Department of Urology, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China.
Int J Exp Pathol. 2018 Apr;99(2):77-86. doi: 10.1111/iep.12267. Epub 2018 Apr 17.
The mechanism of aldosterone-producing adrenocortical adenoma (APA) pathogenesis and the role of microRNAs (miRNAs) in APA pathogenesis have not been completely clarified. We examined the expression and function of miR-140-3p, miR-193a-3p and miR-22-3p, which have binding sites in CYP11B2. Expression of miRNAs and CYP11B2 mRNA was measured by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation was monitored by colorimetric analysis, and cell apoptosis and cell cycle progression were analysed by flow cytometry. ELISA was carried out to detect aldosterone levels in cell culture supernatants. Luciferase reporter assays, qRT-PCR and Western blotting were performed to identify CYP11B2 as a target of miR-193a-3p. Of the three miRNAs examined, miR-193a-3p exhibited a significant decrease and CYP11B2 mRNA exhibited a significant increase in expression in APA compared with adjacent normal adrenal gland tissue. Transfection of miR-193a-3p mimic into the human adrenocortical cell line H295R showed that elevated miR-193a-3p expression inhibits proliferation and aldosterone secretion, induces G1-phase arrest and promotes apoptosis in H295R cells. Furthermore, in luciferase reporter assays, overexpression of miR-193a-3p in H295R cells significantly reduced the luciferase activity of the wild-type CYP11B2 3'-UTR construct, which could be reversed by mutation of the miR-193a-3p-binding site. Moreover, miR-193a-3p overexpression downregulated CYP11B2 mRNA and protein expression. Finally, overexpression of CYP11B2 diminished the effects of miR-193a-3p on H295R cells. Taken together, our results suggest that CYP11B2 levels may be modulated by miR-193a-3p in APA, which could explain, at least partially, why downregulation of miR-193a-3p during APA formation may promote cell growth and suppress apoptosis.
醛固酮产生性肾上腺皮质腺瘤（APA）的发病机制以及 microRNAs（miRNAs）在 APA 发病机制中的作用尚未完全阐明。我们检测了 miR-140-3p、miR-193a-3p 和 miR-22-3p 的表达和功能，这些 miRNA 在 CYP11B2 中有结合位点。通过定量逆转录 PCR（qRT-PCR）测量 miRNA 和 CYP11B2 mRNA 的表达。通过比色分析法监测细胞增殖，通过流式细胞术分析细胞凋亡和细胞周期进程。通过 ELISA 检测细胞培养上清液中的醛固酮水平。进行荧光素酶报告基因检测、qRT-PCR 和 Western blot 分析，以确定 CYP11B2 是 miR-193a-3p 的靶标。在三种检测到的 miRNA 中，与相邻正常肾上腺组织相比，APA 中 miR-193a-3p 表达显著降低，而 CYP11B2 mRNA 表达显著升高。miR-193a-3p 模拟物转染人肾上腺皮质细胞系 H295R 后，结果表明，升高的 miR-193a-3p 表达可抑制增殖和醛固酮分泌，诱导 H295R 细胞 G1 期阻滞并促进凋亡。此外，在荧光素酶报告基因检测中，H295R 细胞中 miR-193a-3p 的过表达显著降低了野生型 CYP11B2 3'-UTR 构建体的荧光素酶活性，而 miR-193a-3p 结合位点的突变可逆转这一结果。此外，miR-193a-3p 过表达下调 CYP11B2 mRNA 和蛋白表达。最后，CYP11B2 的过表达减弱了 miR-193a-3p 对 H295R 细胞的影响。综上所述，我们的研究结果表明，在 APA 中，CYP11B2 水平可能受 miR-193a-3p 调节，这至少可以部分解释为什么在 APA 形成过程中 miR-193a-3p 的下调可能促进细胞生长并抑制凋亡。
