MiR-582-3p participates in the regulation of biological behaviors of A549 cells by ambient PM2.5 exposure

Ambient fine particulate matter (PM2.5) is one of the main environmental air pollutants that is closely related to the development of lung cancer, but the mechanisms are unclear. In this study, A549 cells were exposed to ambient PM2.5 to investigate the alterations of biological behaviors, and the possible role of miR-582-3p in the effects was further explored. The findings showed that PM2.5 exposure could significantly enhance the biological behaviors of A549 cells, and promote their epithelial-mesenchymal transition (EMT) transformation, especially at relatively low doses. Over-activation of Wnt/β-catenin signaling pathway and increased expression of miR-582-3p were also found in A549 cells after PM2.5 exposure. After the knockdown of miR-582-3p in A549 cells, the effects of PM2.5 on malignant biological behavior changes, EMT, and the activation of Wnt/β-catenin signaling pathway were all significantly alleviated. Furthermore, the inhibition of Wnt/β-catenin signaling pathway also inhibited the EMT process of A549 cells, which was rescued by the overexpression of miR-582-3p. Therefore, this study showed that ambient PM2.5 can upregulate the expression of miR-582-3p, consequently activate the Wnt/β-catenin signaling pathway, and thereby enhance EMT transformation and promote the malignant biological behaviors of A549 cells. These findings provide evidence for further research into the mechanisms by which exposure to PM2.5 in the environment promotes lung cancer.


Introduction
Air pollution is a major environmental issue that has substantial impacts on human health throughout the world (Chen et al. 2014). Fine particulate matter (PM 2.5 ), the particulate matter with the aerodynamic diameter of ≤ 2.5 µm, is one of the primary ambient pollutants. Due to the small particle size, light weight, and high dispersion, PM 2.5 can suspend in the air for long time and enter the alveoli through the respiratory tract. Part of the particulate matters can also pass through the alveolar epithelium and enter the blood circulation through the lung tissue gap (Yue et al. 2017). In addition, the small particle size and specific surface area allow PM 2.5 to adsorb a variety of toxic and harmful substances, thus increasing the complexity of effects. The International Agency for Research on Cancer (IARC) has classified particulate matter in ambient air as a class I carcinogen (Loomis et al. 2013).
Lung cancer is still one of the most life-threatening malignancies with high morbidity and mortality. Previous studies have shown that a quarter of cancer deaths in the world are caused by lung cancer (Siegel et al. 2019), and lung cancer metastasis is the main cause of death in lung cancer patients (Okimoto et al. 2017). Lung cancer is the second most common diagnosed cancer and the leading cause of cancer death, accounting for approximately one-tenth (11.4%) of all diagnosed cancers and one-fifth (18.0%) of all deaths worldwide in 2020 with approximately 2.2 million new cases and 1.8 million deaths (Sung et al. 2021). To date, a large number of epidemiological studies have shown that PM 2.5 is closely related to the occurrence and development of lung cancer and the increased risk of lung cancer death (Pun et al. 2017;Raaschou-Nielsen et al. 2013;Tseng et al. 2019), while the underlying biological mechanisms are still unclear. Accumulating evidence has shown that noncoding RNAs play critical roles in the occurrence and progression of lung cancer .
MicroRNAs (miRNAs) are a class of endogenous, noncoding single-stranded small RNAs with the length of about 20-24 nucleotides that widely exist in eukaryotes. It is well known that miRNAs play important roles in regulating protein translation and transcription. Specifically, miRNA mainly inhibits or blocks the translation of a variety of target proteins through complementary binding with the 3′-untranslated region of target messenger RNA, and therefore regulates cell proliferation, differentiation, apoptosis, and metabolism (Mahmood et al. 2016). A large number of studies have shown that many miRNAs act as oncogenes or tumor suppressors, and play vital roles in cancer development and progression. Specifically, miRNAs can regulate the biological processes of a variety of cells, including cell proliferation, differentiation, invasion, and migration, thereby regulating the development of a variety of tumors (Shukla et al. 2020;Sullivan et al. 2017). Recent studies have shown that miR-582-3p is overexpressed in lung cancer cell lines and tissues and has carcinogenic and cancer-promoting effects. Moreover, miR-582-3p is closely related to the pathway of cancer, and its target genes are abundantly enriched in the Wnt signaling pathway (Fang et al. 2015;Jin et al. 2017).
Wnt/β-catenin signaling pathway is a classic cell signal transduction pathway, which is involved in mediating cell growth, migration, differentiation, and apoptosis, and is a key signaling pathway for tumorigenesis and prognosis. In addition, previous study suggested that traffic pollution exposure can induce hypermethylation of the promoter of APC gene, which plays a substantial role in the Wnt/βcatenin signaling pathway, and consequently leads to the inhibition of APC gene expression (Ding et al. 2016). Furthermore, the subsequent detection of pathway-related proteins also confirmed that ambient PM 2.5 exposure would activate the Wnt/β-catenin pathway. In light of these findings, we hypothesized that ambient PM 2.5 exposure can cause the increased expression of miR-582-3p in human lung adenocarcinoma cells (A549), leading to continuous activation of intracellular Wnt/β-catenin pathway, promoting the malignant biological behaviors and EMT of A549 cells, and mediating the development of lung cancer.

Materials and methods
Collection and extraction of ambient PM 2.5 PM 2.5 samples were collected in Shushan District, Hefei City, which is the capital city of Anhui Province and one of the most polluted cities in China. In detail, the ultrafine glass fiber filter membrane with the pore size of 0.2 μm was installed on the PM 2.5 sampler for PM 2.5 collection, with the flow rate of 1 m 3 /min, and the sampling time from 9:00 am to 16:00 pm. The dried filter membrane was cut into 1 cm × 1 cm, put into a beaker containing deionized water, and sonicated for 3 times by an ultrasonic oscillator, with 10 min in each sonication. After filtration, the dry PM 2.5 was dispersed in water for more than 24 h at low temperature, and then dissolved in DMSO to obtain the PM 2.5 stock solution, of which the concentration was 100 mg/mL, and stored at − 20 °C in dark.

Cell culture and transfection
A549 cells (Procell Life Science and Technology Co., Ltd.) were kindly provided by the University of Science and Technology of China. A549 were cultured with DMEM medium containing 10% fetal bovine serum and 1% penicillin streptomycin solution in a constant temperature incubator at 5% CO 2 and 37 ℃. For cell transfection, A549 cells were seeded in 6-well plates and cultured in DMEM containing 10% fetal bovine serum but no antibiotics until cell density reached 60-70%; afterwards, the lipofectamine 2000 kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used for the transfection of the cells, according to the instructions of manufacturers. RNA was extracted 24 h after transfection for further analysis. Cell behaviors were assessed 48-72 h later. All experiments were independently repeated for 3 times. The transfections were designed and synthesized by Gemma Technology (China) Co., Ltd., and the sequences are shown in Table S1.

Cell viability test
3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to assess the toxic effects of PM 2.5 and DKK-1 on A549 cells. The cell suspension at a concentration of 5 × 10 4 cells/mL was seeded into the 96-well plate at 100 μL per hole, and cultured until density reached 70-80%. Then, medium containing pre-defined concentrations of PM 2.5 (0, 25, 50, 100, and 200 μg/mL) or DKK-1 (0, 25, 50, 100, and 200 ng/mL) and blank control (with only culture medium) were placed in the incubator respectively, and the cells were further cultured for 24 h. Afterwards, the culture medium was discarded and 200 μL of MTT solution was added for further incubation for 4 h. Then, the MTT solution was discarded and 150 μL DMSO was added to each well, and oscillated the 96-well plate on a horizontal shaker until the precipitate is completely dissolved. The absorbance value of each well was measured by ELx800 microplate reader at 490 nm.

Plate clone formation assay
The cell concentration was adjusted to 2 × 10 3 cells/mL with the DMEM medium; then, 300 μL cell suspension was added to 6-well cell culture dish (diameter of each well was 3.48 cm), and culture medium was added until the volume was 3 mL. The cells were cultured in the incubator, until the clones were visible. After washing with PBS for 3 times, the clones were fixed with 4% paraformaldehyde for 25 min, stained with 0.1% crystal violet solution for 15-20 min, and then washed and dried. The number of clones was counted under light microscope, and calculated according to the following formula: clone formation rate = (number of cloned cells)/(number of inoculated cells) × 100%.

Wound healing assay
A549 cells were cultured in a 6-well plate to form monolayer, which was scratch by the tip of a 200-μL sterilized pipette perpendicularly to create the wound, and then washed with PBS for 3 times to remove the exfoliated cells. The culture medium of different treatment groups was added to each hole and placed in the incubator for further culture. The migrations of cells at the scratch sites were photographed with an inverted microscope every 12 h. The Image J software (Tanon Gel Image System 4.2) was used to measure the migration distance of cells and the wound healing rate was calculated according to the migration distance.

Transwell cell invasion assay
Transwell cell invasion assay was used to assess the ability of cells invasion. The matrigel was diluted and mixed with pre-chilled serum-free cell culture medium on ice at a ratio of 1:7, and then 50 μL of diluted matrigel was applied evenly in the upper chamber of transwell culture plate. After solidification of the matrigel, 200 μL of the cell suspension prepared in serum-free medium was added to the upper chamber. While in the lower chamber, 600 μL complete medium containing 20% FBS and different concentrations of PM 2.5 (0, 25, 50, 100 μg/mL) was added, respectively. The cells were cultured in a 37 ℃ constant temperature incubator containing 5% CO 2 for 24 h. Then, the matrigel was removed, and the cells were fixed with 4% paraformaldehyde for 25 min. Afterwards, the chamber membrane was stained with 0.1% crystal violet for 20 min, and rinsed with deionized water for 3 times, and the cell numbers in 3 random sights were counted under the inverted microscope. The average number was calculated for the comparison.

Western blot
Western blot was used to detect the expression of epithelialmesenchymal transition (EMT) and Wnt/β-catenin signaling pathway proteins in cells. The cells were lysed and centrifuged, and the supernatant, namely the total cellular protein, was pipetted into a new centrifuge tube, which was quantified with the BCA protein quantitative kit (P0010, Beyotime Biotechnology). Proteins of different molecular weights were separated by electrophoresis, and then transferred to PVDF membranes by SDS-PAGE. The PVDF membranes were blocked in a 5% skim milk blocking solution at room temperature for 2 h, and incubated with a specific primary antibody at 4 °C overnight. After rinsing, the membrane was incubated with pre-diluted secondary antibody for 2 h at room temperature and then developed. The gray scale of the band was analyzed with the Image J gray analysis software (Tanon Gel Image System 4.2).

QRT-PCR
Primers were designed according to the sequences of the genes to detect the corresponding mRNA expression level of the cells. The sequences of the primers are shown in Table S2. RNA was extracted from the cells according to the instructions of the RNA extraction kit, and the RNA concentration in each group was determined by an ultraviolet-visible spectrophotometer (Nanodrop one). RNA was transcribed into cDNA in a reverse PCR instrument (T100™ Thermal Cycler) using the PrimeScript™ RT reagent kit with gDNA Eraser (RR047A, TaKaRa), and cDNA was amplified on a Light Cycler 96 real-time PCR instrument (LightCycler® 96 Instrument) using the TB Green® Premix Ex Taq™ II reagent kit (RR820A, TaKaRa). The relative expression of the target gene was calculated using the 2 −ΔΔCt formula method.

Statistical analysis
SPSS 23.0 was used for statistical analysis. All the data in the experiment were expressed as mean ± standard deviation ( x ± SD), and the quantitative data between the two groups and multiple groups were analyzed by independent-sample t-test and one-way ANOVA of variances. P < 0.05 indicated statistically significant.

Cytotoxic effects
The effect of different concentrations of PM 2.5 (0, 25, 50, 100, 200 μg/mL) on the viability of A549 cells was detected by MTT assay. The A549 cytotoxic effect of PM 2.5 is shown in Fig. 1A. After 24-h exposure, the survival rate of cells decreased with the increase of PM 2.5 concentration in a dose-response relationship. When PM 2.5 exposure reached 100 μg/mL or higher, the cell survival rate was significantly lower than that of the control group (P < 0.01), and the cell survival rate was about 80% when PM 2.5 concentration was 100 μg/mL. For the purpose of investigating the role of Wnt/β-catenin, the specific inhibitor DKK-1 was also used in this study. MTT assay showed the effects of different concentrations of DKK-1 (0, 25, 50, 100, 200 ng/ mL) on the viability of A549 cells. After 24-h exposure, the cell viability of DKK-1 decreased gradually with the increase of DKK-1 concentration. When the concentration of DKK-1 was 50 ng/mL, there was no difference in the effect of DKK-1 on cell viability compared with that of the single solvent group (P > 0.05) (Fig. 1B). Therefore, PM 2.5 doses of 0 (pure solvent control, containing 1‰ DMSO), 25, 50, and 100 μg/mL and DKK-1 dose of 50 ng/mL were selected for the treatment in this study.

PM 2.5 promoted the proliferation, migration, and invasion of A549 cells
According to the MTT assay, A549 cells were treated with different concentrations of PM 2.5 (0, 25, 50, 100 μg/mL) for 24 h, and then the cell proliferation ability was detected by plate clonal formation assay. As shown in Fig. 2A and B, the number of cloned cells increased significantly in the 25 μg/mL PM 2.5 treatment group (P < 0.01), comparing with the control group (0 μg/mL). Interestingly, the number of cell clones in the 50 μg/mL and 100 μg/mL groups also increased, but not as substantial as in the 25 μg/mL group.
To assess the migration of cells, A549 cells were exposed to PM 2.5 (0, 25, 50, 100 μg/mL) for 12, 24, and 36 h, and the migration distance of the cells in the scratched area was measured. As shown in Fig. 2C, the cell migration distances were significantly different among different groups 24 h later. The wound closure rate in the 25 and 50 μg/mL group was 65.4 ± 3.8% and 42.8 ± 2.9%, respectively, which were significantly higher than in the control group (30.3 ± 3.1%) (P < 0.05) (Fig. 2D).
To assess the invasion capability of cells, A549 cells were exposed to PM 2.5 (0, 25, 50, 100 μg/mL) for 24 h, and the invasion capability of the cells was detected by transwell assay. The results showed that the number of invaded cells in the 25 and 50 μg/mL groups increased significantly compared with the control group (0 μg/mL group) (P < 0.05). However, the further increase of PM 2.5 concentration resulted in lower cell invasion ( Fig. 2E and F).

PM 2.5 induced EMT in A549 cells
The biological behaviors of cells after PM 2.5 treatment showed that PM 2.5 with the concentration of 25 μg/mL had the strongest effects. Therefore, A549 cells were exposed to PM 2.5 at concentrations of 0, 6.25, 12.5, and 25 μg/mL for 24 h, and the expression of EMT-related proteins was measured by western blot (Fig. 2G). Quantitative analysis showed that with the increase of PM 2.5 concentrations, the expression of vimentin tended to increase in dose-dependent manner. Specifically, after the cells were exposed to PM 2.5 at 6.25 μg/mL or higher, the protein expression level of vimentin was significantly higher than that of the control group. Similarly, with the increase of PM 2.5 concentration, the protein expression level of E-cadherin showed a dosedependent decrease trend, which were significantly lower than the control group (P < 0.05) ( Fig. 2H and I). Fig. 1 The A549 cytotoxicity of atmospheric PM 2.5 and DKK-1. A The viability of A549 cells after treated with a concentration gradient of atmospheric PM 2.5 (0, 25, 50, 100, 200 μg/ mL) for 24 h. B The viability of A549 cells after exposed to a concentration gradient of DKK-1 (0, 25, 50, 100, 200 ng/mL) for 24 h. In comparison with the control group, *P < 0.05, **P < 0.01

PM 2.5 activated Wnt/β-catenin signaling pathway in A549 cells
After treated with different concentrations of PM 2.5 (0, 25, 50, 100 μg/mL) for 24 h, the levels of Wnt/β-catenin pathway proteins, including APC, β-catenin, GSK-3β, and p-GSK-3β, in A549 cells were detected by western blot. The results are shown in Fig. 2J. Compared with the control group, the expression level of β-catenin and the relative expression level of p-GSK-3β/GSK-3β in A549 cells in the PM 2.5 treatment group were significantly increased (P < 0.05), while the expression level of APC was significantly decreased (P < 0.01), in a dose-response relationship ( Fig. 2K-M).

PM 2.5 altered miR-582-3p expression in A549 cells
After treated with different concentrations of PM 2.5 (0, 25, 50, 100 μg/mL) for 24 h, the QRT-PCR results showed that compared with the control group, the expression levels of miR-582-3p in the PM 2.5 exposure groups were significantly increased (P < 0.01). The expression levels of miR-582-3p of 50 μg/mL and 100 μg/mL PM 2.5 exposure groups were the highest, and there was no significant difference between the two groups (Fig. 3).

Fig. 2
The effects of PM 2.5 on the proliferation, migration, invasion, EMT, and Wnt/β-catenin pathway of A549 cells. A and B The cell cloning situation and column chart of A549 cells after treated with a concentration gradient of atmospheric PM 2.5 (0, 25, 50, 100 μg/ mL) for 24 h (crystal violet staining). C and D The changes of scratch healing degree of A549 cells treated with concentration gradient atmospheric PM 2.5 (0, 25, 50, 100 μg/mL) for different times, as well as the histogram of the wound closure rates of the concentration gradient groups after 24 h of exposure. E and F The invasive A549 cells and column chart after treated with atmospheric PM 2.5 (0, 25, 50, 100 μg/mL) for 24 h (crystal violet staining, 100 times). G-I The expression levels of EMT-related proteins in A549 cells after exposure to a concentration gradient of atmospheric PM 2.5 (0, 6.25, 12.5, 25 μg/mL). J-M The relevant-protein levels of Wnt/β-catenin pathway in A549 cells after exposure to concentration gradient of atmospheric PM 2.5 (0, 25, 50, 100 μg/mL). In comparison with pure solvent group (0 group), *P < 0.05, **P < 0.01
MiR-582-3p inhibitor was transfected into A549 cells to knock down the expression of miR-582-3p, and then the cells were exposed to PM 2.5 (50 μg/mL) for 24 h. The plate clone formation assay showed that comparing with the miR-582-3p inhibitor NC group, the clonal formation ability of A549 cells in the miR-582-3p inhibitor group was significantly decreased (P < 0.05) (Fig. 4B), and the clonal formation ability of A549 cells in the miR-582-3p inhibitor + PM 2.5 group was significantly improved compared with the miR-582-3p inhibitor group (P < 0.01) (Fig. 4C).
The ability of migration was evaluated by wound healing assay. The results showed that after 24 h of exposure, the wound healing rate was significantly different among the groups. Specifically, the miR-582-3p inhibition significantly reduced the wound healing rate of A549 cells, comparing with the miR-582-3p inhibitor NC group (26.2 ± 1.2% vs. 43.8 ± 2.4%) (P < 0.05), while treatment with PM 2.5 significantly rescued the wound healing capability of cells with miR-582-3p inhibition (63.4 ± 2.3% vs. 26.2 ± 1.2%) (P < 0.01) ( Fig. 4D and E).
The invasion capability of the cells was evaluated by transwell invasion assay. The result is shown in Fig. 4F and G. After 24 h of exposure, compared with the miR-582-3p inhibitor NC group, the number of invaded cells in the miR-582-3p inhibitor group was significantly reduced (P < 0.05), and the number of invaded cells in the miR-582-3p inhibitor + PM 2.5 group was significantly increased compared with the miR-582-3p inhibitor group (P < 0.05).

MiR-582-3p mediated PM 2.5 -induced EMT in A549 cells
The role of miR-582-3p in EMT of A549 cells was further investigated. After treated with PM 2.5 (50 μg/mL) for 24 h, the expression of EMT-related proteins, such as vimentin and E-cadherin, was detected by western blot. The results showed that comparing with the miR-582-3p inhibitor NC group, the expression of vimentin in the miR-582-3p inhibitor group was significantly decreased, while the expression of E-cadherin was significantly increased (P < 0.05). In addition, comparing with the miR-582-3p inhibitor group, the expression of vimentin in the miR-582-3p inhibitor + PM 2.5 group significantly increased, while the expression of E-cadherin was decreased significantly (P < 0.05) (Fig. 4H-J).

MiR-582-3p mediated the activation of Wnt/ β-catenin signaling pathway in A549 cells induced by PM 2.5
The role of miR-582-3p in the activation of Wnt/β-catenin signaling pathway in A549 cells was further investigated. After transfection, the A549 cells were treated with PM 2.5 (50 μg/mL) for 24 h, and then the expression of Wnt/β-catenin pathway proteins, including APC, β-catenin, GSK-3β, and p-GSK-3β, was detected by western blot. Comparing with the miR-582-3p inhibitor NC group, the expression of β-catenin and the relative expression of p-GSK-3β/GSK-3β in the miR-582-3p inhibitor group were significantly decreased, while that of APC was significantly increased (P < 0.05) (Fig. 4K-N). In addition, compared with the miR-582-3p inhibitor group, the expression of β-catenin and the relative expression of p-GSK-3β/GSK-3β in the miR-582-3p inhibitor + PM 2.5 group were significantly increased, while the expression of APC significantly decreased (P < 0.05) (Fig. 4K-N).

MiR-582-3p regulates the EMT process of A549 cells through the Wnt/β-catenin signaling pathway
DKK-1, a Wnt/β-catenin inhibitor, was used in this study to explore the mechanism underlying the regulatory effects of Fig. 3 The level of miR-582-3p in A549 cells treated with different concentrations of PM 2.5 (0, 25, 50, 100 μg/mL) was detected by QRT-PCR, using U6 as an internal control. In comparison with pure solvent group, *P < 0.05, **P < 0.01 miR-582-3p on EMT of A549 cells. Cells were transfected with miR-582-3p mimics to induce the overexpression of miR-582-3p (Fig. 4A), and then DKK-1 was used to inhibit the Wnt/β-catenin pathway. As shown in Fig. 5, comparing with the miR-582-3p mimics NC group, the protein expression of vimentin in the miR-582-3p mimics group was significantly upregulated, while the protein expression of E-cadherin was significantly downregulated (P < 0.05). Compared with the miR-582-3p mimics group, the protein expression of vimentin in the miR-582-3p mimics + DKK-1 group was significantly downregulated, while the expression level of E-cadherin was significantly upregulated (P < 0.05).

Fig. 4
MiR-582-3p mediated PM 2.5 -induced proliferation, migration, invasion, EMT, and Wnt/β-catenin pathway of A549 cells. A The levels of miR-582-3p in A549 cells that is equally transfected with miR-582-3p mimics NC, miR-582-3p mimics, miR-582-3p inhibitor NC, or miR-582-3p inhibitor were detected by QRT-PCR method, using U6 as an internal control. B and C The cell cloning situation and column chart in each group (crystal violet staining). D and E The changes of scratch healing degree of A549 cells in each group and different times, as well as the histogram of the wound closure rates of A549 cells in each group after 24 h of exposure. F and G The invasive cells and the column chart of cell invasion number in each group (crystal violet staining, 100 times). H-J The expression levels of EMT-related proteins of A549 cells in different groups. K-N The relevant-protein levels of Wnt/β-catenin pathway of A549 cells in different groups. a is inhibitor NC, b is inhibitor NC + PM 2.5 , c is miR-582-3p inhibitor, and d is miR-582-3p inhibitor + PM 2.5 . In comparison with the inter-group, *P < 0.05, **P < 0.01

Discussion
The results of this study showed that exposure to ambient PM 2.5 significantly enhanced the abilities of proliferation, migration, and invasion of A549 cells. In addition, the EMT marker protein vimentin of A549 cells was significantly upregulated, and E-cadherin was significantly downregulated. The Wnt/β-catenin signaling pathway in the cell was highly activated after PM 2.5 exposure, and the expression level of β-catenin and the relative expression level of p-GSK-3β/GSK-3β increased significantly, while the expression level of APC decreased significantly. In addition, the study also found that the effect of PM 2.5 exposure on the biological behavior changes of A549 cells was most significant at a concentration of 25 μg/mL PM 2.5 . However, when the concentration of PM 2.5 continued to increase, the promoting effects of PM 2.5 on the proliferation, migration, invasion, and EMT of A549 cells all decreased and even MiR-582-3p regulated the EMT of A549 cells through the Wnt/β-catenin signaling pathway. MiR-582-3p mimics NC or miR-582-3p mimics was transfected into A549 cells respectively, and then treated with DKK-1 according to the groups (mimics NC, mimics NC + DKK-1, mimics, mimics + DKK-1). The EMT of A549 cells in each group is detected subsequently. A The protein bands of vimentin, E-cadherin, and β-actin. B The column chart of the relative levels of vimentin and E-cadherin. In comparison with the inter-group, *P < 0.05, **P < 0.01 disappeared. It may be due to the toxic effect of high concentrations of PM 2.5 on A549 cells. Similarly, the study by Zhou et al. (2016) also pointed out that repeated exposure to "safe" concentrations of PM 2.5 induces epigenetic silence of P53, which is an approved tumor suppressor gene, but the epigenetic effect caused by short-term exposure to high-dose PM 2.5 was completely different.
Further studies showed that the expression level of miR-582-3p in A549 cells was increased after PM 2.5 exposure, and knockdown of miR-582-3p expression significantly inhibited cell proliferation, migration, and invasion in A549 cells. In addition, the activation of Wnt/β-catenin signaling pathway and EMT were also inhibited. But the addition of PM 2.5 can obviously rescue these effects. In order to explore the regulatory effect of Wnt/β-catenin signaling pathway on EMT, further experiments were carried out by using DKK-1 to inhibit the Wnt/β-catenin pathway, and then detecting the effect of miR-582-3p overexpression on EMT. The results showed that after overexpression of miR-582-3p in A549 cells, intracellular EMT was significantly increased, but the EMT of the DKK-1 intervention group was significantly downregulated compared with the non-intervention group. The important role of Wnt/β-catenin signaling pathway in promoting EMT in cancer cells has been confirmed by many studies (Puisieux et al. 2014;Zhao et al. 2019), and the results of this study also add new evidence for this. The reduction of E-cadherin during EMT is largely regulated by Wnt/β-catenin signaling. The E-cadherin/β-catenin complex plays an important role in maintaining the stable connection of epithelial cells. Once the E-cadherin/β-catenin complex is disturbed or destroyed, it will eventually lead to the nuclear translocation of β-catenin and the transcriptional activation of EMT-promoting genes. The regulatory role of Wnt/βcatenin-EMT in the development of lung cancer has also been gradually confirmed (Pan et al. 2020;Yang et al. 2017). Therefore, the above results indicated that PM 2.5 exposure leaded to the overexpression of miR-582-3p in A549 cells, which activated the Wnt/β-catenin signaling pathway and further enhanced EMT, and ultimately caused malignant biological behavior changes.
Previous studies have shown that miR-582-3p promotes stem cell characterization of NSCLC cells in vitro and is inversely associated with overall and relapse-free survival in patients (Fang et al. 2015;Siriwardhana et al. 2019). Studies have reported that miR-582-3p can specifically inhibit Axin2, DKK3, and SFRP1-common tumor suppressors in human malignancies including NSCLC, thereby over-activating Wnt/β-catenin signal transduction and playing an important role in the progression of lung cancer (Fang et al. 2015). Jin et al. (2017) also confirmed that miR-582-3p is closely related to the pathway of cancer through pathway enrichment analysis, and its target genes are abundantly enriched in the Wnt signaling pathway. All these studies provide a theoretical basis for our research results. However, some studies have found that the increased expression of miR-582-3p can reduce the proliferation and invasion of some cancers, such as prostate cancer (Huang et al. 2019) and gastric cancer (Xie et al. 2020). Based on these findings, miR-582-3p seems to have both anti-tumor and pro-tumor effects.
In conclusion, this study confirmed that PM 2.5 has a strong effect on causing lung cancer, and also confirmed the regulatory role of miR-582-3p and Wnt/β-catenin signaling pathway in this process. However, there were some limitations in this study. Firstly, the composition of PM 2.5 samples used in this study has not been detected and analyzed. Then, the specific target of Wnt/β-catenin signaling pathway that miR-582-3p is acting on needs to be further explored. In addition, our current study is only established at the cell level in vitro, and verification in vivo experiments are needed in the future.

Conclusion
This study demonstrated that ambient PM 2.5 could upregulate the expression of miR-582-3p, consequently activates the Wnt/β-catenin signaling pathway, and enhances EMT transformation and promotes malignant biological behaviors of A549 cells. These findings provided evidence for further investigating the mechanisms underlying the lung cancer-promoting effects of ambient PM 2.5 exposure.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s11356-021-16801-2. Data availability All data generated or analyzed during this study are included in this article.

Declarations
Ethics approval and consent to participate Not applicable.

Competing interests
The authors declare no competing interests.