谷胱甘肽S转移酶-氧化应激途径在醛固酮分泌调控中的机制研究

KCNJ5 mutation is an important progression of APA development. Aldosterone level is significantly associated with cardiovascular complications Previously we found the unique KCNJ5 mutation characteristics in Chinese APA patients by performing gene sequencing of APA tissue. Based on the previous results, we made the gene microarray analysis of KCNJ5 mutated APA tissues according to aldosteorne level as well as KCNJ5 non-mutated and normal adrenal tissues. We found that Glutathione S Transferase was lower expressed in higher aldosterone level APA than lower aldosterone level APA, and the following results verified the difference. Knock-down of GSTA1 could suppress aldosterone production. This project aims to enlarging sample size to verify the expression of GSTs and investigate its function about proliferation, apoptosis and aldosterone secretion in H295R cells. The interaction between KCNJ5 mutation and GSTs expression and downstream mechanism are also to be explored. The in vivo experiments will be performed to investigate the accurate mechanism about aldosteorne secreting to provide better understanding for APA diagnosis and therapy.

KCNJ5基因突变是肾上腺醛固酮瘤（APA）发生机制的重要进展，醛固酮水平与心血管并发症密切相关，课题组前期在114例APA中进行基因测序发现中国人群中KCNJ5突变的独特特点。基于前期研究，本项目通过KCNJ5突变术前卧位醛固酮水平高低将APA组织分为两组，加上KCNJ5非突变APA及正常肾上腺组织，送检芯片分析，筛选出谷胱甘肽-S-转移酶（GSTs）家族基因在突变高醛固酮组织中表达降低，前期针对芯片结果进行了验证，并且功能试验表明下调GSTA1基因分别可以提高醛固酮合酶基因表达及醛固酮分泌。本课题拟扩大样本量验证GSTs家族基因的表达，在肾上腺H295R细胞中研究GSTs基因对增殖、凋亡、醛固酮分泌及相关基因表达的影响，KCNJ5突变与GSTs基因的调控关系，以及GSTs发挥功能的分子机制；体内实验研究GSTs基因调控APA形成和醛固酮分泌的具体机制，为APA的诊断治疗提供新的依据。

本项目主要研究内容为GSTA1（谷胱甘肽S转移酶1）在肾上腺醛固酮瘤中的表达和功能机制研究。KCNJ5突变为醛固酮瘤中目前发现的最重要突变。前期通过基因芯片分析筛选，通过对比高醛固酮和低醛固酮分泌醛固酮瘤组织发现GSTA1基因为差异表达最大的基因。本课题组首先针对GSTA1基因表达进行研究，发现GSTA1在醛固酮瘤中表达较正常组织低，并且在高醛固酮分泌KCNJ5突变组织中表达更低，应用单细胞测序分析发现，GSTA1基因在肾上腺皮质细胞中相对其他细胞表达最高，并且其表达在醛固酮瘤中低于正常肾上腺组织和肾上腺增生组织，在高醛固酮组织低于低醛固酮组织；功能试验显示过表达GSTA1可以降低醛固酮分泌，而敲低或抑制GSTA1表达可提高醛固酮分泌；上游机制试验显示KCNJ5突变和miRNA92a可调控GSTA1表达并影响其功能；下游机制试验显示，GSTA1可通过改变细胞内活性氧水平和细胞内钙离子水平调控醛固酮分泌。进一步探索发现GSTA1与Wnt/β-catenin信号通路有相互作用。总之，本项目在前期检测醛固酮瘤KCNJ5突变基础之上，发现GSTA1基因为调控醛固酮分泌的关键基因，并针对该基因上下游调控机制进行了深入探索，为后续肾上腺醛固酮瘤发病机制研究及药物研发提供新的理论依据。