MiR-582-3p Participates in the Regulation of Biological Behaviors of A549 Cells by Ambient PM2.5 Exposure

doi:10.21203/rs.3.rs-571917/v1

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MiR-582-3p Participates in the Regulation of Biological Behaviors of A549 Cells by Ambient PM2.5 Exposure

https://doi.org/10.21203/rs.3.rs-571917/v1

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Background

Ambient fine particulate matter (PM_2.5) is one of the main environmental air pollutants that closely related to the development of lung cancer, but the mechanisms are unclear.

Results

In this study, A549 cells were exposed to ambient PM_2.5 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 that PM_2.5 exposure could significantly enhance the biological behaviors of A549 cells, and promote their 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 PM_2.5 exposure. After the knockdown of miR-582-3p in A549 cells, the effects of PM_2.5 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.

Conclusion

This study showed that ambient PM_2.5 can up-regulate the expression of miR-582-3p, consequently activate the Wnt/β-catenin signaling pathway, 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 PM_2.5 in the environment promotes lung cancer.

Toxicology

Atmospheric PM2.5

lung cancer

miR-582-3p

EMT

Wnt/β-catenin signaling pathway

Air pollution is a major environmental issue that has substantial impacts on human health throughout the world[1]. Fine particulate matters (PM_2.5), the particulate matters 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[2]. 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[3].

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[4], and lung cancer metastasis is the main cause of death in lung cancer patients[5]. 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[6]. 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[7–9], while the underlying biological mechanisms are still unclear. Accumulating evidence has shown that non-coding RNAs play critical roles in the occurrence and progression of lung cancer[10].

MicroRNAs (miRNAs) are a class of endogenous, non-coding 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, therefore regulates cell proliferation, differentiation, apoptosis, and metabolism[11]. 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, migration, etc., thereby regulating the development of a variety of tumors[12, 13]. 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[14, 15]. 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. Based on these, this study explored the role of miR-582-3p in biological behavior changes of human lung adenocarcinoma cells (A549) after PM_2.5 exposure.

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 was shown in Fig. 1A. After 24 hours 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 that the effects of different concentrations of DKK-1(0, 25, 50, 100, 200 ng/mL) on the viability of A549 cells. After 24 hours 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. 2B). Therefore, PM_2.5 doses of 0 (pure solvent control, containing 1‰ DMSO), 25, 50, 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 treatment with different concentrations of PM_2.5 (0, 25, 50, 100 µg/mL) for 24 hours, and then the cell proliferation ability was detected by plate clonal formation assay. As shown in Fig. 2A and 2B, 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 hours, 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 hours 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 hours, 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 2F).

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 hours, and the expression of EMT-related proteins were measured by western blot (Fig. 3A). 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 control group. Similarly, with the increase of PM_2.5 concentration, the protein expression level of E-cadherin showed a dose-dependent decrease trend, which were significantly lower than control group (P < 0.05) (Fig. 3B).

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 hours, 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 were shown in the Fig. 4A. 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. 4B).

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 hours, 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. 5).

MiR-582-3p mediated PM_2.5-induced proliferation, migration and invasion of A549 cell

In order to further explore the mechanism of miR-582-3p in the biobehavioral changes of A549 cells caused by PM_2.5, miR-582-3p mimics NC, miR-582-3p mimics, miR-582-3p inhibitor NC and miR-582-3p inhibitor were transfected into A549 cells, and the expression level of miR-582-3p was detected by QRT-PCR assay. Comparing with the miR-582-3p mimics NC group and miR-582-3p inhibitor NC group, the miR-582-3p expression was significantly up-regulated in the miR-582-3p mimics group, and significantly down-regulated in the miR-582 -3p inhibitor group (P < 0.01; Fig. 6). These findings proved that the transfection was successful.

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 hours. 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. 7A), 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. 7B).

The ability of migration was evaluated by wound healing assay. The results showed that after 24 hours 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. 7C and 7D).

The invasion capability of the cells was evaluated by transwell invasion assay. The result was shown in Fig. 7E and 7F. After 24 hours 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), the number of invaded cells in miR-582-3p inhibitor + PM_2.5 group was significantly increased compared with 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 hours, the expression of EMT-related proteins, such as Vimentin and E-cadherin, were 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. 8).

MiR-582-3p mediated the activation of Wnt/β-catenin signaling pathway in A549 cells induced by PM_2.5

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 hours, 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. 9). 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. 9).

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 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. 6), and then DKK-1 was used to inhibit the Wnt/β-catenin pathway. As shown in Fig. 10, comparing with the miR-582-3p mimics NC group, the protein expression of Vimentin in the miR-582-3p mimics group was significantly up-regulated, while the protein expression of E-cadherin was significantly down-regulated (P < 0.05). Compared with miR-582-3p mimics group, the protein expression of Vimentin in miR-582-3p mimics + DKK-1 group was significantly down-regulated, while the expression level of E-cadherin was significantly up-regulated (P < 0.05).

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 up-regulated, and E-cadherin was significantly down-regulated. 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 disappeared. It may be due to the toxic effect of high concentrations of PM_2.5 on A549 cells. Similarly, the study by Zhou W et al [16] 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 difference.

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 down-regulated 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[17, 18], 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 /β-catenin-EMT in the development of lung cancer has also been gradually confirmed[19, 20]. 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, 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[14, 21]. 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[14]. Jin X et al[15] 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[22] and gastric cancer[23]. 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, the specific target of Wnt/β-catenin signaling pathway that miR-582-3p 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.

This study demonstrated that ambient PM_2.5 could up-regulate 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.

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 m³/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 hours 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 store at -20°C in dark.

Cell culture and transfection

A549 cells (Procell Life Science&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% CO₂ 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 hours after transfection for further analysis. Cell behaviors were assessed 48–72 hours 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 assessed the toxic effects of PM_2.5 and DKK-1 on A549 cells. The cell suspension at a concentration of 5×10⁴ 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), as well as blank control (with only culture medium) were placed in the incubator respectively, and the cells were further cultured for 24 hours. Afterwards, 200 µL of MTT solution was added into each well for further incubation of 4 hours, and 150 µL DMSO was added to each well, and the 96-well plate was oscillated 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 2×10³ cells/mL with the DMEM medium, then 300 µL cell suspension was added to 6-well cell-culture dish (diameter of each well was 3.48cm), 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 minutes, stained with 0.1% crystal violet solution for 15–20 minutes, and then washed and dried. The number of clones was counted under light microscope, and calculated according to the formula: clone formation rate = (number of cloned cells)/ (number of inoculated cells) × 100%.

Wound healing assay

After 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 hours. 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% CO₂ for 24 hours. Then the matrigel was removed, and the cells were fixed with 4% paraformaldehyde for 25 minutes. Afterwards, the chamber membrane was stained with 0.1% crystal violet for 20 minutes, 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 epithelial-mesenchymal 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 hours, and incubated with a specific primary antibody at 4°C overnight. After rinsing, the membrane was incubated with pre-diluted secondary antibody for 2 hours at room temperature and then developed. The gray scale of the band was analyzed with 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), the quantitative data between the two groups and multiple groups were analyzed by independent-samples t-test and One-way ANOVA of variances. P < 0.05 indicated statistically significant.

PM_2.5: Fine particulate matter

IARC: International Agency for Research on Cancer

MiRNAs: MicroRNAs

MTT: 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide

DMSO: Dimethyl sulfoxide

EMT: Epithelial-mesenchymal transition

QRT-PCR: Quantitative Real-time Polymerase Chain Reaction

DMEM: Dulbecco's modified Eagle's medium

PBS: phosphate buffer saline

FBS: Fetal bovine serum

PVDF: Polyvinylidene Fluoride

SDS-PAGE: dodecyl sulfate, sodium salt (SDS)-Polyacrylamide gel electrophoresis

Ethics declarations

Ethics approval and consent to participate

not applicable.

Consent for publication

All authors have read and approved the manuscript. The need for publication is not applicable.

Competing interests

The authors declare that they have no competing interest

Availability of data and materials

All data analyzed within this study are included either in the manuscript or in the additional supplementary files.

Funding

This work was supported by the Project Foundation for the Young Talents in Colleges of Anhui Province (No. gxyq2017003), the Grant for Scientific Research of BSKY from Anhui Medical University (No. XJ201621), the National Natural Science Foundation of China (No. 81872580), and the Student’s Platform for Innovation and Entrepreneurship Training Program of China (No. 201810366031).

Author' s contributions

MY and CLL performed the experiments. LLJ, MY and RD prepared the manuscript. QZ, SS and LD analyzed the data and made the graphs. HC, CZ, NL and XMS conducted the statistical analysis. LLJ, MY and RD reviewed and edited the manuscript. RD, JYC and MWY supervised this research project. The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.

Acknowledgements

Not applicable.

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MiR-582-3p Participates in the Regulation of Biological Behaviors of A549 Cells by Ambient PM2.5 Exposure

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Abstract

Figures

Introduction

Results

Cytotoxic effects

PM2.5 promoted the proliferation, migration and invasion of A549 cells

PM2.5 induced EMT in A549 cells

PM2.5 activated Wnt/β-catenin signaling pathway in A549 cells

PM2.5 altered miR-582-3p expression in A549 cells

MiR-582-3p mediated PM2.5-induced proliferation, migration and invasion of A549 cell

MiR-582-3p mediated PM2.5-induced EMT in A549 cells

MiR-582-3p mediated the activation of Wnt/β-catenin signaling pathway in A549 cells induced by PM2.5

MiR-582-3p regulates the EMT process of A549 cells through the Wnt/β-catenin signaling pathway

Discussion

Conclusion

Materials And Methods

Collection and extraction of ambient PM2.5

Cell culture and transfection

Cell viability test

Plate clone formation assay

Wound healing assay

Transwell cell invasion assay

Western blot

QRT-PCR

Statistical analysis

Abbreviations

Declarations

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Availability of data and materials

Funding

Author' s contributions

Acknowledgements

Additional information

Publisher’s Note

References

Supplementary Files

Status:

Version 1

PM_2.5 promoted the proliferation, migration and invasion of A549 cells

PM_2.5 induced EMT in A549 cells

PM_2.5 activated Wnt/β-catenin signaling pathway in A549 cells

PM_2.5 altered miR-582-3p expression in A549 cells

MiR-582-3p mediated PM_2.5-induced proliferation, migration and invasion of A549 cell

MiR-582-3p mediated PM_2.5-induced EMT in A549 cells

MiR-582-3p mediated the activation of Wnt/β-catenin signaling pathway in A549 cells induced by PM_2.5

Collection and extraction of ambient PM_2.5