datasheet1_Astragaloside IV Inhibits Galactose-Deficient IgA1 Secretion via miR-98-5p in Pediatric IgA Nephropathy.zip

Purpose: The factor associated with IgA nephropathy (IgAN) is an abnormality of IgA known as galactose-deficient IgA1 (Gd-IgA1). The purpose of this study was to determine the molecular role played by miRNAs in the formation of Gd-IgA1 in IgAN and investigate the regulatory role of Astragaloside IV (AS-IV) in miRNAs.
Patients and methods: Bioinformatics analysis, along with functional and mechanistic experiments, were used to investigate the relationship and function of miRNA, β-1, 3-galactosyltransferase (C1GALT1), Gd-IgA1, and AS-IV. Analyses involved a series of tools, including quantitative real-time polymerase chain reaction (qRT-qPCR), Western blot, enzyme-linked immunosorbent assay (ELISA), Vicia Villosa lectin-binding assay (VVA), Cell counting kit-8 assay (CCK-8), and the dual-luciferase reporter assay.
Results: miRNA screening and validation showed that miR-98-5p was significantly upregulated in the peripheral blood mononuclear cells (PBMCs) of pediatric patients with IgAN compared with patients diagnosed with mesangial proliferative glomerulonephritis (MsPGN) and immunoglobulin A vasculitis nephritis (IgAV-N), and healthy controls (p < 0.05). Experiments with the dual-luciferase reporter confirmed that miR-98-5p might target C1GALT1. The overexpression of miR-98-5p in DAKIKI cells decreased both the mRNA and protein levels of C1GALT1 and increased the levels of Gd-IgA1 levels; these effects were reversed by co-transfection with the C1GALT1 plasmid, and vice versa. In addition, AS-IV downregulated the levels of Gd-IgA1 level in DAKIKI cells by inhibiting miR-98-5p.
Conclusions: Our results revealed that AS-IV could inhibit Gd-IgA1 secretion via miR-98-5p. Increased levels of miR-98-5p in pediatric IgAN patients might affect the glycosylation of IgA1 by targeting C1GALT1. In addition, our analyses suggest that the pathogenesis of IgAN may differ from that of IgAV-N. Collectively, these results provide significant insight into the pathogenesis of IgAN and identify a potential therapeutic target.

研究背景与目的：IgA肾病（IgA nephropathy, IgAN）的核心致病相关因素为免疫球蛋白A的异常糖基化产物——半乳糖缺陷型IgA1（galactose-deficient IgA1, Gd-IgA1）。本研究旨在明确微小RNA（miRNAs）在IgAN患者Gd-IgA1形成过程中的分子调控作用，并探究黄芪甲苷IV（Astragaloside IV, AS-IV）对miRNAs的调控功能。

研究对象与方法：本研究采用生物信息学分析联合功能与机制实验，探究miRNA、β-1,3-半乳糖基转移酶（β-1, 3-galactosyltransferase, C1GALT1）、Gd-IgA1与AS-IV之间的相互关联及生物学功能。所用实验技术涵盖实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-qPCR）、蛋白质印迹法（Western blot）、酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）、豌豆凝集素结合实验（Vicia Villosa lectin-binding assay, VVA）、细胞计数试剂盒-8实验（Cell counting kit-8 assay, CCK-8）及双荧光素酶报告基因实验。

实验结果：miRNA筛选与验证结果显示，与系膜增生性肾小球肾炎（mesangial proliferative glomerulonephritis, MsPGN）患者、免疫球蛋白A血管炎肾炎（immunoglobulin A vasculitis nephritis, IgAV-N）患者及健康对照者相比，儿童IgAN患者外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中miR-98-5p的表达水平显著上调（p < 0.05）。双荧光素酶报告基因实验证实，miR-98-5p可靶向结合并调控C1GALT1。在DAKIKI细胞中过表达miR-98-5p可同时降低C1GALT1的mRNA及蛋白表达水平，并升高Gd-IgA1的分泌量；该效应可通过共转染C1GALT1质粒逆转，反之亦然。此外，AS-IV可通过抑制miR-98-5p的表达，降低DAKIKI细胞中Gd-IgA1的水平。

研究结论：本研究结果揭示，AS-IV可通过miR-98-5p抑制Gd-IgA1的分泌。儿童IgAN患者体内miR-98-5p水平升高，可能通过靶向调控C1GALT1影响IgA1的糖基化过程。此外，本研究分析提示IgAN的发病机制可能与IgAV-N存在显著差异。综上，本研究结果为IgAN的发病机制提供了重要的理论依据，并确定了一个潜在的治疗靶点。