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.

Keywords: pulmonary fibrosis; epithelial‑mesenchymal transition; A549; miR‑320a‑3p; STAT3; SMAD3.

Figure 1.

Expression levels of miR-320a-3p in PF tissue samples and normal tissue samples. (A-C) The relative expression levels of miR-320a-3p were measured in of three datasets, which were mined from the GEO and ArrayExpress databases. (D) The relative expression levels of miR-320a-3p in clinical samples were determined by reverse transcription-quantitative PCR. miR, microRNA; PF, pulmonary fibrosis; GEO, gene expression Omnibus; NS, no significant difference.

Figure 2.

Expression levels of miR-320a-3p in A549 cells and the effects of miR-320a-3p on EMT in TGF-β1-stimulated A549 cells. (A) A549 cells were treated with different concentrations (0, 1, 3, 5 and 10 ng/ml) of TGF-β1 for 48 h. (B) A549 cells were treated with 10 ng/ml TGF-β1 for 0, 24 and 48 h. The relative expression levels of miR-320a-3p in A549 cells measured by RT-qPCR. (C) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 48 h after transfection with miR-320a-3p mimic or NC. The relative expression levels of CDH1, vimentin and ACTA2 were determined by RT-qPCR. (D) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 48 h following transfection with miR-320a-3p mimic or NC. To confirm the occurrence of EMT, the expression of E-cadherin, vimentin and α-SMA were determined by western blot analysis. (E) Immunofluorescence staining showed that the levels of vimentin and α-SMA were reduced in miR-320a-3p mimic treated cells compared with those in cells treated with TGF-β1, while levels of E-cadherin were increased. E-cadherin, vimentin and α-SMA (red); DAPI-counterstained nuclei (blue). Magnification, ×40, scale bar=50 µm. (F) The transfection efficiency of miR-320a-3p mimic was determined by RT-qPCR. Error bars represent the standard deviation of three experiments. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; EMT, epithelial-mesenchymal transition; NC, negative controls; α-SMA, α-smooth muscle actin; NS, no significant difference; RT-qPCR, reverse transcription-quantitative PCR.

Figure 3.

STAT3 is a target gene of miR-320a-3p and effects of STAT3 on EMT in A549 cells induced by TGF-β1. Dual-luciferase reporter assay. (A) The STAT3 3′-UTR region containing the wild-type (wt) or mutant (mu) binding site for miR-320a-3p. (B) Relative Rluc/fluc activity of cells co-transfected with either wt pSI-Check2-STAT3-3′-UTR or pSI-Check2-STAT3-mut-3′-UTR and miR-320a-3p mimic or corresponding NC. (C) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 48 h after transfection with miR-320a-3p mimic or NC. The expression of p-STAT3, STAT3 were determined by western blot analysis and their ratio were analyzed. (D) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 48 h after transfection with STAT3 siRNA or NC siRNA. To confirm the effects of STAT3 on the EMT in A549 cells, the expression of E-cadherin, vimentin and α-SMA were determined by western blot analysis. (E) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 48 h after co-transfection with miR-320a-3p mimic and HBLV-h-STAT3 or HBLV-NC. The expression of E-cadherin, vimentin, α-SMA, p-STAT3 and STAT3 were determined by western blot analysis. The transfection efficiency of (F) si-STAT3 and (G) HBLV-h-STAT3 were determined by western blot analysis. Error bars represent the standard deviation of three experiments. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; EMT, epithelial-mesenchymal transition; UTR; wt, wild-type; mu, mutant; Rluc/fluc, Renilla luciferase/firefly luciferase; NC, negative controls; p-, phosphorylated; si, short interfering; α-SMA, α-smooth muscle actin; NS, no significant difference.

Figure 4.

miR-320a-3p and STAT3 regulate the p-SMAD3 in A549 cells. (A) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 1 h after transfection with miR-320a-3p mimic or NC. (B) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 1 h after transfection with STAT3 siRNA or NC siRNA. (C) A549 cells were treated with or without TGF-β1 (10 ng/ml) for 1 h after co-transfection with miR-320a-3p mimic and HBLV-h-STAT3 or HBLV-NC. Levels of SMAD3 and p-SMAD3 were determined by western blot analysis. Error bars represent the standard deviation of three experiments. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; p-, phosphorylated; NC, negative controls; si, short interfering; NS, no significant difference.

Figure 5.

Study design and miR-320a-3p protected against EMT in PF through STAT3/SMAD3 signaling pathways. miR, microRNA; EMT, epithelial-mesenchymal transition; PF, pulmonary fibrosis; GEO, Gene Expression Omnibus; AECs, alveolar epithelial cells.