Abstract
OBJECTIVE: To investigate the role of microRNA (miRNA) as posttranscriptional regulators of profibrotic genes in systemic sclerosis (SSc). METHODS: MicroRNA, which target collagens, were identified by in silico analysis. Expression of miRNA-29 (miR-29) was determined by TaqMan real-time polymerase chain reaction analysis of skin biopsy and fibroblast samples from SSc patients and healthy controls as well as in the mouse model of bleomycin-induced skin fibrosis. Cells were transfected with precursor miRNA (pre-miRNA)/anti-miRNA of miR-29 using Lipofectamine. Collagen gene expression was also studied in luciferase reporter gene assays. For stimulation, recombinant transforming growth factor beta (TGFbeta), platelet-derived growth factor B (PDGF-B), or interleukin-4 (IL-4) was used. The effects of inhibiting PDGF-B and TGFbeta signaling on the levels of miR-29 were studied in vitro and in the bleomycin model. RESULTS: We found that miR-29a was strongly down-regulated in SSc fibroblasts and skin sections as compared with the healthy controls. Overexpression in SSc fibroblasts significantly decreased, and accordingly, knockdown in normal fibroblasts increased, the levels of messenger RNA and protein for type I and type III collagen. In the reporter gene assay, cotransfection with pre-miR-29a significantly decreased the relative luciferase activity, which suggests a direct regulation of collagen by miR-29a. TGFbeta, PDGF-B, or IL-4 reduced the levels of miR-29a in normal fibroblasts to those seen in SSc fibroblasts. Similar to human SSc, the expression of miR-29a was reduced in the bleomycin model of skin fibrosis. Inhibition of PDGF-B and TGFbeta pathways by treatment with imatinib restored the levels of miR-29a in vitro and in the bleomycin model in vivo. CONCLUSION: These data add the posttranscriptional regulation of collagens by miR-29a as a novel aspect to the fibrogenesis of SSc and suggest miR-29a as a potential therapeutic target.
Maurer, B