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MiR‑320a‑3P alleviates the epithelial‑mesenchymal transition of A549 cells by activation of STAT3/SMAD3 signaling in a pulmonary fibrosis model.

ABSTRACT: Pulmonary fibrosis (PF) is a common, chronic and incurable lung disease, in which the lungs become scarred over time. MicroRNAs (miRNAs/miRs) serve key roles in various biological processes, including cell proliferation, differentiation, apoptosis and the regulation of epithelial‑mesenchymal transition (EMT) process. The aim of the present study was to investigate the underlying mechanism of miR‑320a‑3p as a potential therapeutic target for PF. Clinical samples and microarray datasets collected from various databases were used to evaluate the expression of miR‑320a‑3p in PF. A549 cells were used to construct an EMT model of PF. A dual‑luciferase reporter assay system was used to identify target genes of miR‑320a‑3p. Western blot analysis and immunofluorescence staining were used to determine the roles of miR‑320a‑3p and its target genes in the EMT process in PF. The present study found that, compared with lung tissue of healthy control subjects, the expression of miR‑320a‑3p in lung tissue of PF patients was significantly reduced. The expression levels of miR‑320a‑3p decreased in TGF‑β1‑stimulated A549 cells in a time‑ and concentration‑dependent manner. The overexpression of miR‑320a‑3p suppressed EMT markers induced by TGF‑β1 in A549 cells and STAT3 was identified as a potential target gene of miR‑320a‑3p. Furthermore, the expression changes of miR‑320a‑3p and STAT3 were found to significantly affect the expression of phosphorylated SMAD3 in TGF‑β1‑stimulated A549 cells. Briefly, overexpression of miR‑320a‑3p could inhibit the EMT process in PF by downregulating STAT3 expression. The mechanism mediating these effects may partly involve crosstalk between the SMAD3 and STAT3.

SUBMITTER: Wang X

PROVIDER: S-EPMC7974326 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundN6-methyladenosine (m6A) is the most common and abundant internal modification of RNA. Its critical functions in multiple physiological and pathological processes have been reported. However, the role of m6A in silica-induced pulmonary fibrosis has not been fully elucidated. AlkB homolog 5 (ALKBH5), a well-known m6A demethylase, is upregulated in the silica-induced mouse pulmonary fibrosis model. Here, we sought to investigate the function of ALKBH5 in pulmonary fibrosis triggered by silica inhalation.MethodsWe performed studies with fibroblast cell lines and silica-induced mouse pulmonary fibrosis models. The expression of ALKBH5, miR-320a-3p, and forkhead box protein M1 (FOXM1) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RNA immunoprecipitation (RIP) assays and m6A RNA immunoprecipitation assays (MeRIP), western bolt, immunofluorescence assays, and 5-ethynyl-2'-deoxyuridine (EdU) fluorescence staining were performed to explore the roles of ALKBH5, miR-320a-3p, and FOXM1 in fibroblast activation.ResultsALKBH5 expression was increased in silica-inhaled mouse lung tissues and transforming growth factor (TGF)-β1-stimulated fibroblasts. Moreover, ALKBH5 knockdown exerted antifibrotic effects in vitro. Simultaneously, downregulation of ALKBH5 elevated miR-320a-3p but decreased pri-miR-320a-3p. Mechanically, ALKBH5 demethylated pri-miR-320a-3p, thus blocking the microprocessor protein DGCR8 from interacting with pri-miR-320a-3p and leading to mature process blockage of pri-miR-320a-3p. We further demonstrated that miR-320a-3p could regulate fibrosis by targeting FOXM1 messenger RNA (mRNA) 3'-untranslated region (UTR). Notably, our study also verified that ALKBH5 could also directly regulate FOXM1 in an m6A-dependent manner.ConclusionsOur findings suggest that ALKBH5 promotes silica-induced lung fibrosis via the miR-320a-3p/FOXM1 axis or targeting FOXM1 directly. Approaches aimed at ALKBH5 may be efficacious in treating lung fibrosis.

Project description:Rationale: Cardiac fibrosis is an integral constituent of every form of chronic heart disease, and persistence of fibrosis reduces tissue compliance and accelerates the progression to heart failure. Relaxin-2 is a human hormone, which has various physiological functions such as mediating renal vasodilation in pregnancy. Its recombinant form Serelaxin has recently been tested in clinical trials as a therapy for acute heart failure but did not meet its primary endpoints. The aim of this study is to examine whether Serelaxin has an anti-fibrotic effect in the heart and therefore could be beneficial in chronic heart failure. Methods: We utilized two different cardiac fibrosis mouse models (ascending aortic constriction (AAC) and Angiotensin II (ATII) administration via osmotic minipumps) to assess the anti-fibrotic potential of Serelaxin. Histological analysis, immunofluorescence staining and molecular analysis were performed to assess the fibrosis level and indicate endothelial cells which are undergoing EndMT. In vitro TGFβ1-induced endothelial-to-mesenchymal transition (EndMT) assays were performed in human coronary artery endothelial cells and mouse cardiac endothelial cells (MCECs) and were examined using molecular methods. Chromatin immunoprecipitation-qPCR assay was utilized to identify the Serelaxin effect on chromatin remodeling in the Rxfp1 promoter region in MCECs. Results: Our results demonstrate a significant and dose-dependent anti-fibrotic effect of Serelaxin in the heart in both models. We further show that Serelaxin mediates this effect, at least in part, through inhibition of EndMT through the endothelial Relaxin family peptide receptor 1 (RXFP1). We further demonstrate that Serelaxin administration is able to increase its own receptor expression (RXFP1) through epigenetic regulation in form of histone modifications by attenuating TGFβ-pSMAD2/3 signaling in endothelial cells. Conclusions: This study is the first to identify that Serelaxin increases the expression of its own receptor RXFP1 and that this mediates the inhibition of EndMT and cardiac fibrosis, suggesting that Serelaxin may have a beneficial effect as anti-fibrotic therapy in chronic heart failure.

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MiR‑320a‑3P alleviates the epithelial‑mesenchymal transition of A549 cells by activation of STAT3/SMAD3 signaling in a pulmonary fibrosis model.

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