Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center and University of Mississippi Medical Center, Jackson, Mississippi.
J Clin Endocrinol Metab. 2018 Mar 1;103(3):965-971. doi: 10.1210/jc.2017-01996.
Aldosterone production is stimulated by activation of calcium signaling in aldosterone-producing adenomas (APAs), and epigenetic factors such as DNA methylation may be associated with the expression of genes involved in aldosterone regulation.
Our aim was to investigate the DNA methylation of genes related to calcium signaling cascades in APAs and the association of mutations in genes linked to APAs with DNA methylation levels.
Nonfunctioning adrenocortical adenoma (n = 12) and APA (n = 35) samples were analyzed. The KCNJ5 T158A mutation was introduced into human adrenocortical cell lines (HAC15 cells) using lentiviral delivery. DNA methylation array analysis was conducted using adrenal tumor samples and HAC15 cells.
The Purkinje cell protein 4 (PCP4) gene was one of the most hypomethylated in APAs. DNA methylation levels in two sites of PCP4 showed a significant inverse correlation with messenger RNA expression in adrenal tumors. Bioinformatics and multiple regression analysis revealed that CCAAT/enhancer binding protein alpha (CEBPA) may bind to the methylation site of the PCP4 promoter. According to chromatin immunoprecipitation assay, CEBPA was bound to the PCP4 hypomethylated region by chromatin immunoprecipitation assay. There were no significant differences in PCP4 methylation levels among APA genotypes. Moreover, KCNJ5 T158A did not influence PCP4 methylation levels in HAC15 cells.
We showed that the PCP4 promoter was one of the most hypomethylated in APAs and that PCP4 transcription may be associated with demethylation as well as with CEBPA in APAs. KCNJ5 mutations known to result in aldosterone overproduction were not related to PCP4 methylation in either clinical or in vitro studies.
醛固酮的产生受到醛固酮瘤（APAs）中钙信号的激活刺激，表观遗传因素，如 DNA 甲基化，可能与参与醛固酮调节的基因表达有关。
我们旨在研究 APA 中与钙信号级联相关的基因的 DNA 甲基化，以及与 APA 相关的基因突变与 DNA 甲基化水平的关联。
分析了无功能肾上腺皮质腺瘤（n=12）和 APA（n=35）样本。使用慢病毒递送将 KCNJ5 T158A 突变引入人肾上腺皮质细胞系（HAC15 细胞）中。使用肾上腺肿瘤样本和 HAC15 细胞进行 DNA 甲基化阵列分析。
浦肯野细胞蛋白 4（PCP4）基因是 APA 中最 hypomethylated 的基因之一。两个 PCP4 位点的 DNA 甲基化水平与肾上腺肿瘤中的信使 RNA 表达呈显著负相关。生物信息学和多元回归分析表明，CCAAT/增强子结合蛋白α（CEBPA）可能与 PCP4 启动子的甲基化位点结合。根据染色质免疫沉淀分析，CEBPA 通过染色质免疫沉淀分析与 PCP4 低甲基化区域结合。在 APA 基因型中，PCP4 甲基化水平没有显著差异。此外，KCNJ5 T158A 并未影响 HAC15 细胞中 PCP4 的甲基化水平。
我们表明 PCP4 启动子是 APA 中最 hypomethylated 的区域之一，并且 PCP4 转录可能与去甲基化以及 APA 中的 CEBPA 有关。已知导致醛固酮过度产生的 KCNJ5 突变与临床或体外研究中的 PCP4 甲基化无关。
