miR-16和miR-100在分泌型IgA诱导系膜细胞分泌炎症因子中的作用及其机制研究

IgA nephropathy(IgAN) is the most common glomerulonephritis in our country, but the exact pathogenesis of IgAN is still obscure. Our previous work demonstrated that secretory IgA（SIgA) could induce human renal mesangial cell (HRMC) producing inflammatory factors, which suggests that SIgA is pathogenic. However, the mechanism remains to be further explored. We hypothesize that SIgA induced HRMC inflammatory factors production was regulated by the role of miRNAs. Then, to verify this hypothesis, miRNA chip detection, bioinformatics analysis and dual-luciferase reporter assay system were used, and it was found that SIgA could promote HRMC secretion of IL-6 and IL-8 respectively through the regulation of miR-16-2-3p and miR-100-3p. To further study, in this work, from molecules, cells, tissues and animal levels, HRMC、blood、urine、kidneys of patients and mouse IgAN model will be used to investigate the role and mechanism of miRNAs in HRMC secretion of inflammatory factors induced by SIgA. All the work will be done by the technologies of affinity chromatography, laser confocal, real-time quantitative PCR, ELISA and Western blot, etc. It will explore the pathogenesis of IgAN from the new viewpoint of miRNA and provide new target for the treatment of IgAN.

IgAN是我国最常见的肾小球肾炎，但确切的发病机制仍不清楚。我们的前期工作证实分泌型IgA（SIgA）能诱导系膜细胞（HRMC)释放炎症因子，提示其具有致病性。但其致病机制有待进一步探讨，为此我们提出假说：SIgA可能通过miRNAs的调控作用，介导HRMC炎症因子的释放。为证实这一假设，我们应用miRNA芯片、生物信息学以及双荧光素酶报告基因检测，发现SIgA可能分别通过调控miR-16-2-3p和miR-100-3p促进HRMC分泌IL-6和IL-8。本课题将通过HRMC、重组慢病毒、患者血、尿、肾组织及小鼠IgAN模型，采用亲和层析、激光共聚焦、实时定量PCR、ELISA、Western blot等技术，从分子、细胞、组织及动物水平明确调控SIgA诱导HRMC炎症因子释放的miRNAs及其作用机制。本研究将从miRNA这个新视点揭示IgAN的发病机制，提供IgAN治疗的新靶点。

IgA肾病（IgAN）是全球最常见的原发性肾小球疾病，但其确切发病机制尚不清楚。我们前期证实分泌型IgA（SIgA）在IgAN中具有致病性，但其致病机制需进一步探讨。研究发现，微小RNA(miRNAs)在肾病中发挥重要作用，因此我们将探讨SIgA能否通过miRNAs调控系膜细胞（HRMC）释放炎症因子。本研究检测了IgAN患者肾组织SIgA沉积并比较其与临床资料的相关性；分离纯化SIgA沉积患者及正常人唾液中SIgA，刺激HRMC后提取总RNA，重复前期芯片结果，qRT-PCR、ELISA比较两组HRMC miR-16-2-3p、miR-100-3p和IL-6、IL-8的差异表达；qRT-PCR、Western blot检测SIgA沉积患者及正常肾组织miR-16-2-3p、miR-100-3p、IL-6和IL-8的表达；双荧光素酶报告基因验证miR-16-2-3p和miR-100-3p与IL-6和IL-8的相互作用；慢病毒转染验证miR-16-2-3p和miR-100-3p可靶向调控HRMC分泌IL-6和IL-8。结果发现，76名患者中有25名肾组织有SIgA沉积，沉积组患者血尿发生率和肾小球滤过率均高于无沉积组，血清胱抑素C、血肌酐、24h尿蛋白均低于无沉积组，血压、血红蛋白及血清白蛋白两组间无差异；正常人和患者SIgA刺激HRMC后不同时间点，HRMC分泌IL-6、IL-8与miR-16-2-3p和miR-100-3p的表达动态相关；患者肾组织中证实miR-16-2-3p、miR-100-3p对IL-6、IL-8具有调控作用，并与肾损伤相关；双荧光素酶报告基因及慢病毒转染证实miR-16-2-3p、 miR-100-3p可靶向调控HRMC分泌IL-6、IL-8。此外，我们还研究了SIgA介导HRMC释放炎症因子的ceRNA机制及相关信号通路，发现SIgA可能通过Lnc-SIP1L3、Lnc-NCEH1吸附miR-302a-5p、miR-592调控HRMC高表达TLR4、TLR9，并进一步证实TLR4高表达后可激活MyD88/NF-kB通路诱导HRMC释放炎症因子。本研究在IgAN领域内首次探讨SIgA通过miRNAs、ceRNA及TLRs相关信号通路介导肾损伤的机制，为IgAN发病机制提供新的重要理论依据，为IgAN的治疗寻找新的靶点。