Department of Laboratory Medicine and Pathobiology, University of Toronto, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 1 King's College Circle, Toronto, ON, Canada.
Oncol Rep. 2011 Mar;25(3):809-15. doi: 10.3892/or.2010.1129. Epub 2010 Dec 30.
Defective mismatch repair leads to the microsatellite instability (MSI) phenotype of colorectal cancer (CRC). We previously showed that the MLH1-93G>A promoter polymorphism is strongly associated with MSI tumours, suggesting a modifier role for this polymorphism in CRC. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. In order to evaluate the functional effects of this polymorphism, we transfected a panel of CRC, endometrial cancer and non-tumourigenic cell lines with MLH1 luciferase promoter constructs. We used constructs in reverse orientation to assess the effect of this polymorphism on EPM2AIP1. The luciferase activities were compared using a two-sided Student's t-test. Electrophoretic mobility shift assays (EMSAs) were used to evaluate whether differential protein binding was responsible for the differences in promoter activity. We observed a higher level of activity with the -93G allele in all the cell lines observed; including the CRC cell line, HCT116 (P=0.002), the endometrial cancer cell line, HEC-1-A (P<0.001) and the normal colonic cell line, CCD-841-CoTr (P=0.002). This polymorphism also affected EPM2AIP1 transcription with the -93A allele demonstrating higher promoter activity in the HCT116 (P=0.007) and HEC-1-A (P=0.004) cells. The EMSA results suggest that this polymorphism alters the affinity of nuclear factors that bind to this region. Our findings indicate that the -93G>A polymorphism modifies the efficiency of MLH1/EPM2AIP1 transcription.
错配修复缺陷导致结直肠癌（CRC）的微卫星不稳定（MSI）表型。我们之前表明，MLH1-93G>A 启动子多态性与 MSI 肿瘤强烈相关，提示该多态性在 CRC 中具有修饰作用。MLH1 启动子是双向的，EPM2AIP1 基因位于反义链上。为了评估该多态性的功能影响，我们用 MLH1 荧光素酶启动子构建体转染了一系列 CRC、子宫内膜癌和非肿瘤细胞系。我们使用反向方向的构建体来评估该多态性对 EPM2AIP1 的影响。使用双侧学生 t 检验比较荧光素酶活性。电泳迁移率变动分析（EMSA）用于评估差异蛋白结合是否是导致启动子活性差异的原因。我们观察到所有观察到的细胞系中，-93G 等位基因的活性水平更高；包括 CRC 细胞系 HCT116（P=0.002）、子宫内膜癌细胞系 HEC-1-A（P<0.001）和正常结肠细胞系 CCD-841-CoTr（P=0.002）。该多态性也影响 EPM2AIP1 的转录，-93A 等位基因在 HCT116（P=0.007）和 HEC-1-A（P=0.004）细胞中表现出更高的启动子活性。EMSA 结果表明，该多态性改变了与该区域结合的核因子的亲和力。我们的研究结果表明，-93G>A 多态性改变了 MLH1/EPM2AIP1 转录的效率。
