Abnormal miR-148b expression promotes aberrant glycosylation of IgA1 in IgA nephropathy

Abstract

Aberrant O-glycosylation in the hinge region of IgA1 characterizes IgA nephropathy. The mechanisms underlying this abnormal glycosylation are not well understood, but reduced expression of the enzyme core 1, β1,3-galactosyltransferase 1 (C1GALT1) may contribute. In this study, high-throughput microRNA (miRNA) profiling identified 37 miRNAs differentially expressed in PBMCs of patients with IgA nephropathy compared with healthy persons. Among them, we observed upregulation of miR-148b, which potentially targets C1GALT1. Patients with IgA nephropathy exhibited lower C1GALT1 expression, which negatively correlated with miR-148b expression. Transfection of PBMCs from healthy persons with a miR-148b mimic reduced endogenous C1GALT1 mRNA levels threefold. Conversely, loss of miR-148b function in PBMCs of patients with IgA nephropathy increased C1GALT1 mRNA and protein levels to those observed in healthy persons. Moreover, we found that upregulation of miR-148b directly correlated with levels of galactose-deficient IgA1. In vitro, we used an IgA1-producing cell line to confirm that miR-148b modulates IgA1 O-glycosylation and the levels of secreted galactose-deficient IgA1. Taken together, these data suggest a role for miRNAs in the pathogenesis of IgA nephropathy. Abnormal expression of miR-148b may explain the aberrant glycosylation of IgA1, providing a potential pharmacologic target for IgA nephropathy.

Figure 1.

Unsupervised hierarchical clustering of miRNA expression profile. miRNA expression pattern of PBMCs of seven patients with IgAN and seven healthy subjects (HSs) were examined using Agilent array composed of 723 human and 76 human viral miRNAs. A total of 147 miRNAs were expressed in all samples, discriminating patients with IgAN from HSs (P<0.0001; FDR<0.01). Two principal clusters were identified on the basis of differential miRNA expression.

Figure 2.

Differential expression validation. Validation of differential expression of miR-148b, miR-188-5p, miR-886-3p, let-7b, and let-7d (A) and miR-361-3p (B) in an independent set of PBMCs from 10 patients with IgAN and 10 healthy subjects (HSs). Expression levels were quantified using qRT-PCR. The miRNA relative expressions were normalized to the expression of U6. Expression levels of miR-148b, miR-188-5p, miR-886-3p, let-7b, let-7d, and miR-361-3p were found to be significantly higher in patients with IgAN than in HSs. The histograms represent the mean ± SEM. *P<0.03; **P<0.01.

Figure 3.

C1GALT1 gene expression levels evaluated by real-time PCR in 10 patients with IgAN and 10 healthy subjects (HSs). C1GALT1 expression levels were significantly lower in patients with IgAN than in HSs. C1GALT1 expression levels were normalized on the housekeeping gene β-actin. The scatter plots represent the mean ± SEM. ^#P<0.0001.

Figure 4.

miR-148b targets C1GALT1. (A) Genomic localization of miR-148b in chromosome 12q13. (B) Sequence alignment of the miR-148b base-pairing sites in the 3′-UTR of C1GALT1 mRNA showing that the regions complementary to miR-148b are highly conserved among human, chimp, mouse, rat, dog, and chicken. The “seed” sequences of miR-148b complementary to C1GALT1 are shown in gray. (C) Linear correlation between the expression of C1GALT1 and the expression of miR-148b in PBMCs of 10 patients with IgAN and 10 healthy subjects (HSs). C1GALT1 mRNA levels inversely correlated with miR-148b expression levels (R²=0.4; P<0.01). (D) Output of bioinformatic analysis performed by MicroSNiPer algorithms showing that the 1365G/A polymorphism (rs1047763) affects miR-148b binding sites in the 3′-UTR of C1GALT1. The 1365G allele (arrow) enhances miR-148b binding, whereas the 1365A allele does not appear in the output as a possible binding site for miR-148b.

Figure 5.

miR-148b expression levels evaluated by real-time PCR in PBMCs from 3 patients with membranoproliferative GN type I (MPGN-I), 5 with focal segmental glomerulosclerosis (FSGS), and 10 with Henoch-Schönlein purpura nephritis (HSPN). miR-148b expression in these diseases and in healthy subjects (HSs) has been compared with that in IgAN. We found that miR-148b levels were higher in patients with IgAN than in those with MPGN-I, FSGS, and HSPN and in HSs. The miRNA relative expressions were normalized to the expression of U6. The histograms represent the mean ± SEM. **P<0.01; ^#P<0.0001.

Figure 6.

miR-148b regulates C1GALT1 mRNA expression. (A) C1GALT1 expression levels were analyzed by real-time PCR in PBMCs of healthy subjects (HSs) after transfection with miR-148b mimic. Increasing the amount of miR-148b within the PBMCs of HSs resulted in a three-fold reduction in endogenous C1GALT1 mRNA levels. (B) C1GALT1 expression levels were analyzed by real-time PCR in PBMCs of five other HSs after transfection with the mutated miR-148b mimic, Δ20-21, and miR-148b mimic wild-type. C1GALT1 mRNA expression decreased only after transfection with the wild-type miR-148b mimic. (C) C1GALT1 expression levels were analyzed by real-time PCR in PBMCs of patients with IgAN after transfection with miR-148b inhibitor. Silencing the activity of miR-148b within IgAN PBMCs led to a three-fold increase in endogenous C1GALT1 mRNA levels. “Mock” indicates mock-transfected cells going through the transfection processes without addition of mimic/inhibitor miRNA. Expression data were normalized on the housekeeping gene β-actin. Data are representative of four independent experiments for A and C and five independent experiments for B (mean ± SEM). **P<0.01.

Figure 7.

miR-148b regulates C1GALT1 expression at protein level. (A) Transfection of PBMCs of healthy participant with 25 nM miR-148b mimic resulted in a 2.2-fold reduction in C1GALT1 protein expression. Increasing the miR-148b levels within PBMCs of healthy participants resulted in the same C1GALT1 protein levels as in IgAN PBMCs, as shown by Western blot. (B) Western blot representing protein levels of C1GALT1 in IgAN PBMCs after transfection with 250 nM miR-148b inhibitor. A significant increase in C1GALT1 protein production was shown in IgAN PBMCs transfected with miR-148b inhibitor (1.7-fold increase). miR-148b loss of function led to the same C1GALT1 protein levels as in PBMCs of healthy participants. “Mock” indicates mock-transfected cells going through the transfection processes without addition of mimic/inhibitor miRNA. In both experiments, β-actin was used as endogenous control. Data are representative of five independent experiments (mean ± SEM). **P<0.01, ^§P≤ 0.001.

Figure 8.

miR-148b regulates Gal-deficient IgA1 levels. (A) The miR-148b expression levels evaluated by real-time PCR in 50 patients with IgAN and 50 healthy subjects (HSs). miR-148b expression levels were significantly higher in patients with IgAN than in HSs. miR-148b expression levels were normalized to the expression of U6. The histograms represent the mean ± SEM. ^§P<0.001. (B) shows the serum levels of Gal-deficient IgA1 in patients with IgAN and HSs. The Gal-deficient IgA1 was significantly higher in sera obtained from the patients with IgAN than in sera from HSs. The relative lectin binding per unit IgA1 was calculated as the OD value of lectin over the OD value of total IgA. (C) The serum level of total IgA in patients with IgAN and HSs determined by ELISA. IgA levels in sera obtained from 50 patients with IgAN and 50 HSs were similar. The histograms represent the mean ±SEM. ^#P<0.0001. (D) Linear correlation between the expression of miR-148b and the Gal-deficient IgA1 levels in 50 patients with IgAN and 50 HSs. miR-148b levels directly correlated with Gal-deficient IgA1 levels (r=0.4,; P<0.0001). (E) The Gal-deficient IgA1 levels in supernatants obtained from DAKIKI cells after transfection with miR-148b mimic and inhibitor. A significant increase in Gal-deficient IgA1 production was shown in DAKIKI cells transfected with 50 nM miR-148b mimic (1.5-fold increase). On the contrary, the transfection with 500 nM miR-148b inhibitor led to a significant reduction in Gal-deficient IgA1 levels (1.8-fold). The relative lectin binding per unit IgA1 was calculated as the OD value of lectin over the OD value of total IgA. The histograms represent the mean ± SEM. ^§P<0.001.