Abstract
Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. MicroRNA (miR)-1946a is predicted to bind to the TGFβ1 3′ untranslated region (UTR), thereby inhibiting its transcription. We hypothesize that alcohol suppresses miR-1946a and induces TGFβ1. Primary murine lung fibroblasts (PLFs) were cultured ± alcohol, miR-1946a mimic or inhibitor, and TGFβ1 signaling inhibitors. miR-1946a was analyzed after alcohol treatment in vitro and in vivo. TGFβ1 expression and TGFβ1 3′UTR-luciferase activity was quantified. We showed that alcohol suppressed miR-1946a in the alcohol-fed mouse lungs and PLFs. MiR-1946a inhibitor increased TGFβ1 expression in the fibroblast. MiR-1946a mimic treatment suppressed TGFβ1 gene expression and TGFβ1 3′UTR activity. Overexpression of miR1946a inhibited alcohol-induced TGFβ1 gene and protein expression as well as alcohol-induced TGFβ1 and α-smooth muscle actin (SMA) protein expression in PLFs. In conclusion, miR-1946a modulates TGFβ1 expression through direct interaction with TGFβ1 3′UTR. These findings identify a novel mechanism by which alcohol induces TGFβ1 in the lung.
Highlights
Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice
We previously showed that chronic alcohol ingestion exaggerated TGFβ1 expression through an augmentation of miR-21 expression, in both the lung and lung fibroblasts[6,17]
By co-transfecting Primary murine lung fibroblasts (PLFs) with the firefly luciferase-expressing plasmid containing wild-type TGFβ1 3′untranslated region (UTR) and miR-1946a mimic, we demonstrated that miR-1946a mimic significantly suppressed luciferase activity in cells transfected with plasmid containing wildtype TGFβ1 3′ untranslated region (3′UTR) (P < 0.05)
Summary
Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. The tissue repair process includes inflammatory cell migration to the injury site, expression of chemokines and cytokines, fibroblast migration to the injury site, generation of a provisional extracellular matrix (ECM), re-growth of epithelial cells, and subsequent apoptosis of activated fibroblasts[2]. When this tightly regulated process is disrupted, the result is unresolved inflammation leading to pathologic wound repair and subsequent organ dysfunction[3]. We hypothesize that alcohol induces TGFβ1 transcription by inhibiting miR-1946a expression
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