impact on TFPI mRNA expression; the C allele being associated with higher TFPI expression, which in turn is associated with reduced risk of thrombosis. In the present study, we aimed to reveal the underlying molecular mechanisms using human embryonic kidney 293 (HEK293) and Michigan Cancer Foundation-7 (MCF7) cells that both express TFPI. Transfecting the cells with luciferase reporter gene constructs containing the TFPI promoter with either the T or the C allele of -287T/C resulted in increased luciferase activity with the C allele relative to the T allele. Three potential candidate transcription factors for binding to the two -287 alleles were predicted using the ALGGEN PROMO software, and results from electrophoretic mobility shift assays indicated that forkhead box protein 3 (FOXP3), initially identified as a functional marker of T regulator cells, bound more specifically to the T allele compared with the C allele. By chromatin immunoprecipitation assays analysis it was confirmed that FOXP3 was able to bind to the DNA region that contains the SNP. Knockdown or overexpression of FOXP3 resulted in increased or decreased TFPI levels, respectively, in both cell types. In conclusion, this study indicates that FOXP3 most likely is involved in the increased levels of TFPI observed with the -287C allele and also that FOXP3 might be a repressor for TFPI expression.
单核苷酸多态性（SNPs）可能在某些疾病的风险中发挥重要作用。我们之前已经表明，组织因子途径抑制剂（TFPI）基因启动子区域的-287T/C SNP 对 TFPI mRNA 表达产生不同的影响；C 等位基因与更高的 TFPI 表达相关，而 TFPI 表达降低又与血栓形成的风险降低相关。在本研究中，我们旨在使用表达 TFPI 的人胚肾 293（HEK293）和密歇根癌症基金会-7（MCF7）细胞揭示潜在的分子机制。用含有 TFPI 启动子的荧光素酶报告基因构建体转染细胞，该启动子具有-287T/C 的 T 或 C 等位基因，导致 C 等位基因相对于 T 等位基因的荧光素酶活性增加。使用 ALGGEN PROMO 软件预测了三个可能与两个-287 等位基因结合的潜在候选转录因子，电泳迁移率变动分析的结果表明，叉头框蛋白 3（FOXP3）最初被鉴定为 T 调节细胞的功能标志物，与 C 等位基因相比，与 T 等位基因的结合更特异。通过染色质免疫沉淀分析证实 FOXP3 能够与包含 SNP 的 DNA 区域结合。在两种细胞类型中，FOXP3 的敲低或过表达分别导致 TFPI 水平增加或减少。总之，本研究表明，FOXP3 很可能参与了与-287C 等位基因相关的 TFPI 水平升高，并且 FOXP3 可能是 TFPI 表达的抑制剂。
