Circular RNA RORβ regulates TGFβR1 in alcohol-induced fibroblast-to-myofibroblast differentiation

Alcohol exposure augments the expression and signaling of transforming growth factor-beta (TGFβ), leading to fibroproliferation. We showed that inhibition of TGFβ receptor type 1 (TGFβR1) mitigates the effect of alcohol in the lung. We further demonstrated that alcohol modulates TGFβ signaling, partly through its ability to modify microRNA (miRNA or miR) expressions in the lung. The mechanism by which alcohol mediates miRNA expressions and how its connection to TGFβ signaling has not been well elucidated. Circular RNAs (circRNAs or circ) have emerged as potential therapeutic targets due to their stability, tissue specificity, and ability to modify miRNAs. Human and murine lung fibroblasts (LF) were treated ± ethanol (60 mM). Samples were analyzed for TGFβR1 and circ-RORβ levels. In silico analysis was performed to identify common miRNAs with binding sites for both TGFβR1 and circ-RORβ. The top 10 miRNA levels in human LF (HLF) ± ethanol were analyzed. Human LF were treated with circ-RORβ anti-sense oligonucleotide (ASO) ± ethanol, and samples were collected for miR-140-3p, TGFβR1, fibronectin (FN1), and α-smooth muscle actin (αSMA) levels, and stress fiber formation analyses. In parallel, HLF were treated with mimic miR-140-3p and analyzed for TGFβR1 mRNA levels. HLF were treated ± RORβ silencing RNA, then analyzed for RORβ, circ-RORβ, miR-140-3p, and TGFβR1 levels. We showed that ethanol upregulates TGFβR1 and circ-RORβ in ethanol-treated LF. In silico analysis revealed that miR-140-3p has putative binding sites for both TGFβR1 3' untranslated region (UTR) and circ-RORβ. We demonstrated that ethanol exposure attenuated LF 's miR-140-3p expression, and inhibition of circ-RORβ abrogated ethanol-mediated miR-140-3p suppression. Inhibition of circ-RORβ attenuated ethanol-induced TGFβR1, FN1, and αSMA expressions, as well as αSMA stress fiber formation in LF, while inhibition of RORβ did not alter circ-RORβ, miR-140-3p, or TGFβR1 levels. Overexpression of miR-140-3p promotes TGFβR1 mRNA degradation and inhibits ethanol-induced TGFβR1 expression while has no impact on circ-RORβ level. Taken together, our findings suggest the potential role of circ-RORβ-miR-140-3p-TGFβR1 axis in alcohol-induced TGFβ signaling and fibroblast-to-myofibroblast differentiation.