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MicroRNA-125b Regulates Fibroblast Apoptosis Proliferation in Systemic Sclerosis


Kozlova, Anastasiia; Pachera, Elena; Maurer, Britta; Jüngel, Astrid; Distler, Jörg H W; Kania, Gabriela; Distler, Oliver (2019). MicroRNA-125b Regulates Fibroblast Apoptosis Proliferation in Systemic Sclerosis. Arthritis and Rheumatology, 71(12):2068-2080.

Abstract

OBJECTIVE
The aim of the study was to analyze the expression, regulation and role of microRNA-125b (miR-125b) in systemic sclerosis (SSc).
METHODS
MiR-125b expression was assessed by qPCR on RNA from dermal fibroblasts and whole skin biopsies of healthy controls (HC) and SSc patients. To identify downstream effectors, RNA from HC fibroblasts after miR-125b knockdown was sequenced and further validated using qPCR and Western blot. Fibrosis, apoptosis and proliferation were assessed by Caspase-Glo 3/7 assay, Western blot, immunofluorescence for cleaved caspase 3 and Annexin V live assay in dermal fibroblasts.
RESULTS
Expression of miR-125b was significantly downregulated in SSc skin biopsies by 53% (median 0.47, Q 0.35, 0.69; p<0.001) and in SSc dermal fibroblasts by 47% (median 0.53, Q 0.36, 0.58; p<0.001) in comparison to HC skin biopsies and fibroblasts, respectively (n=10, each). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin significantly decreased the expression of miR-125b in dermal fibroblasts. MiR-125b knockdown significantly reduced cell proliferation and αSMA expression at RNA and protein levels. RNAseq identified BAK1, BMF and BBC3 as potential targets of miR-125b. qPCR confirmed that knockdown of miR-125b upregulated these genes (n=12, p<0.01). BAK1 showed the strongest induction confirmed on protein level (n=10, p<0.01). Consequently, miR-125b knockdown increased apoptosis compared to scrambled controls. Accordingly, miR-125b overexpression decreased apoptosis.
CONCLUSION
MiR-125b is downregulated in SSc skin and primary SSc dermal fibroblasts. MiR-125b downregulation increases apoptosis, decreases proliferation and αSMA expression in dermal fibroblasts indicating a compensatory, anti-fibrotic mechanism as a potential novel therapeutic option. This article is protected by copyright. All rights reserved.

Abstract

OBJECTIVE
The aim of the study was to analyze the expression, regulation and role of microRNA-125b (miR-125b) in systemic sclerosis (SSc).
METHODS
MiR-125b expression was assessed by qPCR on RNA from dermal fibroblasts and whole skin biopsies of healthy controls (HC) and SSc patients. To identify downstream effectors, RNA from HC fibroblasts after miR-125b knockdown was sequenced and further validated using qPCR and Western blot. Fibrosis, apoptosis and proliferation were assessed by Caspase-Glo 3/7 assay, Western blot, immunofluorescence for cleaved caspase 3 and Annexin V live assay in dermal fibroblasts.
RESULTS
Expression of miR-125b was significantly downregulated in SSc skin biopsies by 53% (median 0.47, Q 0.35, 0.69; p<0.001) and in SSc dermal fibroblasts by 47% (median 0.53, Q 0.36, 0.58; p<0.001) in comparison to HC skin biopsies and fibroblasts, respectively (n=10, each). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin significantly decreased the expression of miR-125b in dermal fibroblasts. MiR-125b knockdown significantly reduced cell proliferation and αSMA expression at RNA and protein levels. RNAseq identified BAK1, BMF and BBC3 as potential targets of miR-125b. qPCR confirmed that knockdown of miR-125b upregulated these genes (n=12, p<0.01). BAK1 showed the strongest induction confirmed on protein level (n=10, p<0.01). Consequently, miR-125b knockdown increased apoptosis compared to scrambled controls. Accordingly, miR-125b overexpression decreased apoptosis.
CONCLUSION
MiR-125b is downregulated in SSc skin and primary SSc dermal fibroblasts. MiR-125b downregulation increases apoptosis, decreases proliferation and αSMA expression in dermal fibroblasts indicating a compensatory, anti-fibrotic mechanism as a potential novel therapeutic option. This article is protected by copyright. All rights reserved.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Rheumatology Clinic and Institute of Physical Medicine
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Health Sciences > Immunology and Allergy
Health Sciences > Rheumatology
Life Sciences > Immunology
Language:English
Date:1 December 2019
Deposited On:16 Aug 2019 08:07
Last Modified:29 Jul 2020 11:08
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:2326-5191
OA Status:Green
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/art.41041
PubMed ID:31309742

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