Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis.
Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis.
复制标题
在大鼠 Thy-1 肾炎中，Sublytic C5b-9 通过 miR-3546/SOX4/Survivin 轴诱导肾小球系膜细胞凋亡。
DOI：
10.1159/000493652
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发表时间：
2018
影响因子：
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通讯作者：
Wang Yingwei
中科院分区：
文献类型：
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作者：
Yao Chunyan;He Fengxia;Liu Longfei;Zhang Zhiwei;Zhao Chenhui;Qiu Wen;Zhao Dan;Zhang Jing;Xie Mengxiao;Gong Yajuan;Yu Tianyi;Xia Lu;Qian Baomei;Wang Yingwei
系膜增生性肾小球肾炎（MsPGN）患者肾小球补体系统激活，补体C5 b-9复合物形成。然而，C5 b-9诱导的肾小球系膜细胞（GMC）损伤的作用和机制知之甚少。大鼠Thy-1 N是研究MsPGN的动物模型。已有研究表明，C5 b-9对大鼠Thy-1 N系膜细胞的攻击是亚溶解性的，亚溶解性C5 b-9可引起系膜细胞凋亡，但其机制尚未完全阐明。为探讨C5 b-9在MsPGN损伤中的作用及其调控机制，本研究采用大鼠Thy-1 N模型，首次检测了Thy-1 N大鼠肾组织（体内）和C5 b-9亚溶解刺激培养的GMCs（体外）microRNA（miRNA）谱的变化。然后，我们确定了miR-3546，这增加了在体内和体外，对GMC凋亡后sublytic C5 b-9以及参与mechanism.MethodsRat Thy-1 N模型建立和GMC与sublytic C5 b-9处理。取大鼠肾皮质和刺激后的GMC进行miRNA芯片检测。随后，通过实时PCR验证增加的miRNA。同时，将相应的miRNA模拟物转染GMC，通过Western blotting和流式细胞术检测，以确定其上调caspase 3的表达和诱导GMC凋亡的能力。此后，使用生物信息学方法预测miR-3546靶向基因（SOX 4），并使用实时PCR和WB检测Thy-1 N组织和GMCs中的SOX 4表达（在亚裂解C5 b-9刺激或miR-3546模拟物/抑制剂转染后）。为了证明miR-3546可以影响SOX 4基因的转录和SOX 4可以调节Survivin的表达，进行了双荧光素酶报告分析、实时PCR、WB和染色质免疫沉淀（ChIP）分析。结果与正常肾组织和未处理的GMCs相比，Thy-1 N组织中有43个和62个miRNAs表达上调（> 2倍），而C5 b-9刺激组中有62个miRNAs表达上调（> 2倍）。在体内和体外共检测到17种miRNAs表达增加，其中11种通过实时荧光定量PCR得到验证。其中，miR-3546能显著促进GMC凋亡，抑制SOX 4和survivin的表达，而SOX 4和survivin的过表达均能显著挽救miR-3546模拟物介导的GMC凋亡。另外，SOX 4过表达可逆转miR-3546模拟物对survivin的抑制，SOX 4可与survivin启动子结合结论miR-3546/SOX 4/survivin轴在亚溶解性C5 b-9介导的GMC凋亡中具有促进作用，本研究为进一步研究Thy-1 N和MsPGN的发病机制提供了新的思路。
Background/AimsThe activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism.MethodsRat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry.ResultsCompared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to-853 nt) and activate survivin gene transcription.ConclusionMiR-3546/SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.