Abstract：
The Mineralocorticoid Receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron, but mechanisms regulating MR expression are still poorly understood. We previously showed that RNA Binding Proteins (RBPs) regulate MR expression at the post-transcriptional level in response to variations of extracellular tonicity. Herein, we highlight a novel regulatory mechanism involving the recruitment of microRNAs (miRNAs) under hypertonicity. RT-qPCR validated miRNAs candidates identified by high throughput screening approaches and transfection of a luciferase reporter construct together with miRNAs Mimics or Inhibitors demonstrated their functional interaction with target transcripts. Overexpression strategies using Mimics or lentivirus revealed the impact on MR expression and signaling in renal KC3AC1 cells. miR-324-5p and miR-30c-2-3p expression are increased under hypertonicity in KC3AC1 cells. These miRNAs directly affect Nr3c2 (MR) transcript stability, act with Tis11b to destabilize MR transcript but also repress Elavl1 (HuR) transcript, which enhances MR expression and signaling. Overexpression of miR-324-5p and miR-30c-2-3p alter MR expression and signaling in KC3AC1 cells with blunted responses in terms of aldosterone-regulated genes expression. We also confirm that their expression is increased by hypertonicity in vivo in the kidneys of mice treated with furosemide. These findings may have major implications for the pathogenesis of renal dysfunctions, sodium retention, and mineralocorticoid resistance.
摘要：
盐皮质激素受体（MR）介导醛固酮在远端肾单位的钠保留作用，但是调节MR表达的机制仍然知之甚少。我们先前表明，RNA结合蛋白（RBP）在转录后水平上调节MR表达，以响应细胞外张力的变化。在这里，我们强调了一种新的调节机制，涉及高张性下microRNAs（miRNAs）的募集。RT-qPCR验证了通过高通量筛选方法和荧光素酶报告构建体与miRNA一起转染鉴定的miRNA候选物。模拟物或抑制剂证明了它们与靶转录物的功能相互作用。使用模拟或慢病毒的过表达策略揭示了对肾KC3AC1细胞中MR表达和信号传导的影响。miR-324-5p和miR-30c-2-3p表达在KC3AC1细胞中的高渗下增加。这些miRNA直接影响Nr3c2（MR）转录物的稳定性，与Tis11b合作破坏MR转录本的稳定性，但也抑制Elavl1（HuR）转录本，增强MR表达和信号传导。miR-324-5p和miR-30c-2-3p的过表达改变了KC3AC1细胞中的MR表达和信号传导，在醛固酮调节的基因表达方面反应减弱。我们还证实，在用呋塞米治疗的小鼠的肾脏中，它们的表达因体内高渗性而增加。这些发现可能对肾功能障碍的发病机理有重要意义。钠潴留，和盐皮质激素抗性。
