NovelmiRNA-25 inhibits AMPD2 in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and represents a promising novel biomarker

Systemic lupus erythematosus (SLE), an autoimmune disease mediated by pathogenic autoantibodies, presents with severe clinical manifestations such as nephritis, multisystem organ failure, or central nervous system disease [1]. Prevalence rates of SLE worldwide vary from approximately 20 to 70 per 100,000 person-years, and incidence rates for women are approximately ten times higher than those for men [2]. As the pathogenesis of SLE remains unclear, treatment, which mainly consists of glucocorticoid-antimalarial drugs and non-steroidal anti-inflammatory drugs (NSAIDs), often focuses on immune suppression to prevent and control disease progression, but flare still occurs [3, 4]. Therefore, the mortality rate of SLE remains high owing to the severity of the disease and the resulting organ damage [1].

Accumulating evidence has indicated that metabolic syndrome is a major co-morbidity of SLE [5]. Of note, there is a growing understanding that cellular metabolic processes result in changes in inflammatory and immune responses in various immune cell populations [6], including macrophages, dendritic cells, neutrophils, and peripheral blood mononuclear cells (PBMCs) such as CD4⁺ T cells and B cells [7‐9]. Specific metabolic processes are critical checkpoints of effector functions in the immune system, with common as well as cell-specific programs [7]. Lupus-prone mice and SLE patients have been found to exhibit activated metabolism of CD4⁺ T cells; the use of metabolic inhibitors was found to effectively normalize these features and was associated with therapeutic effects [10]. Furthermore, a proof-of-concept trial found a reduction in clinical flare-ups following metformin add-on treatment in mild or moderate SLE [11], thereby demonstrating that drugs involved in metabolic regulation may be used as add-on treatment in autoimmune diseases.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate numerous immunologic, inflammatory, and oncogenic pathways by modulating protein translation [12, 13]. miRNAs have been shown to play crucial roles in SLE pathogenesis [14], particularly lupus susceptibility, through their fundamental mechanism of post-transcriptional regulation of gene expression, suggesting their potential utility as diagnostic markers or therapeutic targets in this disease [15]. Dysregulation of miRNAs has been associated with disease activity and major organ involvement in patients with SLE [16]. For example, compared with that in healthy individuals, the expression of miRNA miR-21 is elevated in CD4⁺ T cells from lupus patients; overexpression of miR-21 suppressed the expression of PDCD4, a selective protein translation inhibitor, and regulated aberrant T cell responses [17]. However, data on the relationship between miRNAs in PBMCs and metabolism in SLE has not yet been reported.

In this study, we first evaluated mRNA and miRNA expression profiles in PBMCs from Chinese SLE patients using next-generation sequencing (NGS). Secondly, integrating the results of our bioinformatic analysis of immunometabolism, we sought to identify potential miRNAs associated with the regulation of SLE progression and experimentally validated their differential expression between SLE patients and healthy controls (HCs). Lastly, through dual-luciferase reporter assays and western blotting, we identified the potential metabolism-associated targets that are regulated by miRNAs. The purpose of this study was to elucidate the mechanisms underlying SLE progression and investigate the potential applications of miRNAs in the treatment of this disease.

In the current study, we conducted a multi-tiered integrative analysis using miRNA and mRNA sequencing in PBMCs from subjects with SLE and HCs for the first time. We identified 29 differentially expressed miRNAs, of which 19 were upregulated and 10 downregulated in SLE, with detectable negative regulatory effects on the expression of their target genes in PBMCs from SLE patients. Unlike in previous individual microarray studies [26‐32], we performed NGS analysis of miRNAs and mRNAs from PBMCs of SLE patients simultaneously. Consequently, we identified new differentially expressed miRNAs that were not previously reported in SLE patients.

Metabolomic studies have identified the potential contributions of certain metabolites, including lipids, amino acids, nucleic acids, and carbohydrates, to inflammation; these, therefore, represent promising targets for the diagnosis and disease monitoring of SLE [27‐29]. Therefore, we selected miRNAs involved in metabolic processes to explore potential links between metabolism and SLE. Among the three selected miRNAs associated with metabolic processes, NovelmiRNA-25 and miR-1273h-5p, which were upregulated in SLE, were validated by qRT-PCR in the validation cohort.

miRNAs may be differentially expressed in the PBMCs of SLE patients and healthy individuals. However, various studies have reported conflicting data (studies are listed in Additional file 3: Table S8). Previous studies have found that miR-19b, miR-20a [30], miR-126 [31], miR-125b [32], miR-27a [33], miR-155, miR-181b, miR-17 [34], miR-7 [35], and miR-210 [36] are dysregulated in the PBMCs of SLE patients resulting in the aberrant expression of essential target genes related to the pathogenesis and development of SLE, such as PTEN [35] and HIF-1α [36]. In contrast to these previous studies, we identified two novel miRNAs, NovelmiRNA-25 and miR-1273h-5p, that may be involved in the development of SLE.

According to the known miRNA targets published in several databases, we found that the metabolism-associated target of NovelmiRNA-25 was AMPD2, which is involved in purine nucleotide catabolism [37, 38]. The miRNA-target gene interaction results revealed that AMPD2 participates in the top-scored networks, including essential biological and metabolomic processes. Among metabolic processes, AMPD2, involved in purine biosynthesis and metabolism, is essential for cellular energy homeostasis and nucleic acids synthesis [39]. Based on the GeneCard database [40], we found that AMPD2 was specifically expressed in PBMCs, including CD8⁺ T cells, CD4⁺ T cells, B cells, and monocytes. Notably, these clusters of cells participate in the pathogenesis of SLE [41, 42]. AMPD2, which is mainly produced in the liver and spleen [37], is an enzyme that converts AMP to IMP. As adenosine and ATP are both synthesized from AMP, AMPD2 was involved in several different metabolic pathways by mediating changes in adenosine and ATP levels. Downregulated AMPD2 contributed to AMP accumulation, which mediated higher adenosine and ATP levels. On the one hand, adenosine is known to possess anti-inflammatory and analgesic activities [43]. Koizumi et al. [44] reported that tofacitinib may increase the levels of adenosine in chondrocytes through the downregulation of AMPD2, indicating its anti-inflammatory activity. On the other hand, ATP is known to have pro-inflammatory effects and activates the inflammasome [45]. In patients with RA, high concentrations of ATP were found in the synovial fluid [46], implying that ATP was highly involved in RA. Moreover, some studies have revealed the pathologic roles of increased ATP in the development of inflammatory intestinal disorders [47]. Activated SLE increases the level of ATP, which possess pro-inflammation and immune affect, by regulating AMPD2 expression. Therefore, our results indicated that AMPD2 in SLE was more likely to be involved in the pro-inflammatory pathway by increasing ATP level. As AMPD2 is a metabolism-associated target gene of NovelmiRNA-25, these results indicate that NovelmiRNA-25 is a potential functional target in SLE.

Although upregulated miRNA miR-1273h-5p did not appear to be involved in disease flare-ups or organ injury in SLE, our findings did implicate miR-1273h-5p in the pathogenesis of SLE. miR-1273h-5p, with a length of 21 nt, is encoded by the intergenic sequence on chromosome 16 [48]. miR-1273h-5p was predicted to target five genes associated with metabolic processes; these genes encoded a peroxidase, glycosyltransferase, ATP synthase and hydrolase, carbohydrate phosphatase, and exoribonuclease. Additionally, miR-1273h-5p has been found to be dysregulated in cancers and other autoimmune diseases. For example, the expression of miR-1273 was shown to be increased in the pancreas of a mouse model of pancreatic cancer [49].

Numerous clinical manifestations of SLE are similar to those of other illnesses, making the disease difficult to diagnose or distinguish from other autoimmune diseases. Furthermore, there is no single laboratory indication that can be used for a definitive diagnosis of SLE [50]. In the present study, we found that miRNAs may be promising biomarkers for SLE diagnosis. An ROC curve was used to evaluate the expression of the two candidate miRNAs in the PBMCs of SLE patients, demonstrating that these miRNAs may possess clinical diagnostic value for distinguishing SLE patients from healthy individuals. Moreover, we explored the associations between these miRNAs and the clinical characteristics of SLE patients, including autoantibodies, complement, and other relevant features. The results showed that the expression of the two miRNAs was correlated with complement levels. Further analysis of miRNA levels between patients with active and stable SLE showed that the expression of NovelmiRNA-25 was positively correlated with the degree of SLE activity. This positive correlation suggests that NovelmiRNA-25 may be involved in progression and flare-ups as well as organ damage during SLE and indicates that it is a potential functional target in SLE. The results of a dual-luciferase reporter assay revealed that the overexpression of NovelmiRNA-25 resulted in the downregulation of AMPD2 gene expression in PBMCs through direct targeting of the AMPD2 3′UTR. In addition, the results of western blotting showed that the overexpression of NovelmiRNA-25 downregulated endogenous AMPD2 protein levels in a human cell line. Indicating the function of NovelmiRNA-25 in regulating AMPD2. Moreover, AMPD2 expression in the active SLE group was lower than that in the stable SLE group (Additional file 1B), and AMPD2 protein levels in SLE patients were decreased compared to those in HCs, indicating that AMPD2 was associated with SLE. This evidence further supports the theory that NovelmiRNA-25 is a promising target in the development of therapeutic strategies for SLE.

In summary, in the present work, miRNA and mRNA expression profiling in PBMCs from SLE patients and HCs identified miRNAs that are dysregulated in PBMCs from SLE patients. We further analyzed the regulatory networks of differentially expressed miRNAs and mRNAs and identified NovelmiRNA-25 and miR-1273h-5p as differentially expressed in SLE. The identification of these miRNAs may provide useful insights into the pathophysiology of SLE. Further experimental results point to a novel mechanism underlying SLE pathogenesis involving NovelmiRNA-25 and demonstrated the upregulation of this miRNA in SLE. Our study suggests that miRNAs represent novel diagnostic biomarkers, disease activity markers, and potential therapeutic targets for SLE. Further investigation is needed to clarify the roles of the identified miRNAs in the pathogenesis of SLE, with the aim of developing new methods for the diagnosis and treatment of SLE.