miR-3606-3p alleviates skin fibrosis by integratively suppressing the integrin/FAK, p-AKT/p-ERK, and TGF-β signaling cascades

Abstract

In Brief Current treatment options for systemic sclerosis (SSc) and keloid including surgery and corticosteroids each have their respective limitations, thus commanding the need for new methods of tackling these diseases. Impact: Our findings suggest that miR-3606-3p may be a novel potential target for various skin fibrosis diseases.Our findings add a new dimension to the interventional strategy for skin fibrosis by demonstrating the multiple effects of miR-3606-3p in simultaneously inhibiting fibroblast cell proliferation, inflammation, migration, and collagen deposition. Our study provides a valuable method to measure cell migration in vivo in a humanized keloid mouse model. • Downregulation of miR-3606-3p correlates with the disease severity of skin fibrosis. • miR-3606-3p targets the 3′-UTRs of ITGAV, GAB1 , and TGFBR2 in fibroblasts. • miR-3606-3p downregulation activates ITGAV/FAK, GAB1/AKT/ERK and TGFBR2/SMAD2/3. • These cascades integratively promote proliferation, migration, and inflammation. • miR-3606-3p alleviates skin fibrosis in keloid-bearing humanized mice. Fibroblast abnormalities are crucial causes of skin fibrosis, including systemic sclerosis (SSc) and keloids. However, their mechanisms, including underlying microRNA regulatory mechanisms, remain elusive. This study aimed to evaluate the roles, mechanisms, and therapeutic potential of miR-3606-3p in regulating multiple fibroblast abnormalities. The miR-3606-3p levels were evaluated in skin tissues and primary fibroblasts. RNA-seq and luciferase assays were employed to identify miR-3606-3p targets. Collagen contraction, western blotting, in vivo imaging, and real-time cellular analysis were used to assess fibroblast abnormalities. The therapeutic potential of miR-3606-3p was evaluated in mice. MiR-3606-3p decreased in skin tissues (SSc: Fold Change (FC) = − 2.95, P = 0.0101; keloid: FC = − 3.42, P < 0.0001) and primary fibroblasts (SSc: FC = − 12.74, P = 0.0278; keloid: FC = − 2.08, P = 0.0021) from skin fibrosis patients, and negatively correlated with disease severity. Mechanistically, miR-3606-3p targeted the 3′-untranslated regions (3′-UTRs) of Integrin αV ( ITGAV ), GRB2-associated binding protein 1 ( GAB1 ), and transforming growth factor beta receptor 2 ( TGFBR2 ), all of these three targets increased in skin fibrosis. Simultaneously, miR-3606-3p inhibited fibroblast’s fibrogenesis, migration, inflammation, and proliferation by inhibiting ITGAV/integrin/FAK, GAB1/p-AKT/p-ERK, and TGFBR2/p-SMAD2/3 signaling. ITGAV-mediated integrin/FAK signaling unidirectionally activated the p-AKT/p-ERK and p-SMAD2/3 pathways. Knockdown of GAB1 and TGFRB2 reduced ITGAV-induced p-AKT/p-ERK and p-SMAD2/3 activities. MiR-3606-3p, si-ITGAV , si-GAB1 , and si-TGFBR2 exhibited significant inhibition of fibrogenesis and migration. Inflammation was primarily inhibited by si-ITGAV and si-GAB1, while proliferation was primarily inhibited by si-TGFBR2 . Moreover , miR-3606-3p significantly attenuates skin fibrosis in keloid-bearing mice. MiR-3606-3p is downregulated in skin fibrosis. Moreover, it negatively correlates with disease severity. Functionally, miR-3606-3p inhibits fibrogenesis, migration, inflammation, and proliferation of fibroblasts. Mechanistically, miR-3606-3p inhibits ITGAV , GAB1 , and TGFBR2 by targeting their 3′-UTRs. ITGAV-, GAB1-, and TGFBR2-activated integrin/AKT/ERK/SMAD2/3 signaling induced fibroblast abnormalities. In vivo, miR-3606-3p inhibits skin fibrosis in mice. Therefore, the multi-targeting, multi-phenotypic regulatory properties of miR-3606-3p suggest its potential utility in clinical treatment.

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