miR‑146a‑5p protects against renal injury in MRL/lpr mice via improvement of the Treg/Th17 imbalance by targeting the TRAF6/NF‑κB axis

Abstract

Dysregulated microRNA (miRNA or miR) expression is an important cause of immune homeostasis disorder in patients with systemic lupus erythematosus and lupus nephritis (LN). The present study evaluated the possibility of using miR-146a-5p as a therapeutic target for treating LN. The effects of miR-146a-5p on lupus syndrome in MRL/lpr mice were evaluated. MRL/lpr mice were injected with miR-146a-5p agomir (M146AG) or agomir negative control (NC). Renal function index, pathology and protein expression levels of inflammatory factors in MRL/lpr mice were evaluated after M146AG or agomir NC treatment. Reverse transcription-quantitative PCR, western blotting and immunofluorescence were used to assess the effect of M146AG on mRNA and protein expression levels of (tumor necrosis factor receptor-associated factor 6) TRAF6/NF-κB axis components. A luciferase dual reporter system was used to assess the mechanism of regulation of TRAF6/NF-κB axis expression. Finally, flow cytometry was used to assess the regulatory effect of M146AG on regulatory T cell (Treg)/T helper 17 (Th17) balance. The findings demonstrated that M146AG ameliorated renal lesions and the inflammatory response in MRL/lpr mice. TRAF6 was demonstrated to be targeted and significantly negatively regulated by miR-146a-5p. M146AG intervention significantly increased expression of miR-146a-5p and significantly downregulated the mRNA and protein expression levels of TRAF6 and NF-κB in CD4⁺ T cells of MRL/lpr mice. Furthermore, M146AG intervention alleviated Treg/Th17 imbalance in MRL/lpr mice peripheral blood. The present findings demonstrated that M146AG improved Treg/Th17 imbalance and alleviated renal lesions in MRL/lpr mice by targeting the TRAF6/NF-κB axis. This may provide a new theoretical basis for the clinical diagnosis and treatment of LN.

Keywords: NF-κB; TRAF6; Treg/Th17; lupus nephritis; miR-146a-5p.

Figure 1

M146AG alleviates renal lesions in MRL/lpr mice. (A) Schematic representation of the experimental design. Renal function was assessed using (B) BUN and (C) SCr levels. (D) Representative micrographs of H&E-stained renal sections (scale bar, 50 µm). (E) Glomerular injury score. (F) Representative micrographs of Masson's trichrome-stained renal sections (scale bar, 50 µm). (G) Renal fibrosis score. All data are presented as the mean ± standard deviation. ^**P<0.01. i.v., intravenous; VEH, vehicle; NC, negative control; miR, microRNA; M146AG, miR-146a-5p agomir; H&E, hematoxylin and eosin; BUN, blood urea nitrogen; SCr, serum creatinine.

Figure 2

M146AG inhibits mRNA and protein expression levels of inflammatory factors in serum and renal tissue of MRL/lpr mice. Protein concentrations of (A) IL-6, (B) IL-17A, (C) TNF-α and (D) IFN-γ in serum of MRL/lpr mice. Relative mRNA expression levels of (E) IL-6, (F) IL-17A, (G) TNF-α and (H) IFN-γ in the renal tissue of MRL/lpr mice. All data are presented as the mean ± standard deviation. ^*P<0.05 and ^**P<0.01. VEH, vehicle; NC, negative control; miR, microRNA; M146AG, miR-146a-5p agomir.

Figure 3

M146AG regulates expression of miR-146a-5p and TRAF6/NF-κB axis components in CD4⁺ T cells of MRL/lpr mice. (A) Relative miRNA expression level of miR-146a-5p. The relative mRNA expression levels of (B) TRAF6 and (C) NF-κB. (D) Representative western blotting bands of TRAF6/NF-κB axis protein expression levels. Semi-quantified relative protein expression levels of (E) TRAF6 and (F) NF-kB. (G) Protein expression levels of TRAF6 and NF-κB were assessed using immunofluorescence staining (scale bar, 10 µm). All data are presented as the mean ± standard deviation. ^*P<0.05 and ^**P<0.01. VEH, vehicle; NC, negative control; miR, microRNA; M146AG, miR-146a-5p agomir; TRAF6, tumor necrosis factor receptor-associated factor 6.

Figure 4

miR-146a-5p directly targets TRAF6. (A) Potential binding sites between miR-146a-5p and TRAF6 3'-UTR were predicted using TargetScan. (B) Dual-luciferase reporter assay demonstrated WT TRAF6-3'-UTR expression following transfection with miR-146a-5p mimic or inhibitor. (C) RNA expression levels of miR-146a-5p and TRAF6 mRNA in 293T cells following miR-146a-5p overexpression and inhibition. All data are presented as the mean ± standard deviation. ^*P<0.05 and ^**P<0.01. NC, negative control; miR, microRNA; TRAF6, tumor necrosis factor receptor-associated factor 6; WT, wild-type; MUT, mutant.

Figure 5

M146AG improves the Treg/Th17 imbalance in MRL/lpr mice. (A) Representative bands of Th17A and Foxp3 protein expression levels assessed using western blotting. The relative protein expression levels of (B) Th17A and (C) Foxp3. (D) Representative flow cytometry dot plots for Th17 cells. (E) Percentage of Th17 cells assessed using flow cytometry. (F) Representative flow cytometry dot plots for Treg cells. (G) Percentage of Treg cells assessed using flow cytometry. (H) Th17/Treg ratio. (I) Pearson correlation analysis between expression of miR-146a-5p and the Th17/Treg ratio. (J) Pearson correlation analysis between the relative protein expression of NF-κB and the Th17/Treg ratio. All data are presented as mean ± standard deviation. ^*P<0.05 and ^**P<0.01. VEH, vehicle; NC, negative control; miR, microRNA; M146AG, miR-146a-5p agomir; Treg, T regulatory; Th17, T helper 17.