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. 2020 Nov 26:11:563699.
doi: 10.3389/fimmu.2020.563699. eCollection 2020.

microRNA-630 Regulates Underglycosylated IgA1 Production in the Tonsils by Targeting TLR4 in IgA Nephropathy

Chan Liu  1 , Mu-Yao Ye  2 , Wen-Zhe Yan  3 , Xiao-Fei Peng  4 , Li-Yu He  2 , You-Ming Peng  2
Affiliations

microRNA-630 Regulates Underglycosylated IgA1 Production in the Tonsils by Targeting TLR4 in IgA Nephropathy

Chan Liu et al. Front Immunol. .

Abstract

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 microRNA-630 (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. 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.

Keywords: IgA nephropathy; TLR4; microRNA-630; palatal tonsils; underglycosylated IgA1.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer H. L. declared a shared affiliation with the authors to the handling editor at time of review.

Figures

Figure 1
Figure 1
miR-630 is decreased in the tonsil tissue of the IgAN group. (A) The microarray analysis detected the different expression of microRNAs (miRNAs) in palatine tonsil tissue between IgAN group and CT group. N=2. (B) Quantitative reverse transcription-PCR (qRT-PCR) confirmed the different expression of miRNAs between IgAN group and CT group. (C) The expression of miR-630 was detected in tonsil mononuclear cells (TMCs) derived from the IgAN group and CT group, respectively. (D–H) The correlation between the expression of miR-630 and the clinical parameters including estimated glomerular filtration rate (eGFR), albumin (ALB), Cre, proteinuria, and hematuria were analyzed by Pearson correlation analysis and linear regression analysis. N=14. The data were expressed as mean ± SEM, **p < 0.01. IgAN, IgA nephropathy, N=27; CT, chronic tonsillitis, N=20.
Figure 2
Figure 2
miR-630 regulates the concentration and glycosylation level of IgA1. (A) ELISA detected the concentration of IgA1 in the supernatant of TMCs derived from IgAN and CT group, respectively. (B) The binding force of IgA1 with the broad bean lectin [IgA1-Vicia villosa lectin (VVL)-binding OD value] was measured by enzyme-linked lectin assay. (C) qRT-PCR detected the expression of miR-630 in different subtypes of mononuclear cells including CD4+ T cells, CD8+ T cells, pan B cells, and pan monocytes, which were isolated from TMCs by magnetic beads. (D, E) The correlation between the expression of miR-630 and the concentration of IgA1 as well as the binding force of IgA1 with the broad bean lectin in TMCs were analyzed by Pearson correlation analysis and linear regression analysis. (F) qRT-PCR showed that the expression of miR-630 was increased significantly in TMCs transfected with miR-630 mimics. (G, H) overexpression of miR-630 decreased the concentration of IgA1 and the binding force of IgA1 with the broad bean lectin in TMCs significantly. The data were expressed as mean ± SEM, **p < 0.01.*p < 0.05. IgAN, IgA nephropathy, N=27; CT, chronic tonsillitis, N=20. NC, negative control.
Figure 3
Figure 3
TLR4 is the target gene of miR-630. (A) Bioinformatic analysis showed that the miR-630 putative target sites in TLR4 3’-UTR. The mutated nucleotides were underlined. (B, C) qRT-PCR and western blot analysis detected the level of TLR4 mRNA and protein in the TMCs derived from the IgAN group and CT group, respectively. (D) The WT-TLR4 3’-UTR and the MUT-TLR4 3’-UTR reporters were co-transfected with miR-630 mimic or negative control into TMCs. Forty-eight hours after transfection, the luciferase activities were measured. (E, F) TMCs were transfected with negative miRNA mimics or miR-630 mimic respectively and harvested for the examination of TLR4 protein and messender RNA (mRNA) by qRT-PCR and western blot analysis. The data were expressed as mean ± SEM, *p < 0.05, **p < 0.01. IgAN, IgA nephropathy; CT, chronic tonsillitis. NC, negative control.
Figure 4
Figure 4
TLR4 is involved in miR-630 regulating the concentration and glycosylation levels of IgA1 in IgAN. (A) Semi-quantitative analysis of immunohistochemistry staining showed the expression of TLR4 in the IgAN group and CT group. Representative images were shown. N=5. (B) The expression of TLR4 mRNA was negatively correlated with miR-630 in the TMCs derived from the IgAN group. (C, D) TLR4 mRNA expression was positively correlated with the concentration of IgA1 and the binding force of IgA1 with the broad bean lectin. (E) The validity of TLR4 small interfering RNAs (siRNAs) was verified by western blot analysis. (F, G) Knocking down the expression of TLR4 made the concentration of IgA1 and the binding force of IgA1 with the broad bean lectin decreased significantly in TMCs derived from IgAN. The data were expressed as mean ± SEM, **p < 0.01.***p < 0.001, compared with siRNA-NC; ###p < 0.001, ####p < 0.0005, compared with CT. IgAN, IgA nephropathy; CT, chronic tonsillitis; NC, negative control.
Figure 5
Figure 5
TLR4 might regulate the concentration of IgA1 and the glycosylation level of IgA1 through NF-κB signaling pathway. (A, B) The expressions of IL-1β and IL-8 were detected by RT-PCR in TMC from the IgAN group and CT group. (C, D) The concentrations of IL-1β and IL-8 were measured by ELISA in TMC from the IgAN group and CT group. (E, F) The concentrations of IL-11β and IL-8 were measured by ELISA when knocking down the expression of TLR4 by using TLR4 siRNA or overexpression of TLR4 in TMC. (G) The level of NF-κB p65 was measured by western blot analysis when knocking down the expression of TLR4 by using TLR4 siRNA or overexpression of TLR4 in TMC. (H, I) The effect of TLR4 on the concentration of IL-1β and IL-8 were measured by ELISA when blocking the NF-κB p65 signaling pathway by using BAY 11-7085. (J) The effect of BAY 11-7085 on inhibiting NF-κB p65 signaling pathway was tested by western blot analysis. (K, L) The effect of TLR4 on the concentration of IgA1 and the glycosylation level of IgA1 were abolished by blocking the NF-κB p65 signaling pathway. The data were expressed as mean ± SD. *p < 0.05. **p < 0.01.***p < 0.001. IgAN, IgA nephropathy; CT, chronic tonsillitis; NC, negative control.
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