microRNA-630 regulates underglycosylated IgA1 production in the tonsils by targeting TLR4 in IgA nephropathy

IgA nephropathy (IgAN) is the most common primary glomerular disease. The characteristic pathology involves immune complexes formed by the deposition of IgA1 and underglycosylated IgA1 aggregates in the mesangial area, which may be accompanied by the deposition of IgG and/or IgM and complement components. However, the molecular mechanisms of IgAN remain unclear. In the present study, microarray analysis showed that the expression of miR-630 was significantly reduced in palatal tonsils from IgAN patients compared with chronic tonsillitis. Additionally, bioinformatic analysis showed that Toll-like receptor 4 (TLR4) was the predicted target gene of miR-630 and was regulated by miR-630. When miR-630 was overexpressed in palatal tonsil mononuclear cells from IgAN patients, the expression of TLR4 was reduced and the content of IgA1 in the cell culture supernatant was decreased, and the level of galactosylation in the IgA1 hinge region was increased. Moreover, immunohistochemical analysis showed that the expression of TLR4 in IgAN patients was significantly increased and. After knocking down the expression of TLR4, both the concentration of IgA1 and the binding force of IgA1 with broad bean lectin were significantly reduced in IgAN. Furthermore, the mechanism study demonstrated that TLR4 might regulate the expression of IL-1β and IL-8 through NF-κB signaling pathway to modulate the concentration of IgA1 and the glycosylation level of IgA1. This interesting finding may offer new insight into the molecular mechanism of IgAN. To identify the differentially expression of microRNAs in tonsil of patients with IgA nephropathy. Tonsillar tissue specimens were obtained from 24 patients with IgA nephropathy (IgAN) whom had been diagnosed by renal biopsy, used as experimental group. The tonsillar tissue specimens which were obtained from20 patients with chronic tonsillitis (CT) lacking renal diseases were used as control group. Chose two specimens randomly from the above two groups respectively, and use the Agilengt Human miRNA microarrays V19 to detect the microRNAs (miRNAs) profile differentially expressed in tonsil of IgAN. The miRNA microarray differential analysis resulted in 29 significantly deregulated miRNAs after applying a fold change threshold >2, either up regulated or down regulated. Among the 29 significantly deregulated miRNAs, 7 miRNAs were up regulated and 22 miRNAs were down regulated. Tonsillar mononuclear cells (TMCs) were separated by density-gradient centrifugation using Lymphocyte Separation Medium, and we performed quantitative real-time PCR (qRT-PCR) to validate 4 deregulated miRNAs showed in the microarray results. The deregulated miRNAs were miR-630, miR-513b, miR-135a-3p, miR-642-3p respectively.

IgA肾病（IgA nephropathy, IgAN）是最常见的原发性肾小球疾病。其特征性病理表现为IgA1及低糖基化IgA1聚集形成的免疫复合物沉积于肾小球系膜区，可伴随IgG和/或IgM及补体成分的沉积。但目前IgAN的分子机制仍未明确。本研究经芯片分析发现，与慢性扁桃体炎患者相比，IgAN患者的腭扁桃体组织中miR-630的表达水平显著降低。此外，生物信息学分析显示，Toll样受体4（Toll-like receptor 4, TLR4）是miR-630的预测靶基因，并受其调控。当在IgAN患者腭扁桃体单核细胞中过表达miR-630时，TLR4的表达水平下调，细胞培养上清液中的IgA1含量降低，且IgA1铰链区的半乳糖基化水平升高。免疫组化分析进一步证实，IgAN患者体内TLR4的表达显著升高。敲低TLR4的表达后，IgAN患者体内的IgA1浓度以及IgA1与蚕豆凝集素的结合力均显著降低。机制研究表明，TLR4可能通过NF-κB信号通路调控IL-1β和IL-8的表达，进而调节IgA1的浓度及其糖基化水平。这一发现为阐明IgAN的分子机制提供了新的研究视角。
本研究旨在鉴定IgA肾病患者扁桃体组织中差异表达的微小RNA（microRNAs, miRNAs）。研究纳入24例经肾活检确诊的IgAN患者的扁桃体组织标本作为实验组，另纳入20例无肾脏疾病的慢性扁桃体炎（chronic tonsillitis, CT）患者的扁桃体组织标本作为对照组。分别从两组中随机选取2份标本，采用安捷伦人类miRNA芯片V19（Agilent Human miRNA microarrays V19）检测IgAN患者扁桃体组织中差异表达的miRNA谱。以倍数变化（fold change）>2作为筛选阈值，芯片差异分析共得到29个显著异常表达的miRNA，其中7个miRNA表达上调，22个miRNA表达下调。采用淋巴细胞分离液通过密度梯度离心法分离扁桃体单核细胞（tonsillar mononuclear cells, TMCs），并通过实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）对芯片结果中筛选出的4个异常表达miRNA进行验证，这4个miRNA分别为miR-630、miR-513b、miR-135a-3p及miR-642-3p。