miR-7213-5p-mediated suppression of CCL19 in fibroblast cells may attenuate lupus nephritis

Abstract

Lupus nephritis (LN) is an immune-complex nephritis and one of the most severe organ manifestations of systemic lupus erythematosus. To elucidate the mechanisms underlying LN, we firstly performed comprehensive RNA sequencing and microRNA (miRNA) sequencing analyses on the kidneys of female lupus-prone MRL/lpr mice and female C57BL/6 mice. Our results revealed significant renal impairment in 17-week-old female MRL/lpr mice, as evidenced by elevated 24-hour urinary protein, serum creatinine, and blood urea nitrogen levels, along with severe renal pathology. RNA sequencing identified 100 upregulated and 59 downregulated genes in the kidneys of 17-week-old MRL/lpr mice, which were enriched in immune response, transcriptional regulation, and metabolic reprogramming. MiRNA sequencing further identified 23 upregulated and 9 downregulated miRNAs in MRL/lpr mice. Interaction network analysis revealed that the upregulated miRNAs (miR-3473b and miR-204-3p) were linked to transcriptional regulation and DNA repair, while the downregulated miRNA (miR-7213-5p) was closely associated with immune cell trafficking, immune function regulation, and metabolism. Subsequent validation confirmed the significant downregulation of miR-7213-5p in MRL/lpr kidneys, whereas the levels of its predicted target, CC motif chemokine 19 (CCL19), were significantly elevated in both renal fibroblasts and serum. Mechanistically, miR-7213-5p directly targeted the 3'-untranslated region of CCL19, thereby suppressing both the expression and secretion of CCL19 induced by TNF-α in L929 fibroblasts. These findings highlight the anti-inflammatory role of miR-7213-5p via the regulation of CCL19, suggesting its potential as a therapeutic target for LN.

Keywords: CCL19; Lupus nephritis; MiR-7213-5p; MiRNA-mRNA interaction.

Fig. 1

Renal functional and pathological characterization of MRL/lpr mice. (a-c) Comparison of 24hUP (a), SCr (b), and BUN (c) between MRL/lpr and C57BL/6 mice. (d) Representative images of HE (scale bars, 50 μm), Masson (scale bars, 50 μm), PAS staining (scale bars, 50 μm), and TEM ultrastructure (scale bars, 1 μm). Data are shown as mean ± SD, and individual data are presented. P values were analyzed by two-way ANOVA with Tukey’s multiple comparisons test, n = 6 per group. P value legend: * < 0.05, *** < 0.001, **** < 0.0001, ns not significant

Fig. 2

RNA sequencing profiling of DEGs in the kidneys of MRL/lpr and C57BL/6 mice. (a) Heatmap illustrating DEGs in the kidneys of MRL/lpr mice compared to C57BL/6. (b, c) Venn diagram displaying upregulated (b) and downregulated (c) genes numbers. (d, e) GO enrichment analysis of biological processes for upregulated (d) and downregulated (e) gene sets. n = 3 per group

Fig. 3

MiRNA sequencing profiling of differentially expressed miRNAs in the kidneys of MRL/lpr mice and C57BL/6 Mice. (a) Heatmap illustrating differentially expressed miRNAs in the kidneys of MRL/lpr mice compared to C57BL/6. (b) GO enrichment analysis of predicted target genes of differentially expressed miRNAs. n = 3 per group

Fig. 4

The interaction network between differentially expressed miRNAs and DEGs in the kidneys of MRL/lpr mice. (a) Network of 23 upregulated miRNAs and 59 downregulated mRNAs. (b) GO analysis of targets for miR-3473b and miR-204-3p (downregulated mRNAs). (c) Network of 9 downregulated miRNAs and 100 upregulated mRNAs. (d) GO analysis of targets for miR-7213-5p (upregulated mRNAs). n = 3 per group

Fig. 5

RT-qPCR analysis of miR-3473b, miR-204-3p, and miR-7213-5p expression in the kidneys of MRL/lpr and C57BL/6 mice. (a) Relative expression levels of miR-3473b. (b) Relative expression levels of miR-204-3p. (c) Relative expression levels of miR-7213-5p. P values were analyzed by two-tailed unpaired t tests, n = 6 per group. P value legend: ** < 0.01, *** < 0.001

Fig. 6

miR-7213-5p downregulates fibroblast CCL19 via 3’-UTR binding in lupus nephritis. (a) RT-qPCR analysis of CCL19 mRNA expression in the kidneys of MRL/lpr mice. (b, c) Immunofluorescence staining for CCL19 (red) and vimentin (green) proteins in kidney sections from MRL/lpr and C57BL/6 mice. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. b: representative images; c: quantitative analysis. (d) Serum CCL19 protein levels in MRL/lpr mice measured by ELISA. (e) Predicted binding sites of miR-7213-5p within the 3’-UTR of CCL19, with the seed sequence highlighted. (f) Luciferase reporter assay validating miR-7213-5p-CCL19 3’-UTR interaction in L929 fibroblasts transfected with mimics/inhibitors. (g, h) CCL19 mRNA (g) and protein (h) expression levels in TNF-α-stimulated L929 cells following miR-7213-5p modulation. I ELISA quantification of CCL19 secretion in culture supernatants from treated L929 cells. Data are shown as mean ± SD, and individual data are presented. P values of a, c, and d were analyzed by two-tailed unpaired t tests, n = 6 per group. P values of f-i were analyzed by two-way or one-way ANOVA with Tukey’s multiple comparisons test, n = 3 per group. P value legend: ** < 0.01, *** < 0.001, **** < 0.0001, ns not significant