Search

DNA methylation and demethylation regulate the transcription of genes. DNA methylation-associated gene expression of adrenal steroidogenic enzymes may regulate cortisol production in cortisol-producing adenoma (CPA). We aimed to determine the DNA methylation levels of all genes encoding steroidogenic enzymes involved in CPA. Additionally, the aims were to clarify the DNA methylation-associated gene expression and evaluate the difference of CPA genotype from others using DNA methylation data. Twenty-five adrenal CPA and six nonfunctioning adrenocortical adenoma (NFA) samples were analyzed. RNA sequencing and DNA methylation array were performed. The methylation levels at 118 methylation sites of the genes were investigated, and their methylation and mRNA levels were subsequently integrated. Among all the steroidogenic enzyme genes studied, CYP17A1 gene was mainly found to be hypomethylated in CPA compared to that in NFA, and the Benjamini-Hochberg procedure demonstrated that methylation levels at two sites in the CYP17A1 gene body were statistically significant. PRKACA mutant CPAs predominantly exhibited hypomethylation of CYP17A1 gene compared with the GNAS mutant CPAs. Inverse associations between CYP17A1 methylation in three regions of the gene body and its mRNA levels were observed in the NFAs and CPAs. In applying clustering analysis using CYP17A1 methylation and mRNA levels, CPAs with PRKACA mutation were differentiated from NFAs and CPAs with a GNAS mutation. We demonstrated that CPAs exhibited hypomethylation of the CYP17A1 gene body in CPA, especially in the PRKACA mutant CPAs. Methylation of CYP17A1 gene may influence its transcription levels.

DNA methylation alteration is tissue-specific and play a pivotal role in regulating gene transcription during cell proliferation and survival. We aimed to detect genes regulated by DNA methylation, and then investigated whether the gene influenced cell proliferation or survival in adrenal cells. DNA methylation and qPCR analyses were performed in nonfunctioning adrenocortical adenoma (NFA, n = 12) and aldosterone-producing adenoma (APA, n = 35) samples. The VDR gene promoter was markedly hypomethylated in APA with ATP1A1 mutation, and the promoter methylation levels showed a significant inverse association with the transcripts in APA. ATP1A1 mutation led to VDR transcription in HAC15 cells, and VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. We demonstrated that APA with ATP1A1 mutation showed entire hypomethylation in the VDR promoter and abundant VDR mRNA and protein expression. VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. Abundant VDR expression would be essential for ATP1A1 mutation-mediated cell proliferation.