Cyclosporine A promotes cell proliferation, collagen and α-smooth muscle actin expressions in rat gingival fibroblasts by Smad3 activation and miR-29b suppression.

Abstract

Transforming growth factor (TGF)-β/Smad3 signaling promotes tissue fibrosis, and miR-29b is a downstream inhibitor of TGF-β/Smad3-mediated fibrosis, both of which may be involved in the pathogenesis of cyclosporine A-induced gingival overgrowth. The aim of this study is to investigate the effect of miR-29b and TGF-β/Smad3 signaling in cyclosporine A-stimulated rat gingival fibroblasts. We explored the effect of cyclosporine A on cell proliferation, type Ι collagen (COL1) and alpha-smooth muscle actin (α-SMA) expressions in rat gingival fibroblasts with the Cell Counting Kit-8 assay, real-time polymerase chain reaction, western blot and immunofluorescence. The expressions of TGF-β1, Smad3 and miR-29b were also evaluated after cyclosporine A treatment. The effects of Smad3 and miR-29b on cyclosporine A-induced alterations were further determined by Smad3 knockdown and miR-29b overexpression using transient transfection experiments. We found that cyclosporine A increased cell proliferation, COL1 and α-SMA expressions in rat gingival fibroblasts. TGF-β1 mRNA and protein expressions were upregulated and phosphorylated Smad3 was activated by cyclosporine A treatment, while miR-29b expression was significantly suppressed. Moreover, Smad3 knockdown and miR-29b overexpression reversed cyclosporine A-enhanced cell proliferation, COL1 and α-SMA expressions in gingival fibroblasts. In addition, the suppression of miR-29b upon cyclosporine A exposure was significantly elevated by Smad3 knockdown, whereas cyclosporine A-induced Smad3 activation was not altered by miR-29b overexpression. These results suggest that cyclosporine A promotes cell proliferation, collagen and α-SMA expressions in rat gingival fibroblasts by Smad3 activation and miR-29b suppression.