microRNA激活NFAT5通路在肾髓细胞高渗应激中的作用

Cells in the renal inner madulla are normally exposed to hyperosmotic stress. Accumulating evidence indicates that nuclear factor of activated T cell-5 (NFAT5) plays a critical role in osmoregulation of renal medullary cells; however, the mechanism by which NFAT5 is regulated remains largely unknown. To systematically identify the specific microRNAs (miRNAs) that regulate the NFAT5 pathway, a human primary miRNA library was applied for cell-based high throughput screening with the NFAT luciferase reporter system. Four miRNAs were found to efficiently modulate NFAT activity and the process is calcineurin-independent, consistent with the manner of NFAT5 activation. We hypothesize that these miRNAs might significantly regulate the NFAT5 pathway. In this proposal, we will focus on each selected miRNA in regulating NFAT5 activity and recognize the possible mechanisms by which miRNA modulates it. Furthermore, we will assess the role of these miRNAs in responding to hyperosmotic stress in inner medullary collecting duct (IMCD) cells. It will also examine the role of the miRNAs in osmoregulation and urinary concentration mechanism by using miRNA deficient mouse. Our study will help clarify the role of miRNAs in the regulation of NFAT5 pathway and reveal the underlying mechanisms by which renal medullary cells survive and function under hyperosmotic stress.

正常生理下肾内髓细胞暴露在极有害的高渗胁迫环境中，活化T细胞核因子-5（NFAT5）在肾髓细胞高渗应激过程中发挥重要的保护性调节作用，但NFAT5受调控的机制未完全阐明。为系统鉴定调控NFAT5通路的miRNA，前期运用miRNA过表达文库及NFAT报告系统，在人胚肾HEK293A细胞进行高通量筛选，非钙信号刺激条件下发现4个可显著调控NFAT活性的miRNA。鉴于NFAT5功能的发挥不依赖钙信号途径，我们提出这些miRNA能特异激活NFAT5通路。本项目将从不同层面验证4个miRNA对NFAT5通路的调控作用，并阐明其分子机制；体外研究这些miRNA在肾髓细胞高渗应激中的功能；通过miRNA敲除小鼠，从在体水平评估这些miRNA在肾脏高渗胁迫应答及尿液浓缩机制中的作用。本课题将深化认识miRNA对NFAT5信号通路的调控机制，并进一步阐明肾髓细胞在高渗环境下存活的分子机理。

正常生理下肾内髓细胞暴露在极有害的高渗(高盐、高尿素浓度)胁迫环境中。细胞通过合成大量的有机渗透物、热休克蛋白HSP70来维持存活。但细胞高渗调控HSP70的分子机制未完全阐明。本研究发现，高渗情况下，HSP70受到miR-23a-5p的转录后水平调节。高盐刺激抑制miR-23a-5p的表达水平，从而提高HSP70的蛋白水平，进而促进细胞的活性及增殖。此外，动物禁水实验表明，在小鼠内髓、肾乳头等高渗组织中，miR-23a-5p与HSP70的表达水平呈现负相关。本研究揭示了miR-23a-5p通过调控HSP70在肾髓细胞高渗应激中的重要作用。