To validate our technology and development of a prototype diagnostic assay we investigated miRNAs in Systemic sclerosis as potential biomarkers, and their potential role in SSc pathogenesis.
The aetiopathology of systemic sclerosis is poorly understood and the molecular mechanisms that underlie the excessive ECM deposition have not been truly identified yet. Representing a new class of regulatory molecules, miRNAs have recently been investigated as potential key factors in the pathogenesis of SSc. Moreover, the fact that miRNAs can reflect the pathogenic states in autoimmunity, suggests these molecules to be promising disease markers and potential therapeutic targets. We examined the miRNA signature in the serum and a cellsubset (pDC) of SSc patients. To our knowledge this is the first study investigating a comprehensive profiling in SSc patients, including subjects with or without clinically overt skin fibrosis.
Using miRNA expression profiling and validation in multiple cohorts, we demonstrate that expression of certain miRNAs are specific for SSc patients and mediate regulatory mechanisms that may contribute to the derailment of pDCs in this disease. According to prediction analysis and reporter assays, these miRNAs are able to target genes involved in pDC differentiation/activation or previously associated with SSc pathogenesis.
Our profiling analysis highlighted that the serum of SSc patients is markedly different than healthy controls in terms of miRNA expression, with 30 miRNAs altered in one or more SSc clinical subtypes. We show that one specific miRNA is incrementally upregulated in eaSSc patients, in SSc patients without fibrosis, and in subjects with overt fibrosis, including those with dcSSc with a disease duration < 2 years. Considering that these patients could progress to a more severe phenotype and develop cutaneous and/or internal organ fibrosis over time, the upregulation of this miRNA expression may represent one of the multiple molecular aberrances occurring during SSc evolution.
The data presented here support the concept that molecular aberrances occurring early in SSc pDCs could impact the entire immune system, thus inducing autoimmunity and ultimately favoring the onset of SSc and its progression. While in-vivo studies are necessary to prove the role of miRNA in SSc development, this miRNA may represent a potential novel epigenetic target for restoring immune system homeostasis in SSc and in other diseases characterized by a type I IFN signature. We show that our miRNA measurements and outcome were reproducible in an independent cohort of SSc patients, confirming that these two molecules are robustly dysregulated in SSc patients regardless of the genetic background and the disease heterogeneity. In addition, the altered miRNA demonstrated stable levels of expression over time in multiple samplings of patients and healthy controls, suggesting the validated miRNA as a potential epigenetic disease marker. Noteworthy, this miRNA was specifically up-regulated both in localized scleroderma and systemic sclerosis, but not in other systemic autoimmune diseases like systemic lupus erythematosus and primary Sjögren’s syndrome, indicating that this miRNA could be implicated in the fibrosis characterizing scleroderma patients.
Additionally, our study demonstrated that this miRNA is circulating in SSc serum enclosed in exosomes. Since the miRNA was co-expressed together with its host gene, is embedded in exosomes and is predicted to regulate fibrotic-related signaling, we hypothesize that it may be involved in a feed-forward loop that promotes ECM deposition and hence the development of fibrosis in SSc. Supporting our hypothesis, the overexpression of this miRNA in fibroblasts and endothelial cells regulated expression of fibrosis-related genes. Our data also demonstrates a possible role of the miRNA in myofibroblast differentiation, a process characterizing fibrotic tissue.
In conclusion, this study proposes that the miRNA serves as a new molecular marker for early stages of the disease and is implicated in the molecular circuity leading to fibrosis in SSc.
However, although significantly elevated in SSc as compared to other autoimmune diseases (SLE, pSS & LoS), the expression levels of the miRNA also largely overlapped with the levels in HC. For this reason we found this miRNA expression not sensitive enough to use as diagnostic candidate, and further validation and optimisation was discontinued. However, this miRNA could represent a potential biomarker for SSc, but only when combined with other specific miRNAs this could be developed as new tool for diagnosis.
