DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells
DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells
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DOI：
10.1677/jme.1.01560
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发表时间：
2004-12-01
影响因子：
3.5
通讯作者：
Voutilainen, R
中科院分区：
文献类型：
--
作者：
Liu, J;Li, XD;Voutilainen, R
异常的DNA甲基化可能与人类肾上腺皮质肿瘤的发生有关，这通常伴随着异常的激素产生。在本研究中，我们旨在以人肾上腺皮质 NCI-H295R 细胞系为模型，阐明 DNA 甲基化对类固醇生成的影响。用 DNA 甲基化抑制剂 5-aza-2'-deoxycytidine（Azad；10 muM，持续 7 天）处理可将增殖率降低至对照的约 20%，细胞数量降低至对照的 60%，同时细胞周期蛋白依赖性激酶抑制剂 p57(KIP2) 基因的表达增加。此外，Azad 治疗增加皮质醇分泌剂量和时间依赖性，而硫酸脱氢表雄酮分泌不受影响。 Azad 治疗降低了基础和 (BU)(2)cAMP 诱导的低密度脂蛋白和高密度脂蛋白受体、类固醇生成急性调节蛋白 (StAR)、胆固醇侧链裂解酶、类固醇 17α-羟化酶/17,20-裂解酶和类固醇 21-羟化酶 mRNA 的表达，以及 StAR 蛋白水平。相反，Azad 处理增加了类固醇 11β-羟化酶和 3β-羟基类固醇脱氢酶/Delta(5)-Delta(4)-异构酶基因的基础表达，尽管它抑制了 (Bu)(2)cAMP 诱导的这两个基因的表达。 Azad 处理降低了类固醇生成因子 1 (SF-1) 和 DAX-1（X 染色体 1 上剂量敏感的性逆转 - 先天性肾上腺发育不全关键区域）基因（均在其启动子中包含假定的 CpG 岛）的表达以及 SF-1 基因启动子区域中 HpaII 识别位点的甲基化程度。 Azad 处理也改变了甲基 CpG 结合蛋白 MeCP2 的免疫染色模式。这些结果表明 DNA 甲基化可能与人肾上腺皮质细胞的细胞增殖和类固醇生成的调节有关。
Aberrant DNA methylation may be involved in human adrenocortical tumorigenesis, which is often accompanied by abnormal hormone production. In this study, we aimed to clarify the effects of DNA methylation on steroidogenesis using the human adrenocortical NCI-H295R cell line as a model. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (Azad; 10 muM for 7 days) decreased the proliferation rate to approximately 20% and the cell number to 60% of the control, with a simultaneous increase in the expression of the cyclin-dependent kinase inhibitor p57(KIP2) gene. In addition, Azad treatment increased cortisol secretion dose and time dependently, whereas dehydroepiandrosterone sulfate secretion was not affected. Azad treatment decreased basal and (BU)(2)cAMP-induced expression of low- and high-density lipoprotein receptor, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, steroid 17alpha-hydroxylase/17,20-lyase and steroid 21-hydroxylase mRNA, as well as the StAR protein level. In contrast, Azad treatment increased the basal expression of steroid 11beta-hydroxylase and 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase genes, although it inhibited the (Bu)(2)cAMP-induced expression of these two genes. The expression of steroidogenic factor-1 (SF-1) and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome 1) genes (both harboring putative CpG islands in their promoters) and the methylation degree of the HpaII recognition site(s) in the SF-1 gene promoter region were reduced by Azad treatment. The immunostaining pattern of the methyl-CpG-binding protein MeCP2 was also modified by Azad treatment. These results suggest that DNA methylation may be implicated in the regulation of cell proliferation and steroidogenesis in human adrenocortical cells.