miR-148b modulates the C1GALT1 expression explaining the abnormal glycosylation process in IgA Nephropathy

Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide characterized by aberrant O-glycosylation in the hinge region of IgA1. The basis for the abnormal glycosylation in IgAN is still unknown, but an important involvement of the enzyme core 1, beta 1,3-galactosyltransferase 1 (C1GALT1) is known. However, the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in the IgAN pathogenesis has not yet been reported. In this study, by high-throughput microRNA profiling, we identified 37 miRNAs differentially expressed in peripheral blood mononuclear cells (PBMCs) from IgAN patients compared to healthy subjects. Among them, upregulated miR-148b potentially targeted C1GALT1, INVS and PTEN, three genes notably downregulated in IgAN patients. C1GALT1 expression levels in IgAN patients were reduced and negatively correlated with the upregulated miR-148b expression. We demonstrated the biological relationship between miR-148b and C1GALT1 by transient transfection experiments ex vivo. When we reduced the upregulated miR-148b function in PBMCs of IgAN patients an increase of the C1GALT1 mRNA and protein levels was observed. We validated biologically also the miR-148b targeting of INVS , involved in the altered modulation of the WNT–β-catenin and PI3K/Akt pathways in IgAN patients. All together our data evidence an important role of miR-148b in the pathogenesis of IgAN, which could explain the aberrant glycosylation of IgA1 in the pathogenesis and should light on a potential target for the theraphy of the disease. The role of miRNAs expression in the pathogenesis of IgAN has not been well explored. To identify miRNAs differentially expressed in IgAN respect to healthy subjects (HS), we analyzed the global miRNA expression profile in PBMCs from IgAN patients and HS using the miRNA microarray approach. Among 723 human miRNAs represented on the microarrays, 147 were expressed in each sample. Unsupervised hierarchical clustering analysis generated a tree with IgAN and HS clearly separated into two group (Fig. 1). This separation was further confirmed by displaying the relationships among miRNAs expression patterns using principal component analysis (PCA) (Fig. S1). Applying a fold change threshold > 2 (false discovery rate < 0.01), 35 miRNAs were found to be significantly up-regulated and 2 were significantly down-regulated in IgAN. Complete list of differentially expressed miRNAs is shown in Table S1. To validate the microarray results we performed real-time PCR (q-RT-PCR) for miR-148b, miR-188-5p, miR-361-3p, miR-886-3p, let-7b, let-7d on miRNAs isolated from PBMCs of an independent set of 10 IgAN patients and 10 HS with the same clinical and demographic characteristics of the population used for microarray experiments. The expression of all analyzed miRNAs was significantly higher in IgAN patients confirming microarray results