Regulation of Fibroblast Apoptosis and Proliferation by Micro RNA ‐125b in Systemic Sclerosis

Objective To analyze the expression, regulation, and role of micro RNA ‐125b (miR‐125b) in systemic sclerosis ( SS c). Methods MiR‐125b expression was assessed by quantitative polymerase chain reaction ( qPCR ) of RNA from dermal fibroblasts and whole skin biopsy specimens from healthy controls and SS c patients. To identify downstream effectors, RNA from healthy control fibroblasts was sequenced after miR‐125b knockdown and further validated using qPCR and Western blotting. Fibrosis, apoptosis, and proliferation were assessed by Caspase‐Glo 3/7 assay, Western blotting, immunofluorescence staining for cleaved caspase 3, and annexin V real‐time assay in dermal fibroblasts. Results Expression of miR‐125b was significantly down‐regulated in SS c skin biopsy specimens by 53% (median fold change 0.47 [interquartile range 0.35–0.69]; P < 0.001) and in SS c dermal fibroblasts by 47% (median fold change 0.53 [interquartile range 0.36–0.58]; P < 0.001) compared to healthy control skin biopsy specimens and fibroblasts, respectively (n = 10 samples per group). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin A significantly decreased the expression of miR‐125b in dermal fibroblasts. MiR‐125b knockdown significantly reduced cell proliferation and α‐smooth muscle actin (α‐ SMA ) expression at the messenger RNA ( mRNA ) and protein levels. RNA ‐Seq identified BAK 1 , BMF , and BBC 3 as potential targets of miR‐125b. Quantitative PCR confirmed that knockdown of miR‐125b up‐regulated these genes ( P < 0.01; n = 12). Bcl‐2 homologous antagonist killer 1 showed the strongest induction confirmed at the protein level ( P < 0.01; n = 10). Consequently, miR‐125b knockdown increased apoptosis compared to scrambled control. Accordingly, miR‐125b overexpression decreased apoptosis. Conclusion Our findings indicate that miR‐125b is down‐regulated in SS c skin and primary dermal fibroblasts. MiR‐125b down‐regulation increases apoptosis and decreases proliferation and α‐ SMA expression in dermal fibroblasts, indicating that its compensatory, antifibrotic mechanism may be a potential novel therapeutic option.