Background/Purpose: MicroRNAs (miRs) are a class of small, noncoding RNAs that regulate many biological processes. Some microRNAs are involved in skin fibrosis. Here, we aimed to analyze the differential expression and regulation of miR-125b and its pathophysiological role in systemic sclerosis (SSc).

Methods: Low density array was run on pooled RNA from 3 SSc and 3 healthy controls’ fibroblasts. Further validation was performed by qPCR on RNA derived from cultured fibroblasts as well as from whole skin biopsies. Fibroblasts were stimulated with pro-inflammatory cytokines such as TGFβ, IL-1β, -4, -13, -17A, and PDGF. In order to identify downstream effects of miR-125b, knockdown with anti-miR-125b (or scrambled controls) in healthy controls’ (HC) fibroblasts was performed to mimic the condition of SSc patients. RNA was isolated from healthy fibroblasts (n=4) after 24 hours of knockdown and was proceeded to deep sequencing using Ilumina HiSeq2000. After bioinformatic analysis, validation of the sequencing data was performed with qPCR on the RNA of the same as well as additional HC fibroblasts. Apoptosis was assessed by Caspase-Glo 3/7 assay.

Results: Screening presented miR-125b as one of the candidate miRs differently expressed in SSc. That was further validated by qPCR in primary dermal fibroblasts (SSc patients = 11, HC = 8), where it was downregulated by 47% (median 53%, first quartile 33%, third quartile 70%; p<0.01). Additionally, miR expression was measured in skin biopsies of both SSc patients (n=4) and HC (n=5). In patients’ samples, miR-125b was downregulated by 35.5% (median 64.5%, first quartile 60.5%, third quartile 77.5%; p<0.05). To localize the expression of miR-125b, we separately measured the expression in dermis and epidermis of paraffin fixed tissues. In both cases, expression of miR-125b was downregulated. MiR-125b expression appeared to be independent from main pro-inflammatory cytokines action. RNA sequencing identified > 3500 differentially expressed genes with p<0.05. More than half of the highly expressed genes with at least 15% change were predicted targets of miR-125b by TargetScan and MiRWalk, indicating successful functional inhibition of miR-125b. Bioinformatic analysis revealed extracellular matrix organization and apoptosis regulation as the two main clusters of differentially expressed genes. Among them, BAK1 and BMF are participants of the BCL2 apoptosis pathway and predicted targets of miR-125b. Consistent with the sequencing results, qPCR showed upregulation of these genes after knockdown of miR-125b. Interestingly, BCL2 which by itself is an anti-apoptotic factor and also a target of miR-125b, was not changed after knockdown. This indicates that physiological miR-125b expression prevents cells form apoptosis. Accordingly, miR-125b knockdown resulted in 70% more apoptosis compared to either untreated cells or scrambled controls.

Conclusion:  This is the first time microRNA-125b is shown to be differently expressed in SSc skin and primary dermal fibroblasts. Its downregulation increases apoptosis and might be a potential compensatory mechanism to prevent fibrosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microrna-125b-as-a-potential-anti-fibrotic-and-anti-apoptotic-regulator-in-systemic-sclerosis/