Single nucleotide polymorphisms in the <i>MYLKP1</i> pseudogene are associated with increased colon cancer risk in African Americans

Introduction Pseudogenes are paralogues of functional genes historically viewed as defunct due to either the lack of regulatory elements or the presence of frameshift mutations. Recent evidence, however, suggests that pseudogenes may regulate gene expression, although the functional role of pseudogenes remains largely unknown. We previously reported that MYLKP1, the pseudogene of MYLK that encodes myosin light chain kinase (MLCK), is highly expressed in lung and colon cancer cell lines and tissues but not in normal lung or colon. The MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. In this study, we further validate MYLKP1 as an oncogene via elucidation of the functional role of MYLKP1 genetic variants in colon cancer risk. Methods Proliferation and migration assays were performed in MYLKP1-transfected colon and lung cancer cell lines (H441, A549) and commercially-available normal lung and colon cells. Fourteen MYLKP1 SNPs (MAFs >0.01) residing within the 4 kb MYLKP1 promoter region, the core 1.4 kb of MYLKP1 gene, and a 4 kb enhancer region were selected and genotyped in a colorectal cancer cohort. MYLKP1 SNP influences on activity of MYLKP1 promoter (2kb) was assessed by dual luciferase reporter assay. Results Cancer cell lines, H441 and A549, exhibited increased MYLKP1 expression, increased MYLKP1 luciferase promoter activity, increased proliferation and migration. Genotyping studies identified two MYLKP1 SNPs (rs12490683; rs12497343) that significantly increase risk of colon cancer in African Americans compared to African American controls. Rs12490683 and rs12497343 further increase MYLKP1 promoter activity compared to the wild type MYLKP1 promoter. Conclusion MYLKP1 is a cancer-promoting pseudogene whose genetic variants differentially enhance cancer risk in African American populations.引言 假基因（Pseudogenes）是功能基因的旁系同源基因，既往因缺乏调控元件或携带移码突变而被认为是失活的。然而，近年研究证据表明，假基因可能参与调控基因表达，但其具体功能仍在很大程度上未明确。我们先前的研究报道，编码肌球蛋白轻链激酶（myosin light chain kinase，MLCK）的MYLK基因的假基因MYLKP1，在肺癌、结肠癌细胞系及组织中呈高表达，而在正常肺与结肠组织中表达极低。MYLKP1启动子在正常支气管上皮细胞中活性极弱，而在肺腺癌细胞中活性显著升高。本研究通过解析MYLKP1遗传变异与结肠癌发病风险的关联，进一步验证MYLKP1作为癌基因的功能。 方法 本研究对转染MYLKP1的结肠癌细胞系、肺癌细胞系（H441、A549）以及商业化正常肺、结肠细胞开展增殖与迁移实验。我们选取了14个位于MYLKP1 4kb启动子区域、MYLKP1基因核心1.4kb区域及4kb增强子区域内的单核苷酸多态性（Single Nucleotide Polymorphisms，SNPs），其次要等位基因频率（Minor Allele Frequency，MAFs）>0.01，并在结直肠癌队列中完成基因分型。通过双荧光素酶报告基因实验，评估MYLKP1单核苷酸多态性对MYLKP1 2kb启动子活性的影响。 结果 H441与A549癌细胞系的MYLKP1表达水平、MYLKP1启动子荧光素酶活性、细胞增殖与迁移能力均显著上调。基因分型研究筛选出两个MYLKP1单核苷酸多态位点（rs12490683、rs12497343），与非裔美国人对照组相比，这两个位点可显著升高非裔美国人的结肠癌发病风险。相较于野生型MYLKP1启动子，rs12490683与rs12497343可进一步增强MYLKP1启动子的活性。 结论 MYLKP1是一种促癌性假基因，其遗传变异可在非裔美国人群中差异性升高癌症发病风险。