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Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis.
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.
Affiliation
  • Yao C; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • He F; Department of Pathology, Second Affiliated Hospital of Nanjing Medical University, Nanjing, China.
  • Liu L; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Zhang Z; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Zhao C; Department of Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
  • Qiu W; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Zhao D; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Zhang J; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Xie M; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Gong Y; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Yu T; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Xia L; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Qian B; Department of Immunology, Nanjing Medical University, Nanjing, China.
  • Wang Y; Department of Immunology, Nanjing Medical University, Nanjing, China.
Cell Physiol Biochem ; 49(5): 1898-1917, 2018.
Article in En | MEDLINE | ID: mdl-30235450
ABSTRACT
BACKGROUND/

AIMS:

The 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.

METHODS:

Rat 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.

RESULTS:

Compared 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.

CONCLUSION:

MiR-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.
Subject(s)
Key words

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Complement Membrane Attack Complex / Apoptosis / MicroRNAs / SOXC Transcription Factors / Isoantibodies / Microtubule-Associated Proteins Type of study: Prognostic_studies Limits: Animals Language: En Journal: Cell Physiol Biochem Journal subject: BIOQUIMICA / FARMACOLOGIA Year: 2018 Document type: Article Affiliation country: China

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Complement Membrane Attack Complex / Apoptosis / MicroRNAs / SOXC Transcription Factors / Isoantibodies / Microtubule-Associated Proteins Type of study: Prognostic_studies Limits: Animals Language: En Journal: Cell Physiol Biochem Journal subject: BIOQUIMICA / FARMACOLOGIA Year: 2018 Document type: Article Affiliation country: China
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