Sueblinvong Viranuj, Fan Xian, Kartmouty-Quintana Harry, Kopp Benjamin T, Kang Bum-Yong
Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, GA, 30322, USA.
University of Texas Health Science Center at Houston, Houston, TX, 77030, USA.
Sci Rep. 2025 Sep 2;15(1):32295. doi: 10.1038/s41598-025-15040-6.
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.
酒精暴露会增强转化生长因子-β（TGFβ）的表达和信号传导，导致纤维组织增生。我们发现抑制1型TGFβ受体（TGFβR1）可减轻酒精对肺部的影响。我们进一步证明，酒精可调节TGFβ信号传导，部分是通过其改变肺中微小RNA（miRNA或miR）表达的能力。酒精介导miRNA表达的机制及其与TGFβ信号传导的联系尚未完全阐明。环状RNA（circRNA或circ）因其稳定性、组织特异性以及修饰miRNA的能力而成为潜在的治疗靶点。对人及小鼠肺成纤维细胞（LF）进行±乙醇（60 mM）处理。分析样本中TGFβR1和circ-RORβ的水平。进行计算机分析以鉴定同时具有TGFβR1和circ-RORβ结合位点的常见miRNA。分析人LF（HLF）±乙醇处理后的前10种miRNA水平。用circ-RORβ反义寡核苷酸（ASO）±乙醇处理人LF，并收集样本分析miR-140-3p、TGFβR1、纤连蛋白（FN1）和α平滑肌肌动蛋白（αSMA）的水平以及应力纤维形成情况。同时，用模拟miR-140-3p处理HLF并分析TGFβR1 mRNA水平。用±RORβ沉默RNA处理HLF，然后分析RORβ、circ-RORβ、miR-140-3p和TGFβR1的水平。我们发现乙醇上调了乙醇处理的LF中的TGFβR1和circ-RORβ。计算机分析显示miR-140-3p在TGFβR1的3'非翻译区（UTR）和circ-RORβ上均有假定的结合位点。我们证明乙醇暴露会减弱LF中miR-140-3p的表达，而抑制circ-RORβ可消除乙醇介导的miR-140-3p抑制。抑制circ-RORβ可减弱乙醇诱导的LF中TGFβR1、FN1和αSMA的表达以及αSMA应力纤维的形成，而抑制RORβ并未改变circ-RORβ、miR-140-3p或TGFβR1的水平。miR-140-3p的过表达促进TGFβR1 mRNA降解并抑制乙醇诱导的TGFβR1表达，而对circ-RORβ水平无影响。综上所述，我们的研究结果表明circ-RORβ-miR-140-3p-TGFβR1轴在酒精诱导的TGFβ信号传导和成纤维细胞向肌成纤维细胞分化中具有潜在作用。
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