Background MicroRNAs (miRs) are post-transcriptional negative regulators that have been implicated in Systemic Sclerosis (SSc). MiR-34a has been linked to endothelial senescence and myeloid cell driven inflammation. However its role in SSc hasn't been investigated yet.
Objectives To investigate miR-34a expression in peripheral blood (PB) CD14 cells and in paired lesional and non-lesional SSc skin biopsies.
Methods Healthy controls (HC)(n=7) and 27 patients with Raynaud phenomenon (RP) were enrolled to the study. The latter were divided into 3 groups: long standing SSc (lsSSc)(n=10), early SSc (eSSc)(n=9) and primary RP (n=8) respectively. Immunostaining (IHC) for CD68 and in situ hybridization (ISH) for miR-34a were performed on paired lesional and non-lesional skin tissues from SSc patients. In addition, miR-34a expression was evaluated by qPCR on CD14 cells isolated from PB and paired lesional and non-lesional skin samples. IL-6 receptor (IL6-R) was selected as a potential miR-34a target (TargetScan) and experimentally confirmed by qPCR. IL-6 and IL-6R plasma levels were determined by ELISA. CD14 cells from PB of HC (n=5) were stimulated with IL-6 (30ng/ml) in vitro and miR-34a expression assessed by qPCR.
Results MiR-34a expression was increased in lsSSc compared to HC (p=0.01). Early SSc and RP patients showed pattern of expression similar to HC (p=0.63 and p=0.71). IHC showed that CD68 cells are over-represented in lesional skin compared to non-lesional skin (p=0.02) and ISH revealed that miR-34a is over-expressed in lesional skin compared to non-lesional skin (p=0.001). In addition qPCR confirmed higher expression of miR-34a in lesional than in non-lesional skin (p=0.001). Consistently, IL-6R expression in PB CD14+ cells was lower in lsSSc and eSSc patients compared to primary RP (p=0.0001 and p=0.0002) whereas IL-6R expression was similar comparing primary RP and HC (p=0.73). In addition, IL-6R inversely correlated with miR-34a expression in lsSSc in PB CD14+ cells (r=-0.81; p=0.01). IL-6 plasma levels were higher in lsSSc (p=0.002) as well as in eSSc (p=0.04) compared to HC whereas no significant difference was found in soluble IL-6R plasma levels. MiR-34a expression in CD14+ cells directly correlated with the skin score value (R=0.52, p=0.03) and with IL-6 plasma levels (R=0.42; p=0.01) in SSc patients. Anti-Scl70+ patients have higher expression of miR-34a than anti-centromere+ patients (p=0.04). Considering SSc related vasculopathy, SSc patients with digital ulcers had higher miR-34a expression than SSc patients without ulcers (p=0,01). Finally, miR-34a was inducible in healthy CD14 cells by IL-6 in vitro stimulation.
Conclusions MiR-34a is IL-6 inducible miR, which expression in PB CD14+ cells positively correlates with skin fibrosis and vascular manifestations. It negatively correlates with the expression of its target IL-6R in patients with severe disease suggesting for the role of this miR in the regulation of IL-6/IL-6R pathway. MiR-34a could represent a possible biomarker to identify patients with SSc associated RP and organ involvement.
Disclosure of Interest None declared