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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10238-025-01960-0.

Introduction

Lupus nephritis (LN) is a renal inflammatory disorder caused by the autoimmune disease systemic lupus erythematosus (SLE) [1]. SLE exhibits a higher prevalence among women, with a female-to-male ratio ranging from 6.1:1 to 13.3:1 [2]. Approximately 10% of LN patients may progress to end-stage kidney disease [3, 4]. Despite extensive research, the exact pathogenesis of LN remains unclear. Therefore, substantial efforts are still needed to investigate the pathogenesis of LN and identify specific, sensitive biomarkers to aid in early diagnosis, disease activity assessment, and optimization of clinical treatment strategies.

MicroRNAs (miRNAs) are small (19–23 nucleotides) non-coding RNA molecules that regulate gene expression at the post-transcriptional level by binding to the 3’-untranslated region (3’-UTR) of target mRNAs, leading to translational repression or mRNA degradation [5]. As crucial post-transcriptional regulators, miRNAs are involved in various physiological and pathological processes. Several miRNAs, including miR-10, miR-103a-3p, miR-192, miR-150, and miR-378a-3p, are strongly associated with the onset and prognosis of renal diseases [6–10]. Deletion of Dicer or Drosha, which specifically alters miRNA expression in mouse podocytes, leads to progressive glomerular damage, along with proteinuria and podocyte dysfunction. For instance, miR-217 has been shown to reduce podocyte apoptosis in membranous nephropathy by targeting TNF superfamily member 11 [11]. These findings underscore the critical regulatory roles of miRNAs in kidney diseases. Although emerging evidence suggests that certain miRNAs may play a role in the pathogenesis of LN [12–15], the precise molecular mechanisms by which miRNAs influence the progression of LN remain poorly understood. Further investigations are essential to elucidate the detailed contributions of miRNAs to the initiation and progression of LN, which could provide critical insights into their potential as diagnostic biomarkers and therapeutic targets.

In this study, we integrated RNA sequencing and miRNA sequencing data from age-matched lupus-prone MRL/MpJ-Fas < lpr>/J (MRL/lpr) and C57BL/6 mice to identify potential critical regulatory signaling axes. Our analyses revealed miR-7213-5p as a key immune-related miRNA in LN pathogenesis. Further mechanistic validation demonstrated that CCL19 is a direct target of miR-7213-5p, suggesting its potential role in LN immunoregulation.

Materials and methods

Mice

Female MRL/MpJ-Fas < lpr>/J (MRL/lpr) mice and female C57BL/6 mice were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). The mice were housed in a temperature-controlled room (22 °C) at the Peking Union Medical College Hospital animal centre, with a 12-hour light/dark cycle. At 8 and 17 weeks of age, MRL/lpr and C57BL/6 mice were anesthetized and then euthanized to collect blood and kidney samples. All animal experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Biochemical measurements in blood and urine

Serum creatinine (SCr) and blood urea nitrogen (BUN) concentrations were quantified using an automated biochemistry analyzer in the clinical laboratory. Additionally, 24-hour urinary protein (24hUP) levels were determined using commercially available assay kits (Jiancheng Bioengineering Institute).

Histological analysis

Kidney tissues were collected and fixed in 4% formaldehyde for preservation. Subsequently, paraffin-embedded Sect. (2 μm thick) were prepared and stained using hematoxylin and eosin (HE), Masson’s trichrome (Masson), and periodic acid-Schiff (PAS) to assess renal morphology and structural alterations.

Transmission electron microscopy (TEM)

For ultrastructural analysis, kidney tissues were fixed in 2.5% glutaraldehyde at 4 °C for 24 h, rinsed with phosphate buffer, and then post-fixed with 1% osmium tetroxide/1.5% potassium ferrocyanide solution for 1 h. After graded ethanol dehydration (50–100%, 8 min/step), samples were embedded in SPON12 resin (polymerized at 60 °C, 48 h). Ultrathin Sect. (70 nm) were collected on Formvar-coated copper grids, double-stained with uranyl acetate and lead citrate, and imaged using a TEM-1400Plus at 80 kV.

Total RNA extraction

Total RNA was extracted from kidney tissues using TRIzol reagent (Invitrogen) following standard protocol. Briefly, homogenized tissue samples were mixed with chloroform (1:5 v/v) and centrifuged (12,000 × g, 20 min, 4 °C). The aqueous phase was collected for RNA precipitation with isopropanol, followed by 75% ethanol wash. RNA pellets were finally resuspended in 20 µL RNase-free water and stored at −80 °C.

RNA sequencing

Library preparation was performed using the Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China). RNA was denatured and mRNA enriched with oligo(dT)-attached magnetic beads. After fragmentation, first-strand cDNA was synthesized by random hexamer-primed reverse transcription, followed by second-strand synthesis, end repair, A-tailing, and adaptor ligation. The library was amplified by PCR and subjected to quality control. Single-stranded library products were denatured, circularized, and amplified by rolling circle amplification (RCA) with phi29, generating DNA nanoballs (DNBs) with over 300 copies of the original molecule. The DNBs were loaded onto a patterned nanoarray, and paired-end 100/150 base reads were generated on the DNBSEQ platform (BGI-Shenzhen, China).

Raw sequencing data were processed using SOAPnuke [16] to remove reads containing adapters, those with > 20% low-quality bases (Phred score ≤ 15), or those with > 5% unknown bases (“N”). The resulting high-quality clean reads were stored in FASTQ format. All subsequent analyses were conducted using the Dr. Tom Multi-omics Data Mining System (https://biosys.bgi.com). Clean reads were aligned to the reference genome with HISAT2 [17]. Transcript identification was performed by mapping reads to a comprehensive gene set—including known and novel, coding and noncoding transcripts—using Bowtie2 [18]. Gene expression levels were estimated with RSEM [19]. Differential expression analysis was carried out using DESeq2 [20], with significantly differentially expressed genes defined as those with a Q-value ≤ 0.05. Expression patterns were visualized via heatmaps generated with pheatmap (v1.0.12). To explore phenotypic associations, differentially expressed genes were subjected to Gene Ontology (GO) enrichment analyses using Phyper, based on a hypergeometric test. Significantly enriched terms and pathways were identified using a Q-value threshold of ≤ 0.05.

MiRNA sequencing

The library was constructed using the MGIEasy MiRNA Library Prep Kit (BGI-Shenzhen). RNA was sequentially ligated with 3’ and 5’ adapters, followed by reverse transcription into cDNA and amplification via PCR. Fragment size selection was performed using polyacrylamide gel electrophoresis to ensure quality control. The single-stranded library was then denatured and circularized, with uncyclized linear DNA molecules removed. The circularized DNA was amplified through phi29 and RCA to generate DNBs, each containing over 300 copies of the original single-stranded circularized molecule. DNBs were deposited onto a patterned nanoarray, and paired-end 50-base sequencing reads were generated using the DNBSEQ (BGI-Shenzhen, China).

The raw sequencing data, referred to as raw tags, were processed to obtain clean tags using SOAPnuke [21]. The filtering criteria were as follows: low-quality tags were removed, as well as those containing 5’ primer contamination, lacking a 3’ primer, missing insertion sequences, exhibiting poly-A tails, or shorter than 15 nucleotides. After filtering, clean tags were mapped to the reference genome and MiRbase (v22) database, using Bowtie2 [22]. The expression levels of miRNAs were quantified by counting the absolute number of molecules using unique molecular identifiers (UMIs) [23]. MiRNA expression levels were quantified by the ratio of C to T, where C is the UMI count of a specific miRNA and T is the total UMI count (library size) of the sample. For differential expression analysis, we employed the DEGseq package [24] via the Dr. Tom Multi-omics Data Mining System (https://biosys.bgi.com). Differences in expression were considered statistically significant when the Q-value was ≤ 0.05. Target genes of miRNAs were predicted using RNAhybrid [25], miRanda [26], and TargetScan [27]. To annotate gene functions, all target genes were aligned with the GO databases. GO enrichment analyses were performed using the phyper function in R via the Dr. Tom Multi-omics Data Mining System (https://biosys.bgi.com).

Quantitative real-time polymerase chain reaction (RT-qPCR)

For miRNA quantification, miR-3473b, miR-204-3p, miR-7213-5p, U6 snRNA and miR-16 levels were measured using TaqMan miRNA Assay Probes (# 4427975, # A25576, Thermo Fisher Scientific, USA). For mRNA analysis, transcript levels of CCL19, GAPDH, 18 S, and ACTB were measured using SYBR Green dye (Biotium, USA) in combination with gene-specific primers. All primer sets were purchased from TianYi Hui Yuan Biotechnology. The primer sequences used in this study are detailed in Table S1. The expression stability of candidate reference genes was systematically evaluated using the NormFinder algorithm (Table S2). Based on the NormFinder algorithm and previous studies [28, 29], U6 snRNA and GAPDH were selected as internal controls for miRNA and mRNA normalization, respectively. Relative gene expression was then calculated using the 2^−ΔΔCT method.

Immunofluorescence

Three-micrometer sections were prepared from formalin-fixed, paraffin-embedded mouse kidney tissues. After standard deparaffinization and rehydration procedures, antigen retrieval was performed by heat-mediated treatment in citrate buffer (pH 6.0) for 30 min. Tissue sections were then blocked with hydrogen peroxide followed by 5% serum, each for 20 min at room temperature. Primary antibody incubation was carried out overnight at 4 °C using anti-CCL19 (sc-74233, Santa Cruz Biotechnology) and anti-Vimentin (sc-66002, Santa Cruz Biotechnology) antibodies. Detection was performed using the TSAPLus fluorescence double-labeling three-color staining kit (G1226-50T, Servicebio). Nuclei were counterstained with DAPI, and representative images were acquired using an AXR confocal microscope (Nikon, Japan). The fluorescence intensity was analyzed using ImageJ.

Cell culture

Mouse Fibroblasts Cells (L929) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). We selected the L929 cell line, a typical fibroblast model which has been applied for renal fibrosis at studies [30, 31], as an in vitro model to study the direct interaction between miR-7213-5p and CCL19. The L929 cell lines was cultured in DMEM medium, supplemented with 10% FBS (10099-141, Gibco) and 100 U/mL penicillin-streptomycin, in an atmosphere of 5% CO₂ at 37 °C.

Transfection

Cell transfection was performed using Lipofectamine 2000 (11668027, Invitrogen) according to the manufacturer’s instructions. For miR-7213-5p overexpression, cells were transfected with miR-7213-5p mimics, while miR-7213-5p knockdown was achieved using miR-7213-5p inhibitors. Scrambled RNAs served as negative controls. Transfections were conducted at ~ 60% cell confluency with equal RNA concentrations across all conditions. Following overnight incubation, cells were treated with TNF-α (10 ng/mL; P6020, Beyotime Biotechnology) for 24 h and subsequently analyzed by RT-qPCR or ELISA.

Luciferase assay

Luciferase vectors used in this study were purchased from GENEray (China). For miRNA binding site analysis, pmirGLO luciferase vectors containing the miR-7213-5p binding sites on the 3’-UTR of CCL19 were constructed. A mutant plasmid was also purchased to test the binding specificity. The miR-7213-5p binding site was mutated from AAGAGA to TTCTCT. Luciferase vectors and miR-7213-5p mimics/inhibitors were transfected into L929 cells, and luciferase activity was measured using a luciferase assay kit (E1910, Promega).

ELISA

The CCL19 levels in serum, cells, and cell culture supernatants were quantified using commercial ELISA kits (KT2150-B, Kete Biotechnology) in strict accordance with the manufacturer’s protocol.

Statistical analysis

All values are expressed as means ± SD. Statistical analysis was conducted using an unpaired t-test for two groups, and one-way or two-way ANOVA for multiple groups (GraphPad Prism 10). A P value < 0.05 was considered statistically significant.

Results

The renal impairment observed in MRL/lpr mice

To assess LN progression, we compared renal parameters between age-matched female MRL/lpr and C57BL/6 mice. Eight-week-old MRL/lpr mice showed normal renal function (24hUP, SCr, BUN) and histology compared to 8-week-old female C57BL/6 mice (Fig. 1a-d), while the 17-week-old female MRL/lpr mice exhibited significantly elevated 24hUP (Fig. 1a), SCr (Fig. 1b), BUN (Fig. 1c), and renal pathology damage, including glomerular endothelial proliferation, crescent formation, neutrophil infiltration, fibrinoid necrosis, interstitial fibrosis, and podocyte foot process disruption compared to both 17-week-old female C57BL/6 mice and 8-week-old female MRL/lpr mice (Fig. 1d).

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

RNA sequencing in the kidneys of MRL/lpr mice

To investigate the molecular mechanisms of LN, we performed RNA sequencing on the kidneys of 8- and 17-week-old female MRL/lpr and C57BL/6 mice. Heatmap analysis revealed distinct transcriptional profiles between the four groups (Fig. 2a). To eliminate the influence of genetic background, we focused on the intersection of significantly differentially expressed genes (DEGs) between 17-week-old female MRL/lpr lupus mice versus age-matched C57BL/6 controls and between 17-week-old versus 8-week-old female MRL/lpr lupus mice. We further excluded DEGs identified in 17-week-old female C57BL/6 mice and 8-week-old female C57BL/6 mice to eliminate potential age-related differences. Ultimately, we identified 100 upregulated genes (Fig. 2b, Table S3) and 59 downregulated genes (Fig. 2c, Table S3) in the kidneys of 17-week-old female lupus MRL/lpr mice, which may represent key regulatory factors in the pathogenesis of LN.

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

To characterize the functional roles of DEGs in LN, we performed GO analysis of the 100 upregulated and 59 downregulated genes identified in MRL/lpr mouse kidneys. The upregulated genes were predominantly enriched in immune cell activation, differentiation, and inflammatory regulation (Fig. 2d), while the downregulated genes were mainly associated with transcriptional regulation and metabolic reprogramming (Fig. 2e).

MiRNA-mediated pathways and gene enrichment in the kidneys of MRL/lpr mice

To elucidate the post-transcriptional mechanisms underlying gene regulation in LN, we performed miRNA sequencing to characterize the miRNA profile of the kidneys from MRL/lpr mice. We identified 23 upregulated miRNAs (Table S4) and 9 downregulated miRNAs (Table S5) in 17-week-old female MRL/lpr mice compared with 17-week-old female C57BL/6 mice (Fig. 3a). The dysregulated miRNA targets were functionally enriched in transcriptional control, developmental processes, and phosphorylation/Wnt-mediated cell communication and metabolism (Fig. 3b).

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

The interaction network of DEGs and MiRNAs in the kidneys of MRL/lpr mice

To delineate the miRNA-mRNA regulatory networks underlying LN pathogenesis, we performed interaction networks connecting 23 upregulated miRNAs to 59 downregulated genes, as well as 9 downregulated miRNAs to 100 upregulated genes in MRL/lpr mice. Finally, among the 23 upregulated miRNAs, miR-3473b and miR-204-3p exhibited the most extensive interactions with 36 downregulated genes, with enrichment predominantly in transcriptional regulation and DNA repair processes (Fig. 4a and b). On the other hand, among the 9 downregulated miRNAs, miR-7213-5p exhibited the most extensive interactions with 15 upregulated genes, with target genes significantly enriched in immune cell trafficking, immune function regulation, and metabolic processes (Fig. 4c and d). These findings position miR-3473b, miR-204-3p and miR-7213-5p as central regulators of gene expression networks in LN, potentially orchestrating critical pathological processes through distinct transcriptional and post-transcriptional mechanisms.

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

Further confirmation of MiRNAs in the kidneys of MRL/lpr mice

To confirm the regulatory network between DEGs and miRNAs, we measured the expression levels of miR-3473b, miR-204-3p, and miR-7213-5p in the kidney tissues of MRL/lpr and C57BL/6 mice. Consistent with our miRNA sequencing results, miR-3473b and miR-204-3p were significantly upregulated in the kidneys of MRL/lpr mice (Fig. 5a and b), while the expression of miR-7213-5p was lower compared to the C57BL/6 control group (Fig. 5c). Among the three differentially expressed miRNAs, miR-7213-5p exhibited the most pronounced dysregulation and was therefore selected for further analysis. This focus was further justified by the strong enrichment of its predicted target genes in pathways critically implicated in LN immunopathology. Although miR-3473b and miR-204-3p were also upregulated, their lower fold-changes (< 3) suggest a potentially less central role in the regulatory network. Thus, miR-7213-5p was prioritized for functional validation.

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

MiR-7213-5p targets CCL19 to regulate immune dysregulation in LN

Considering the central role of immune disorder in LN and the GO analysis showing that miR-7213-5p may regulate immune cell trafficking and immune function regulation, we further investigated the interaction between miR-7213-5p and its immune related target. Our comprehensive miRNA-mRNA network analysis indicated CCL19, a cytokine critical for immune cell trafficking and lymphoid tissue development [32], to be among the 15 predicted target genes of miR-7213-5p (Table S6). Considering the pivotal role of immunity in LN progression, CCL19 was therefore chosen as the prime candidate for subsequent experimental validation. Subsequent validation in the MRL/lpr mice showed that CCL19 mRNA expression was significantly upregulated in renal tissue (Fig. 6a). Consistent with this, immunofluorescence analysis revealed a significant increase in CCL19 protein expression in the kidneys of MRL/lpr mice compared with the C57BL/6 control group, with primary localization observed in vimentin-positive renal fibroblasts (Fig. 6b and c). Additionally, serum CCL19 protein levels were significantly elevated in MRL/lpr mice compared to C57BL/6 controls (Fig. 6d). Our bioinformatics analysis predicted that miR-7213-5p could bind to the 3’-UTR of CCL19 mRNA, with a minimal free energy of − 24.1 kcal/mol, suggesting a strong binding potential between miR-7213-5p and CCL19 (Fig. 6e). Based on these findings, we further investigated the regulatory relationship between miR-7213-5p and CCL19.

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

To verify whether miR-7213-5p directly targets CCL19, we cloned a fragment of the CCL19 3’-UTR containing the miR-7213-5p binding sites into a dual luciferase assay system. Our results demonstrated that overexpression of miR-7213-5p significantly suppressed luciferase reporter activity, while inhibition of this miRNA enhanced luciferase reporter activity (Fig. 6f). Mutating the binding site abolished these effects, confirming that miR-7213-5p efficiently targets the CCL19 3’-UTR (Fig. 6f). Since TNF-α is a known key mediator of lupus-associated kidney damage [33], we stimulated L929 fibroblast cells with TNF-α, and it markedly upregulated CCL19 expression and secretion. Further functional experiments demonstrated that overexpression of miR-7213-5p significantly reduced both the TNF-α-induced CCL19 mRNA and protein levels in L929 cells (Fig. 6g and h), along with its secretion (Fig. 6i). Conversely, inhibition of miR-7213-5p enhanced the expression and secretion of CCL19 in these fibroblasts (Fig. 6g-i). This study confirms that miR-7213-5p suppresses CCL19 expression and secretion by targeting its 3’-UTR. Collectively, these findings suggest that the miR-7213-5p/CCL19 regulatory axis may play an important role in the progression of LN.

Discussion

In this study, we investigated the molecular mechanisms underlying LN by integrating differential expression data of miRNAs and mRNAs. We identified 159 DEGs in lupus-prone MRL/lpr mice compared with controls. Functional enrichment analysis of these DEGs revealed their primary involvement in immune response, transcription regulation, and metabolic reprogramming. Additionally, the differentially expressed miRNAs were significantly enriched within the DEG network, providing partial insight into the molecular mechanisms of LN. Interaction network analysis revealed that upregulated miR-3473b and miR-204-3p were linked to transcriptional regulation and DNA repair, while downregulated miR-7213-5p was closely associated with immune cell trafficking, immune function regulation, and metabolic processes. Ultimately, our in vitro experiments confirmed that CCL19, a pro-inflammatory chemokine highly expressed in fibroblasts, is a direct target of miR-7213-5p. The results revealed that miR-7213-5p exerts inhibitory effects on fibroblast-mediated inflammatory responses, and its downregulation contributes to the pathogenesis of LN.

Our study confirmed that 17-week-old female MRL/lpr mice precisely replicate the key pathological features of human LN. Next, we performed RNA and miRNA sequencing to analyze 17-week-old female MRL/lpr mice in comparison with age-matched female C57BL/6 mice. The controlled experimental conditions enhance the reproducibility and reliability of the research. Despite the well-established role of miRNAs in the pathogenesis of various diseases, research focusing on their involvement in LN remains limited. One study used chips to analyze miRNA and mRNA expression in kidney tissues of NZBWF1 mice with mild and severe renal damage due to lupus [34]. They compared 28-week-old and 8-week-old NZBWF1 mice and found that only miR-1968-5p was significantly decreased in the 28-week-old NZBWF1 mice. However, this analysis only focused on a limited number of miRNA changes in the LN mouse model, lacked a comparative analysis with healthy control mice, and thus cannot fully elucidate the relationship between baseline miRNA and mRNA levels and disease progression. Our research uses RNA and miRNA sequencing, which analyzed the entire transcriptome, not limited to known genes and miRNAs. Comparing age-matched lupus-prone MRL/lpr mice with C57BL/6 mice enabled us to identify more specific miRNAs. This approach offers a more robust and detailed understanding of the molecular dynamics involved in LN.

Hyperactive immune responses and chronic inflammatory states are key characteristics of LN [35, 36]. Through GO enrichment analyses of upregulated genes in MRL/lpr mice, we demonstrated their significant involvement in immune cell activation, differentiation, and inflammatory regulation. These processes particularly mediate immune cell infiltration into renal tissues, which amplifies inflammatory responses and promotes tissue injury. CCL19, a major CCR7 ligand, plays a critical role in immune cell trafficking and inflammatory responses [32, 37]. Studies have shown that binding of CCL19 to CCR7 not only activates the NF-κB pathway but also establishes a positive feedback loop with it, while engaging in bidirectional crosstalk with the TNF-α signaling pathway [38–41]. This synergistic mechanism effectively initiates and amplifies inflammatory signals, disrupts immune homeostasis, and thereby drives the progression of chronic inflammation. In LN, the NF-κB and TNF-α signaling pathways act as central hubs in the immunoinflammatory network, collectively mediating renal immunopathological injury [33, 42–45]. Therefore, CCL19 and its regulated NF-κB/TNF-α inflammatory axis may represent potential therapeutic targets for LN. Our research found that CCL19 is significantly elevated in both renal fibroblasts and serum of LN mice. However, although CCL19 is upregulated as a proinflammatory factor in renal tissues of LN [46], the molecular mechanisms behind its dysregulation remain poorly understood. Notably, our study identified miR-7213-5p as a critically downregulated regulatory miRNA that may play a central role in modulating immune cell trafficking and immune function regulation. We further demonstrated that miR-7213-5p directly targets the 3’-UTR of CCL19 to suppress TNF-α-driven CCL19 expression and secretion in fibroblasts. This discovery reveals a previously unrecognized anti-inflammatory pathway that may mitigate LN progression. Together, our findings highlight the potential role of the miR-7213-5p/CCL19 axis in LN pathogenesis and its promise as a therapeutic target for disease intervention.

A limitation of this study is the use of the L929 fibroblast cell line, a non-renal model, despite its utility as a controlled platform. Consequently, further validation using primary renal fibroblasts is warranted to confirm the pathophysiological relevance of the miR-7213-5p/CCL19 axis in LN.

Conclusion

To our knowledge, this is the first study to simultaneously investigate miRNA-mRNA regulatory network in the kidneys of lupus-prone MRL/lpr mice, revealing that miR-7213-5p mediates the inhibition of fibroblast CCL19 expression in LN. Further research on miR-7213-5p and CCL19 is crucial to advancing our understanding of the molecular mechanisms of LN and developing novel molecular therapeutic strategies for its treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

LN

Lupus nephritis

miRNA

microRNA

CCL19

CCL19 CC motif chemokine 19

3’-UTR

3’-untranslated region

MRL/lpr

MRL/MpJ-Fas < lpr>/J

SCr

Serum creatinine

BUN 

Blood urea nitrogen

24hUP

24-hour urinary protein

HE

Hematoxylin and eosin

Masson

Masson’s trichrome

PAS

Periodic acid-Schiff

TEM

Transmission electron microscopy

RT-qPCR

Quantitative real-time polymerase chain reaction

Author contributions

X.L. and Y.Q. conceived and designed the study. Z.G. conducted the experiments, performed data analysis, and drafted the manuscript. J.W. and L.L. assisted with the experiments. L.Z. and K.Z. contributed to data analysis and interpretation. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by grants from Natural Science Foundation of Beijing Municipality (7232128), National High-Level Hospital Clinical Research Funding under Grants (2022-PUMCH-B-020), China Postdoctoral Science Foundation (2022M710450), Tianjin Municipal Health Commission (2023037), National Key Research and Development Program of China (2022YFC2703900 and 2022YFC2703901), National Natural Science Foundation of China (82373158), and the 12th National Science and Technology Support Program (2011BAI10B02).

Data availability

The RNA sequencing and miRNA sequencing datasets generated in this study are openly available in the Gene Expression Omnibus (GEO) at accession number [GSE296205 and GSE296302], https://www.ncbi.nlm.nih.gov/geo/.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

All animal procedures received approval from the PUMCH Institutional Ethics Committee of Animal Care and Use (No. XHDW-2024-05).

Consent to participate

Not applicable.