LncMEG3/miR-21/Orail-SOC轴调节肾系膜细胞ECM分泌致糖尿病肾病纤维化的作用研究

In China, the diabetic patients and incidence increased dramatically over the past years. Furthermore, the tendency of adiposity becomes more evident. Either renal dysfunction or kidney function impairment accounts for one of the most severe complications of diabetic mellitus, as well the leading cause of patient mortality. Mechanically, glomerular mesangial stromal hyperplasia is an important well-defined cause of renal dysfunction. Our previous study found that store-operated calcium entry (SOCE), of which Orai1 is one of the mainstay constituents, modulates matrix secretion from mesangial cells. However, the expression of Orai1 protein in mesangial cells treated with high glucose was negatively regulated by miR-21 and positively correlated with the level of long non-coding RNA MEG3 which was suppressed in diabetes. The inducible expression of miR-21 in glomerular mesangial cells by high glucose then inhibits Orai1 expression and enhances mesangial cell matrix production that leading to renal injury ultimately. To this end, in this project, we planned to use cell and animal module, i.e. in vitro and in vivo experiments to elucidate the molecular mechanism of miR-21 regulating SOCE and mesangial cell matrix secretion, its role in the pathogenesis of renal dysfunction in diabetic mice as well. By the way, we are out to certify that all of them can interact with each other physically. Also, we sought to further evaluate the potential application of miR-21 or MEG3 in the treatment of renal injury diabetic nephropathy by delivery targeting miR-21 antagomirs to renal mesangial cells. Intriguingly, the delivery system was composed of the non-coding RNA wrapping in nanoparticle, and they can be localized precisely in the glomerular area through intricate but not yet defined mechanism. The present study will provide a theoretical basis and potential drug target for clinical precision treatment of diabetic nephropathy renal injury, but also lay the underpinnings for individualized targeted therapy, and potent molecular marker screening as well.

糖尿病肾病（DN）是糖尿病重要并发症之一，也是造成糖尿病病人死亡主要原因；DN机制未明，肾系膜细胞（MCs）细胞外基质（ECM）增生是导致肾功能障碍重要原因。我们前期发现:高糖组MCs中LncRNA MEG3（MEG3）和Orai1表达均下降、miR-21表达上调；MEG3和miR-21表达调控MCs内Orai1和钙水平；DN小鼠肾小球ECM含量明显上升、miR-21表达改变影响肾小球ECM水平。本项目拟采用①RIP和荧光报告系统验证MCs内MEG3/miR-21/Orai1存在相互作用及调控关系②qRT-PCR、WB和IHC等实验阐明MEG3/miR-21调控Orai1-SOCE（钙库操纵钙内流）和MCs基质分泌的分子机制及其在DN肾损伤中的作用③纳米靶向递送MEG3表达载体和miR-21抑制物至肾小球MCs，评价其在治疗DN中潜在应用价值，为临床精准治疗DN提供理论依据和潜在药物靶点。

糖尿病肾病(DN)是世界范围内最常见和最致命的糖尿病并发症之一，与高死亡率相关。然而，其发展背后的确切机制尚不清楚。系膜细胞(MCs)和非编码RNA对DN至关重要，但MCs中是否存在MEG3/miR-21/ORAI1调控轴尚不清楚。因此，在本研究中，我们旨在了解MEG3/miR-21/ORAI1调节轴是否在DN的病理生理学中起作用。我们证明高糖刺激下调MEG3和ORAI1的表达，同时增强miR-21的表达。外源性miR-21模拟物抑制ORAI1表达，MEG3过表达部分挽救或逆转ORAI1表达。此外，RIP实验表明，AGO2抗体标记的珠粒比对照组IgG抗体标记的珠粒更能富集miR-21和MEG3;它们都形成了RNA诱导的沉默复合体。此外，使用miR-21抑制剂，db/db小鼠的生化指标显著改善，肾纤维蛋白坏死得到改善。因此，MEG3/miR-21/ORAI1轴调控糖尿病小鼠和MCs中DN的表现，一旦在人类中发现这种轴的存在，miR-21抑制剂可能是缓解DN的潜在治疗策略。

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