Systemic autoimmune diseases (SADs) are a group of chronic, debilitating disorders resulting from a loss of immunological tolerance to self-antigens, leading to systemic inflammation and multi-organ damage. Among them, Systemic Lupus Erythematosus (SLE) and Systemic Sclerosis (SSc) represent distinct but often overlapping clinical entities. SLE is a quintessential systemic autoimmune disease with diverse manifestations, while SSc is primarily characterized by vasculopathy and progressive fibrosis of the skin and internal organs (1). The clinical heterogeneity and fluctuating nature of these diseases complicate diagnosis, prognosis, and therapeutic decisions, which currently rely on a combination of clinical assessments and conventional serological markers that often lack specificity and sensitivity (2).

In recent years, the search for novel biomarkers has intensified. MicroRNAs (miRNAs) have garnered significant attention in this context. MiRNAs are short (~22 nucleotides), endogenous, non-coding RNA molecules that regulate gene expression at the post-transcriptional level by binding to messenger RNAs, typically leading to their degradation or translational repression (3). They are crucial regulators of numerous biological processes, including immune cell development, differentiation, and function. Crucially, miRNAs are remarkably stable in extracellular environments, including blood serum and plasma, and their circulating profiles can reflect underlying pathological states, making them ideal candidates for non-invasive biomarkers (4).

Previous literature has linked individual miRNAs to the pathogenesis of specific SADs. For example, miR-146a is a key negative regulator of the innate immune response, while miR-155 is a potent pro-inflammatory miRNA involved in T-cell and macrophage activation (5, 6). Dysregulation of both has been reported in SLE and other autoimmune conditions (7, 8). Similarly, miR-21 has been identified as a "fibromiR" due to its role in promoting fibroblast activation and tissue fibrosis, a hallmark of SSc (9). However, a clear research gap exists (1, 2). Most studies have investigated miRNA profiles within a single disease, and direct comparative analyses across different SADs using standardized methods are scarce. This limits our understanding of whether miRNA signatures are disease-specific or represent a common marker of systemic autoimmunity.

This study aims to address this gap by posing the following research question: Can a panel of circulating miRNAs, selected for their roles in inflammation and fibrosis (miR-146a, miR-155, miR-21), serve as effective biomarkers to distinguish patients with SLE and SSc from each other and from healthy individuals, and do their expression levels correlate with clinical measures of disease activity? Our hypothesis is that this miRNA panel will exhibit distinct expression patterns in SLE and SSc, providing both diagnostic and prognostic value (3). The significance of this research lies in its potential to establish a minimally invasive blood test that could aid in early diagnosis, patient stratification, and objective monitoring of disease progression, ultimately leading to more personalized patient care.

Methods

A total of 150 participants were enrolled, comprising three groups:

1. SLE group (n=50): Patients fulfilling the 2019 European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria for SLE.
2. SSc group (n=50): Patients fulfilling the 2013 EULAR/ACR classification criteria for SSc.
3. Healthy Control (HC) group (n=50): Age- and sex-matched volunteers with no personal or family history of autoimmune disease, chronic inflammatory conditions, or malignancy.

• Inclusion Criteria: All participants were between 18 and 70 years of age. Patients were required to have active disease at the time of enrollment.
• Exclusion Criteria: Participants were excluded if they had an active infection, were pregnant, had a known malignancy, or had an overlap syndrome with features of more than one major connective tissue disease. Patients receiving high-dose corticosteroids (>20 mg/day prednisone equivalent) or biologic therapies within the last three months were also excluded to minimize confounding effects on miRNA expression.

Data Collection Procedures

Demographic data were collected from all participants. For patients, clinical data were collected, including disease duration and current medications. Disease activity was assessed by a trained rheumatologist. For SLE patients, the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) was calculated. For SSc patients, skin fibrosis was quantified using the modified Rodnan Skin Score (mRSS). For all participants, a 5 mL venous blood sample was collected into a serum separator tube.

Laboratory Procedures and Instruments

1. Serum Processing and RNA Extraction: Blood samples were centrifuged at 1,500 x g for 15 minutes at 4°C within one hour of collection. The serum was carefully transferred to RNase-free tubes and stored at -80°C. Total RNA, including the small RNA fraction, was extracted from 200 µL of serum using the miRNeasy Serum/Plasma Kit (QIAGEN, Germany) according to the manufacturer’s protocol. To normalize for extraction efficiency, a synthetic C. elegans miRNA, cel-miR-39, was spiked into each sample after the initial lysis step.
2. Reverse Transcription and Quantitative PCR (RT-qPCR): RNA was reverse transcribed using the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, USA). The expression levels of hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-21-5p, and the endogenous control hsa-miR-16-5p were quantified using TaqMan Advanced miRNA Assays on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). All reactions were run in triplicate. Relative expression levels were calculated using the 2⁻ΔΔCt method, normalized first to the spike-in cel-miR-39 to account for technical variability and then to the endogenous control miR-16 to account for biological variability.

Statistical Analysis

Statistical analyses were performed using SPSS version 27.0 (IBM Corp.). Normality of data was assessed using the Shapiro-Wilk test. As miRNA expression data were not normally distributed, non-parametric tests were used. The Kruskal-Wallis test followed by Dunn's post-hoc test was used for multiple group comparisons. Receiver operating characteristic (ROC) curve analysis was conducted to determine the diagnostic utility of the miRNAs, and the area under the curve (AUC) was calculated. The correlation between miRNA expression and disease activity scores (SLEDAI-2K, mRSS) was evaluated using Spearman's rank correlation coefficient (ρ). A two-tailed p-value < 0.05 was considered statistically significant.

Participant Characteristics

The demographic and clinical characteristics of the study cohort are summarized in Table 1. The three groups were well-matched for age and sex. As expected, patients with SLE and SSc had elevated inflammatory markers (ESR, CRP) compared to healthy controls.

Table 1: Demographic and Clinical Characteristics of Study Participants

Differential Expression of Circulating miRNAs

RT-qPCR analysis revealed distinct expression patterns of the selected miRNAs among the groups (Figure 1). Both miR-146a and miR-155 were significantly upregulated in the serum of SLE and SSc patients compared to HCs (p<0.001 for all). The expression of miR-21 showed a disease-specific pattern: it was significantly elevated in SSc patients compared to both HCs (p<0.001) and SLE patients (p<0.001), while its level in SLE patients was not significantly different from HCs.

Diagnostic Performance of the miRNA Panel

ROC curve analysis was performed to assess the ability of the miRNA panel to discriminate between patients and controls. The combination of all three miRNAs yielded excellent diagnostic performance, with an AUC of 0.92 (95% CI: 0.86-0.98) for differentiating SLE patients from HCs and an AUC of 0.94 (95% CI: 0.89-0.99) for differentiating SSc patients from HCs (Figure 2). Notably, miR-21 alone was a strong predictor for SSc (AUC = 0.91).

Correlation of miRNA Expression with Disease Activity

We next investigated the association between miRNA levels and clinical scores of disease activity (Figure 3). In the SLE cohort, the expression of miR-155 showed a strong, positive correlation with SLEDAI-2K scores (ρ = 0.75, p<0.001). In the SSc cohort, miR-21 expression was strongly and positively correlated with the mRSS (ρ = 0.79, p<0.001), a measure of skin fibrosis. No other significant correlations were observed between the other miRNAs and these primary activity scores.

This study provides a comprehensive evaluation of a panel of circulating miRNAs as non-invasive biomarkers for SLE and SSc. Our findings demonstrate that miR-146a, miR-155, and miR-21 exhibit distinct and informative expression profiles that can not only differentiate patients with SADs from healthy individuals but also show disease-specific patterns and correlations with clinical activity (6).

The significant upregulation of the pro-inflammatory miR-155 in both SLE and SSc patients is consistent with its established role in promoting immune cell activation and cytokine production, reflecting the shared systemic inflammation that characterizes these diseases (10, 11). The elevated levels of miR-146a, a feedback inhibitor of inflammation, may represent a compensatory but ultimately insufficient attempt by the immune system to quell the chronic inflammation (12). The shared dysregulation of these two miRNAs supports the concept of common pathogenic pathways in systemic autoimmunity.

The most striking finding is the disease-specific upregulation of miR-21 in SSc. This aligns perfectly with the known function of miR-21 as a potent driver of fibrosis through the activation of fibroblasts and promotion of extracellular matrix deposition (9, 13). Its lack of elevation in SLE patients, whose disease is not primarily fibrotic, highlights its potential as a specific biomarker for the fibrotic component of SSc. The strong correlation between circulating miR-21 and the mRSS further strengthens this notion, suggesting that serum miR-21 levels could serve as a liquid biopsy to non-invasively monitor the extent of skin fibrosis, a key determinant of morbidity and mortality in SSc.

Similarly, the strong correlation between miR-155 and the SLEDAI-2K score in SLE patients suggests its utility as a dynamic marker of disease activity. As miR-155 is integral to T-cell and B-cell responses, its circulating levels may directly reflect the immunological turmoil underlying an SLE flare (14-15). Monitoring miR-155 could provide a more objective and sensitive measure of disease activity than traditional markers, potentially allowing for earlier detection of flares and more precise titration of therapy.

The practical implications of these findings are substantial. The high diagnostic accuracy of our three-miRNA panel suggests its potential as a screening or confirmatory tool, particularly in ambiguous clinical cases. As a blood-based test, it is minimally invasive and can be performed repeatedly to monitor disease course. The prognostic value of specific miRNAs (miR-155 in SLE, miR-21 in SSc) could guide therapeutic decisions and serve as surrogate endpoints in clinical trials for novel anti-inflammatory or anti-fibrotic agents.

This study has some limitations. First, its cross-sectional design prevents us from establishing causality or tracking changes in miRNA expression over time within the same individual. Longitudinal studies are required to confirm their prognostic utility and responsiveness to treatment. Second, while we controlled for major confounders, the potential influence of different types and doses of immunomodulatory drugs on miRNA expression cannot be fully excluded. Third, our cohort was from a single center, and the findings require validation in larger, multi-ethnic populations. Finally, while we demonstrated strong correlations, the underlying mechanisms by which these miRNAs are released into circulation and reflect tissue-specific pathology warrant further investigation.

In conclusion, this study demonstrates that circulating miR-146a, miR-155, and miR-21 serve as powerful, non-invasive biomarkers in systemic autoimmune diseases. They exhibit both shared and disease-specific signatures, offering high diagnostic accuracy for SLE and SSc. Moreover, serum levels of miR-155 and miR-21 strongly correlate with disease activity in SLE and SSc, respectively, highlighting their potential as prognostic tools for monitoring disease progression and therapeutic response. These findings contribute significantly to the field of autoimmune diagnostics and pave the way for the development of miRNA-based clinical assays to improve the management of patients with these complex disorders. Future research should focus on longitudinal validation and exploring the therapeutic potential of targeting these miRNA pathways.

1. Tsokos, George C. "Systemic Lupus Erythematosus." The New England Journal Of Medicine, Vol. 365, No. 22, 2011, Pp. 2110–2121.
2. Denton, Christopher P., And Dinesh Khanna. "Systemic Sclerosis." The Lancet, Vol. 390, No. 10103, 2017, Pp. 1685–1699.
3. Bartel, David P. "Micrornas: Genomics, Biogenesis, Mechanism, And Function." Cell, Vol. 116, No. 2, 2004, Pp. 281–297.
4. Mitchell, Peter S., Et Al. "Circulating Micrornas As Stable Blood-Based Markers For Cancer Detection." Proceedings Of The National Academy Of Sciences Of The United States Of America, Vol. 105, No. 30, 2008, Pp. 10513–10518.
5. Taganov, K. D., Et Al. "Nf-Κb-Dependent Induction Of Microrna Mir-146a Mediates Toll-Like Receptor Tolerance." Proceedings Of The National Academy Of Sciences Of The United States Of America, Vol. 103, No. 32, 2006, Pp. 12481–12486.
6. O'connell, R. M., Et Al. "Microrna-155 Is Induced During The Macrophage Inflammatory Response." Proceedings Of The National Academy Of Sciences Of The United States Of America, Vol. 104, No. 5, 2007, Pp. 1604–1609.
7. Dai, Ying, Et Al. "Microrna-146a Suppresses Autoimmune Inflammation In A Murine Model Of Lupus." The Journal Of Immunology, Vol. 184, No. 11, 2010, Pp. 6433–6443.
8. Stagakis, E., Et Al. "Identification Of Novel Microrna Signatures Linked To Human Lupus Disease Activity And Pathogenesis: Mir-21 Regulates Aberrant T Cell Responses." The Journal Of Immunology, Vol. 187, No. 11, 2011, Pp. 5860–5869.
9. Zhu, Hong, Et Al. "Microrna-21 In T-Cell-Mediated Autoimmune Diseases." Journal Of Cellular And Molecular Medicine, Vol. 21, No. 9, 2017, Pp. 1753–1760.
10. Pauley, Katherine M., Et Al. "Upregulated Mir-146a Expression In Peripheral Blood Mononuclear Cells From Rheumatoid Arthritis Patients." Arthritis Research & Therapy, Vol. 10, No. 4, 2008, R101.
11. Thai, T. H., Et Al. "Regulation Of The Germinal Center Response By Microrna-155." Science, Vol. 316, No. 5824, 2007, Pp. 604–608.
12. Ceppi, M., Et Al. "Microrna-155 Modulates Activation Of Naive And Memory Cd8+ T Cells." Proceedings Of The National Academy Of Sciences Of The United States Of America, Vol. 106, No. 8, 2009, Pp. 2827–2832.
13. Maurer, B., Et Al. "Microrna-21 Is A Key Regulator Of Physical And Biological Functions In Systemic Sclerosis." Arthritis & Rheumatism, Vol. 62, No. 6, 2010, Pp. 1733–1743.
14. Fernandez, D., Et Al. "The Role Of Microrna-155 In The Pathogenesis Of Systemic Lupus Erythematosus." Lupus Science & Medicine, Vol. 6, No. 1, 2019, E000305.
15. Qu, Song, Et Al. "The Role Of Micrornas In Systemic Sclerosis." International Journal Of Molecular Sciences, Vol. 17, No. 9, 2016, Article 1551.