The relevant expression level data of miR-320a-3p in PF tissues and normal tissue specimens were obtained from the GEO (http://www.ncbi.nlm.nih.gov/gds/) and the ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) databases. The following key words were used as search terms: ‘lung fibrosis’, ‘idiopathic pulmonary fibrosis (IPF)’, ‘microRNA (miRNA)’, ‘miR320a’. The following criteria were strictly followed: i) Study type: Expression profiling by microarray; ii) datasets were derived from Homo sapiens; iii) datasets included interstitial lung fibrosis tissues and normal tissues; iv) the datasets included >5 samples in each group; and v) the expression data of miR320a from the patients and control subjects were clearly defined and provided. Potentially relevant datasets were further screened by reviewing titles, summaries and overall design. Relevant full articles and samples information were evaluated in order to identify studies that met the eligibility criteria. The data of eligible datasets and series were current to June 2020. Basic information about enrolled datasets are listed in Table SI.

The PF tissue samples (n=12; sex ratio between male and female was 1:2; age range, 39–78 years) were collected by bronchoscopic lung cryobiopsy performed in the Department of Respiratory Medicine of the First Affiliated Hospital of Chongqing Medical University. Normal control samples (n=17; sex ratio between male and female was 1:1.43; age range, 47–77 years) were obtained from normal tissues adjacent to cancer tissues collected in the Department of Cardiothoracic Surgery of the First Affiliated Hospital of Chongqing Medical University. The recruitment dates for the patients were between June and December 2020. The two sets of samples were collected and frozen in liquid nitrogen for subsequent RNA extraction and reverse transcription-quantitative (RT-q) PCR. The collection and analysis procedures of all samples were approved by the Ethics Research Institute of the First Affiliated Hospital of Chongqing Medical University (approval no. 2020-147-2) and written informed consent was obtained from all the participants. Information on patients is listed in Table SII.

The adenocarcinomic human alveolar basal epithelial cell line, A549, was provided by the Stem Cell Bank, Chinese Academy of Science (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum Premium (PAN-Biotech GmbH) and 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with a 5% CO₂ atmosphere. A549 cells were transfected with miR-320a-3p mimic, signal transducer and STAT3 short interfering (si)RNA or corresponding negative control (NC; Shanghai GenePharma Co., Ltd.) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Following transfection for 24 h, the cells were treated with or without TGF-β1 (PeproTech, Inc.) for 48 h and then harvested for subsequent experiments. The si-NC and mimic-NC were non-targeting sequences. The HBLV-h-STAT3-3×flag-mcherry-PURO (HBLV-h-STAT3) and HBLV-mcherry-PURO (HBLV-NC) were constructed by Hanbio Biotechnology Co., Ltd. A549 cells were transfected with HBLV-h-STAT3 or HBLV-NC and puromycin was used to select cells with functional transfection status (DIYIBio Inc.). The transfection sequences are listed in Table I.

A549 cells (1×10⁵ per well) were seeded in the 6-well plate for total RNA extraction. Total RNA was isolated from clinical samples and cultured cells using RNAiso Plus (Takara Bio, Inc.) according to the manufacturer's instructions. RNA was reverse transcribed using the PrimeScript RT Reagent kit with genomic DNA (gDNA) Eraser (Takara Bio, Inc.). The gDNA elimination reaction was performed for 2 min at 42°C. Reverse transcription was conducted for 15 min at 37°C and 5 sec at 85°C. The qPCR analysis was performed using the CFX96 real-time PCR system (Bio-Rad Laboratories, Inc.) and the TB Green Premix EX Taq II PCR kit (Takara Bio, Inc.). Amplification consisted of an initial 30 sec incubation at 95°C, followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. Data were expressed as the relative differences between the control and treated cells after normalization to GAPDH expression. To determine the relative expression levels of miRNAs, the U6 small nuclear RNA was used for normalization. The relative expression levels of the mRNA and miRNA was calculated using the 2^−ΔΔCq method (12). Primers for miR-320a-3p and U6 were purchased from GenePharma Co. Ltd., while those for GAPDH, ACTA2, vimentin, CDH1 were purchased from Takara Bio, Inc. The sequence of the primers used are listed in Table II. All experiments were repeated three times.

The total protein content of the cells was extracted by using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) which contained 1% phosphatase inhibitor (Wuhan Boster Biological Technology, Ltd.) and 1% phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). Total protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). For western blotting, 20 µg of protein was loaded into each lane of the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, followed by electrophoresis and protein transfer to polyvinylidene fluoride membranes (EMD Millipore). Following transfer, the membranes were blocked with QuickBlock™ blocking buffer for western blotting (Beyotime Institute of Biotechnology) for 15 min at room temperature. The primary antibodies rabbit anti-E-cadherin (1:10,000; cat. no. ab40772), rabbit anti-vimentin (1:3,000; cat. no. ab92547), rabbit anti-α-SMA (1:3,000; cat. no. ab32575) and rabbit anti-GAPDH (1:10,000; cat. no. ab181602) were purchased from Abcam. The rabbit anti-phosphorylated (p-)STAT3 (Tyr705, 1:2,000; cat. no. 9145), mouse anti-STAT3 (1:1,000; cat. no. 9139), rabbit anti-p-SMAD3 (Ser423/425, 1:1,000; cat. no. 9520) and rabbit anti-SMAD3 (1:1,000; cat. no. 9523) were purchased from Cell Signaling Technology, Inc. Immunoblots were probed with the corresponding primary antibody at 4°C overnight and subsequently with the appropriate secondary antibody (1:8,000; cat. no. ZB-2301; OriGene Technologies, Inc.) for 1 h at room temperature. The protein bands on the membranes were visualized with enhanced chemiluminescence reagent (Vazyme Biotech Co., Ltd.) and images were captured using an automatic Fusion FX Edge chemiluminescence image analysis system (Vilber Lourmat Sa). Densitometric analysis of the blot images was performed using the Fusion Capt software v.18.06 (Vilber Lourmat Sa). GAPDH was used as an internal normalizer.

Cells (5×10⁴ per well) were seeded and grown in 6-well plates with cell culture silicon slides. Following treatment, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature, then permeabilized with 0.2% Triton X-100 in PBS for 20 min at room temperature (rinsed three times with PBS between each step). Non-specific binding sites were blocked with 3% BSA (Wuhan Servicebio Technology Co., Ltd.) for 30 min at room temperature. The cells were then incubated overnight at 4°C with the corresponding primary antibody at a 1:200 dilution. Afterwards, the cells were incubated with the appropriate fluorescein-conjugated secondary antibody for 1 h in the dark at 37°C. Primary antibodies and secondary antibodies were purchased from Wuhan Servicebio Technology Co., Ltd. The fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei for 10 min in the dark at room temperature before image acquisition. The images were acquired using a fluorescence microscope (Nikon Corporation) with ×40 magnification. The red fluorescence indicated positive antibody expression and the blue fluorescence indicated nuclear DAPI staining. Mean gray value analysis was performed using the ImageJ software v1.53e (National Institutes of Health).

The target genes of miR-320a-3p were identified using the miRanda (http://www.microrna.org/microrna/home.do), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html) algorithms. The dual-luciferase reporter system was used to validate the target relationship between miR-320a-3p and STAT3. Human STAT3 3′-UTR containing a 3′-UTR with mutated (mu) sequences complementary to the seed sequence of miR-320a-3p was inserted into the pSI-Check2 vector (Promega Corporation). The wild type (wt) STAT3 3′-UTR sequence was also inserted into the pSI-Check2 vector. Luciferase reporter plasmid was constructed and provided by Hanbio Biotechnology Co., Ltd. The A549 cells were grown in 96-well plates and then co-transfected with miR-320a-3p mimic or NC and pSI-Check2-STAT3-mu-3′-UTR or wt pSI-Check2-STAT3-3′-UTR using transfection reagent LipoFiter (Hanbio Biotechnology Co., Ltd.). Then, 6 h after transfection, the medium was replaced with fresh medium and after 48 h, the dual-luciferase reporter system (Promega Corporation) was used to measure luciferase activity. The normalized luciferase activity was expressed as the ratio of Renilla luciferase activity to firefly luciferase activity (Rluc/fluc).

Data were expressed as the mean ± standard deviation and SPSS v25 software (IBM Corp.) was used for data analysis. Independent-samples t-test was used for the analysis of data between two groups. Differences among three or more groups were compared using one-way analysis of variance followed by Tukey's post hoc test. GraphPad Prism v8.0 (GraphPad Software, Inc.) was used to draw the box plot, symbols and lines plot and column bar graph. P<0.05 was considered to indicate a statistically significant difference.

A total of 3 datasets were selected for the present study, according to the inclusion criteria (GSE21394/E-GEOD-21394, GSE32538/E-GEOD-32538 and GSE27430/E-GEOD-27430). With the combination of datasets from the GEO and ArrayExpress database, a total of 174 PF samples were included. Sample sizes ranged from 13–142 subjects and the pathological types include the usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF), cryptogenic organic pneumonia (COP), desquamative interstitial pneumonia (DIP), nonspecific interstitial pneumonia (NSIP), connective tissue disease-associated interstitial lung disease (CTD-ILD) and respiratory bronchiolitis-associated interstitial lung disease (RB-ILD). It was found that two results in the analysis of three datasets showed that the relative expression levels of miR-320a-3p were significantly lower in PF tissue samples compared with normal tissue samples (P<0.05, P<0.0001; Fig. 1A and B). The remaining analyses showed that the relative expression levels of miR-320a-3p decreased in the PF group, but the difference was not statistically significant (Fig. 1C). Based on the results of the analysis of the datasets, clinical tissue samples of PF patients (n=12) and normal control subjects (n=17) were collected. The pathological types of samples of PF included UIP/IPF, NSIP, COP and CTD-ILD. The tissue of the control group was confirmed to be normal tissue by pathology. The data showed that the relative expression levels of miR-320a-3p were markedly decreased in PF tissue samples compared with normal tissue samples (Fig. 1D).

The A549 cell lines is widely used as a model system to study the mechanisms of the EMT process in PF. In the present study, qPCR analysis was first used to determine the relative expression levels of miR-320a-3p in A549 cells stimulated for 48 h with TGF-β1 at different concentrations (0, 1, 3, 5 and 10 ng/ml). It was found that TGF-β1 significantly reduced the miR-320a-3p expression levels compared with the control group and the optimal inhibitory effect was obtained at a concentration of 10 ng/ml (Fig. 2A). Subsequently, the levels of miR-320a-3p in A549 cells stimulated with 10 ng/ml of TGF-β1 was determined at various time points (0, 24 and 48 h) and it was found that the optimal inhibition was achieved at 48 h (Fig. 2B). After transfecting A549 cells with miR-320a-3p mimic and NC, followed by treatment with TGF-β1 (10 ng/ml) for 48 h, qPCR analysis was used to determine the expression levels of mRNA of CDH1, vimentin and ACTA2. The results showed that compared with the TGF-β1 treatment group, miR-320a-3p mimic treatment increased the expression of mRNA of CDH1 and significantly decreased the expression of mRNA of vimentin and ACTA2. Western blot analysis and immunofluorescence staining was also used to examine the expression of the epithelial phenotype marker E-cadherin and the mesenchymal phenotype markers vimentin and α-SMA. The results showed that compared with the corresponding control group, TGF-β1-induced EMT increased the expression of vimentin and α-SMA and significantly reduced the expression of E-cadherin. miR-320a-3p mimic treatment attenuated the above effects, that is, it restored the expression of E-cadherin and reduced the expression of vimentin and α-SMA (Fig. 2C-E). These findings indicated that TGF-β1 can induce EMT changes in A549 cells and miR-320a-3p mimic can inhibit those EMT changes. The transfection efficiency of miR-320a-3p mimic is shown in Fig. 2F.

To further investigate the mechanism, several bioinformatics analyses were conducted and the results revealed that STAT3 is a potential target of miR-320a-3p. A dual-luciferase reporter assay was then conducted and revealed that the miR-320a-3p mimic significantly inhibited the luciferase activity in the wt pSI-Check2-STAT3-3′-UTR group compared with the mimic-NC group (P<0.01; Fig. 3A and B). However, the miR-320a-3p mimic did not influence the luciferase activity of pSI-Check2-STAT3-mut-3′-UTR. Western blot analysis also showed that miR-320a-3P inhibited the expression of both STAT3 and p-STAT3 in TGF-β1-stimulated A549 cells, but not the ratio of p-STAT3 and STAT3 (Fig. 3C), indicating that miR-320a-3p directly regulated STAT3 expression. STAT3 is a member of a family of cytokine-responsive transcription factors and has been identified as being hyperphosphorylated during EMT (13). Accordingly, it was investigated whether the expression of STAT3 is involved in TGF-β1-induced EMT in A549 cells. Therefore, A549 cells were transfected with STAT3 siRNA and NC siRNA and then treated them with TGF-β1 (10 ng/ml) for 48 h. The results showed that, compared with the control group, STAT3 siRNA reduced the expression of vimentin and α-SMA, while the expression of E-cadherin was significantly increased (Fig. 3D). To further confirm that miR-320a-3p mimic inhibited TGF-β1-induced EMT in A549 cells through a STAT3-dependent mechanism, miR-320a-3p mimic was co-transfected with HBLV-h-STAT3 or HBLV-NC into A549 cells. Compared with mock-transfected cells, HBLV-h-STAT3 significantly increased the expression of STAT3. In addition, the upregulation of STAT3 inhibited the effect of the miR-320a-3p mimic on the expression of E-cadherin and significantly increased the expression of vimentin, α-SMA and p-STAT3 (Fig. 3E). In summary, the above data indicated that miR-320a-3p mimic can attenuate TGF-β1-induced EMT in A549 cells by targeting STAT3 and p-STAT3. The transfection efficiency of si-STAT3 and HBLV-h-STAT3 are shown in Fig. 3F and G.

Following transfection of A549 cells with miR-320a-3p mimic, the expression of p-SMAD3 induced by TGF-β1 was significantly reduced, while total SMAD3 was unaffected (Fig. 4A). Therefore, it was investigated whether the inhibitory effect of miR-320a-3p on the phosphorylation of SMAD3 was mediated by STAT3. To this end, the A549 cells were first transfected with STAT3 siRNA and it was confirmed that knocking down STAT3 led to reduced expression of p-SMAD3 induced by TGF-β1 (Fig. 4B). Then, transfecting A549 cells with HBLV-h-STAT3 revealed that overexpression of STAT3 can significantly increase the expression of p-SMAD3 induced by TGF-β1 (Fig. 4C). Taken together, these findings suggested that miR-320a-3p inhibited TGF-β1-induced EMT in A549 cells partly by modulating the expression of STAT3 and p-SMAD3.