CircZNF609等4个环状RNAs在狼疮性肾炎中的发病机制与防治作用研究

Circular RNAs (circRNAs) have recently been demonstrated to play very important roles in the pathogenesis of several diseases by titration of microRNAs (miRNAs or miRs). This function is described as miRNA sponges. However, there is no report of circRNAs in lupus nephritis (LN) or in any other glomerular and tubulointerstitial diseases. We have recently identified 177 differentially expressed circRNAs in six frozen renal biopsy tissue from LN patients by second generation high robust RNA-sequencing technique through comparing to normal control kidney tissue (5 cm far from tumor tissue). Six out of ten selected differentially expressed circRNAs in sequencing were validated by SYBR green qPCR. The six validated circRNAs including circZNF609, circ188A, circUBR5, circPDE4B, HLA-C, and ELK4 were significantly increased and correlated with the clinical indices of kidney injury in LN patients. To apply the six circRNAs to investigate the mechanism of LN development in LN model mice, the expanded analysis of the 6 circRNAs identified the conservation of circZNF609, circ188A, circUBR5, and circPDE4B between human and mouse. In this project, we aim to verify whether circRNAs are involved in the pathogenesis of LN and whether circRNAs can serve as novel therapeutic targets to improve outcome of LN. To achieve this goal, we propose the following step-by-step approach: 1) We will up-regulate the expression of circZNF609, circ188A, circUBR5, and circPDE4B with transfection of the vector containing each target circRNA in human podocytes, mesangial cells, and proximal tubular cells. Then, we will examine the expression of the top 5 miRs regulated by each circRNA based on circRNA-miRNA network analysis and the level of miR-150 that we reported previously in LN patients. Finally, we will examine the expression of inflammatory and fibrosis associated genes including IL-17, IFN-α, monocyte chemotactic protein-1 (MCP-1), TGF-β1, Collagen-3, and SOCS1. According to the strongest statistical significance in circRNA-miRNAs-mRNAs-proteins regulation pathway and the most clinical relevance to LN pathogenesis, we will select one circRNA (circ-X) as therapeutic target to move into LN mice study. 2) We will administrate the circ-X siRNA to fcgr2b-/- mice, which is a spontaneous LN animal model, to observe the effect of circ-X siRNA on acute and chronic kidney injury including proteinuria, renal dysfunction, and histopathological abnormality. We will also examine the expression of circ-X, miRNAs, mRNAs, and proteins described in the above cell culture. We anticipate that the findings of circRNAs-miRNAs-mRNAs-proteins from three types of kidney resident cells and LN model mice will provide novel insights into the pathogenesis of LN, and may discover new therapeutic targets and tools for improving LN outcome.

环状RNAs(circRNAs)被证实可通过海绵作用调控microRNAs(miRs)参与疾病的发病机制，但在狼疮性肾炎（LN）及其他肾病中尚无报道。我们在初发未治LN患者肾组织中用二代RNA测序发现、用qPCR证实circZNF609 等4个circRNAs显著高表达并与LN疾病活动显著相关；且与小鼠具有同源性。本项目拟在人类肾脏足细胞、系膜细胞与肾小管细胞中分别转染4个circRNAs使其高表达，观察各自调控的5个miRs与miR-150、炎症与纤维化基因IL-17、SOCS1等mRNAs与蛋白的表达变化。然后从体外实验优选一个circ-X，给予自发LN动物模型Fcgr2b-/-小鼠circ-X抑制剂下调其肾内表达，观察LN小鼠肾脏急慢性损伤程度与miRs-mRNAs-蛋白表达变化。旨在：1)探明circRNAs在LN中的发病机制;2)发现LN防治新靶点，为LN精准医疗提供理论基础。

慢性肾病（CKD）患病率在我国高达10.8%。CKD主要由原发与继发肾小球疾病及急性肾损伤（AKI）慢性化所致。其共同病理特征为肾脏纤维化。研究证实非编码RNA微小RNA（miR）与环状RNA（circRNA）参与人类多种疾病的发生发展。其中circRNA对miR的“海绵”作用在医学领域成为近5年的研究热点。但非编码RNA在肾脏纤维化中的作用机制尚不清楚。本项目首先在肾活检证实的狼疮肾炎（LN）患者肾组织中应用RNA二代测序发现171个差异性表达的circRNAs，其中上调幅度最大者为circHLA-c，下调幅度最大者为circMTND5，还发现circZNF609等四个人鼠同源的circRNAs。qPCR验证结果与测序结果一致。在此基础上，我们1）首次报道了LN患者肾组织中circHLA-c可能通过吸附miR-150（2014年面上项目的核心基因）参与LN的发生发展。2）发现LN患者肾组织中肾小管细胞内线粒体损伤，位于线粒体上的circMTND5在LN肾组织线粒体损伤中可能通过“海绵”miR-6812发挥作用，将circRNA研究深入到亚细胞器水平。通过在肾小管上皮细胞与其线粒体组分中，通过RIP, Pulldown及转染与沉默circMTND5证实上述结论。同时课题组将继发的LN延伸至原发的局灶阶段肾小球硬化症（FSGS）与AKI慢性化的动物模型中研究circRNAs在肾脏纤维化中的机制。发现3）在FSGS小鼠肾组织中circZNF609上调，miR-615下调，在细胞水平上通过转染circZNF609证实其通过下调miR-615参与FSGS的发病机制。另外在叶酸诱导肾小管间质纤维化小鼠模型及肾小管细胞中发现4）circHIPK3可能通过海绵miR-30a与circ-15698/miR-185/TGF-β1通路参与肾脏纤维化。5）MFACR/miR-652-3p/MTFP1轴参与缺血再灌注诱导的AKI发生。另外，6）在自发LN模型Fcgr2b敲除小鼠肾组织中证实了circMTND5下调，其RNA蛋白结合效应仍在研究中。上述研究结果说明circRNAs在肾脏病的发病机制中存在疾病特异性，同一个circRNA在LN中可能通过多种途径发挥其致病机制。能够干预特异性circRNA的RNAi可能成为未来治疗肾脏病的潜在打靶新药。为RNAi类的新药研发提供了理论依据。