HBE cells were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). Lung adenocarcinoma cells (A549) were obtained from the Laboratory of Neuro-oncology, Tianjin Neurological Institute (Tianjin, China). The cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin and 1% glutamine. All of the cells were cultured in six-well plates in a 37°C humidified incubator supplied with 5% CO₂.

The DEPs were a gift from the State Key Laboratory of Internal Combustion Engine Fuel Science of Tianjin University (Tianjin, China). DEPs (100 µg/ml) were freshly prepared and suspended in 100 ml RPMI-1640 with 10% FCS, 1% penicillin-streptomycin and 1% glutamine by sonication. The suspended particles were then stored at 4°C in the dark. The cells were cultured in six-well plates and then treated with 3 ml RPMI-1640 in the experimental group.

The inhibitors were chemically synthesized by Shanghai GenePharma (Shanghai, China). The 2′-O-Me oligonucleotides were composed entirely of 2′-O-methyl bases and contained the following sequences: 5′-GTCCAC TCT TGT CCT CAA TG-3′; scrambled sequences were 5′-AAG GCA AGC UGA CCC UGA AGU-3′. The oligonucleotides were purified using a high-pressure liquid chromatography system, dissolved in diethylpyrocarbonate (DEPC) water and then frozen at −20°C. Oligonucleotides (50 nm/l) were transfected into HBE cells at 70% confluence using Oligofectamine according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA).

Total RNA was extracted using the TRIzol reagent (Invitrogen Life Technologies) according to the standard procedures. A NanoDrop spectrophotometer (Gene Co., Ltd., Hong Kong, China) was used to detect the total RNA concentration. Total RNA (1 µg) was used to synthesize cDNA by reverse transcription with MMLV reverse transcriptase (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. qPCR analysis was performed to determine the miR-21 expression in HBE cells, A549 and DEP-treated HBE cells 48 h following transfection with the miR-21 inhibitor or scrambled negative control. The expression of u6 was used as an internal control. A total of 40 cycles of qPCR were performed, consisting of 95°C for 3 min, 95°C for 15 sec and 60°C for 30 sec. RT and PCR primers were purchased from GenePharma, 5S was used for normalization. The relative quantification was conducted using amplification efficiencies derived from cDNA standard curves. The data are expressed as the fold change (2^−ΔΔCt) and initially analyzed using Opticon Monitor Analysis Software V2.02 software (MJ Research Inc., Waltham, MA, USA).

Following treatment with DEPs and DEPs in combination with the miR-21 anti-sense oligonucleotide (ASODN) for 48 h, the adherent cells in the six-well plate were rinsed with phosphate-buffered saline (PBS), and the cell extracts were prepared by suspension in ice-cold lysis buffer. Floating cells from the same well were collected, lysed and mixed with SDS loading buffer (100 mM Tris-Cl, pH 6.8; 200 mM DTT; 4% SDS; 0.2% bro-P-40 lysis buffer [20 mM Tris, pH 8.0; 137 mM NaCl; 1% Nonidet P-40; 10% glycerol; 1 mM CaCl2; 1 mM MgCl2; 1 mM phenylmethylsulfonyl fluoride; 1 mM sodium fluoride; 1 mM sodium orthovanadate; and a protease inhibitor mixture]). Homogenates were clarified by centrifugation at 12,000 × g for 15 min at 4°C, and protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The next 40 mg of lysates was subjected to SDS-PAGE on 8% SDS-acrylamide gels. The separate proteins were transferred onto PVDF membranes (Millipore, Bedford, MA, USA) and incubated with primary antibodies against AKT (1:1,000 dilution; rabbit polyclonal; Signalway Antibody, College Park, MD, USA), PI3K (1:1,000 dilution; mouse monoclonal; Cell Signaling Technology, Inc., Beverly, MA, USA), PTEN, caspase 3, PCNA, cyclin D1, Bcl-2 (1:100 dilution; mouse polyclonal; Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) and GAPDH (1:100 dilution; rabbit polyclonal; Zhongshan Goldenbridge Biotechnology Co., Ltd.). The membranes were then incubated with an HRP-conjugated secondary antibody (Zymed Laboratories Inc., San Francisco, CA, USA). Specific proteins were detected using a Super Signal protein detection kit (Pierce Biotechnology, Inc.). The membrane was reprobed with an antibody against GAPDH (1:100 dilution; rabbit polyclonal; Zhongshan Goldenbridge Biotechnology Co., Ltd.) using the procedures described above.

For cell cycle analysis using FCM (flow cytometry), log phase transfected and control cells were harvested, washed with PBS, fixed with 90% ethanol overnight at 41°C, then incubated with RNase at 37°C for 30 min. The cell nuclei were stained with propidium iodide (PI) for an additional 30 min. A total of 104 nuclei were examined using a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The samples were analyzed using flow cytometry for the FL-2 area, DNA histograms were analyzed using Modifit software. The experiments were performed in triplicate. The results are presented as the percentage of cells in a given phase. The apoptosis assays were conducted using annexin staining and TUNEL methods.

To quantify DEP-induced apoptosis, annexin V/PI staining was performed, and apoptosis was evaluated by flow cytometry analysis. Following DEP treatment for 48 h, the floating and attached cells were collected and subjected to annexin V/PI staining using an annexin V-FITC Apoptosis Detection kit (BioVision, Palo Alto, CA, USA), according to the manufacturer’s instructions. The resulting fluorescence was measured by flow cytometry using a FACS flow cytometer (Becton-Dickinson).

The invasive ability of treated and untreated HBE and A549 cells was assessed using a 24-well transwell chamber and a reconstituted extracellular matrix membrane (Matrigel, BD Biocoat; BD Bioscience Franklin Lakes, NJ, USA). The cell invasion chambers were prepared by placing 100 µl of a 1:5 dilu tion of Matrigel onto the upper filter and then incubating the cells for ~30 min at 37°C to allow for Matrigel polymerization. The untreated HBE and A549 cells, as well as the HBE and A549 cells that were treated with DEPs for 48 h, were removed from the culture flasks and resuspended at 1×10⁵ cells/ml in serum-free medium. Then, 0.1 ml of the cell suspension was added to the upper chambers. The medium supplemented with serum was used as a chemoattractant in the lower chamber. The chambers were incubated for 48 h at 37°C in a humid atmosphere of 5% CO₂/95% air. The filters were then fixed in 95% ethanol and stained with hema toxylin. The upper surfaces of the filters were scraped thrice with cotton swabs to remove the non-invasive cells. The experi ments were repeated thrice and the migrated cells were microscopically counted in five different fields per filter.

The HBE and A549 cells were cultured in six well plates and the clones were grown to confluency. A linear wound was made by scraping a closed Pasteur pipette across the confluent cell layer, following 24 h DEP treatment in the experimental group. The cells were washed twice to remove the detached cells and debris. Next, the wounds sizes were observed and measured at set times.

First, the miR-21 expression in HBE cells, A549 and treated HBE cells was profiled. The analysis demonstrated that miR-21 is overexpressed by greater than 16-fold in untreated A549 cells compared with the untreated HBE cells. The miR-21 expression in the HBE cells treated with DEPs for 48 h was 6-fold higher than the untreated HBE cells (Fig. 1A).

The effect of DEPs on PTEN/PI3K/AKT activity was then measured. PTEN, PI3K, phosphorylated AKT (p-AKT) and AKT were immunoprecipitated from the total cell extracts derived from the HBE cells treated with 100 µg/ml DEPs. The PTEN, PI3K, p-AKT and AKT expression in untreated and treated HBE cells were examined (Fig. 1B). As determined by western blot analysis, the PTEN expression was significantly reduced in the untreated HBE cells. However, PI3K, AKT and p-AKT expression were markedly increased in the HBE cells following treatment with DEPs for 48 h (Fig. 1C).

To evaluate the effect of the miR-21 inhibitor on the experimental group treated with DEPs, qPCR was used to compare the expression of miR21 in HBE cells transfected with the miR-21 inhibitor in the experimental and control groups. The measurements were performed 48 h following transfection. The data were analyzed from triplicate samples. DEPs alone increased the miR-21 expression in the studied cells. However, following transfection of the DEP-treated group with the miR-21 inhibitor, miR-21 expression was significantly reduced (Fig. 2A).

Studies have demonstrated that PTEN is a miR-21 target gene, and PTEN regulates the PI3K/AKT pathway (16). PTEN frequently inhibits PI3K/AKT pathway activation in lung cancer (15,17). Therefore, one potential function of spliced miR-21 may be to upregulate the PTEN tumor suppressor gene. However, the above analysis demonstrated that increasing the expression of miR-21 suppressed PTEN in HBE cells. Given that the inhibition of gene expression by microRNAs is well understood, as-miR-21 was transfected into the experimental group above, and an increase in the PTEN protein expression and a reduction in the PI3K, p-AKT and AKT expression in the HBE cells was subsequently observed (Fig. 2B).

To analyze whether increased miR-21 expression in HBE and A549 cells was able to alter cellular apoptosis, DEP-induced apoptosis was measured via annexin V-PI staining in HBE and A549 cells. The cells were exposed to DEPs (100 µg/ml) for 48 h. As demonstrated in Fig. 3A and B, a significant increase in early phase apoptotic cells was observed in the DEP-treated HBE cells (9.64%) compared with the untreated cells (3.24%) (P<0.05); however, no significant differences were observed between the treated (2.84%) and untreated A549 cells (3.32%; P>0.05; Fig. 3A).

DEP has been reported to induce growth arrest at the G1/G0 phase in a wide variety of cells (18–20). To understand the effects of DEP treatment on cell growth in the present study, the cell cycle distribution of HBE and A549 cells 48 h following treatment with 100 µg/ml DEPs was measured via PI staining. The untreated cells served as negative controls. DEPs induce a significant increase (P<0.05) in the number of S-phase cells in the A549 cell lines. DEPs markedly enhanced the number of G0/G1-phase cells in HBE cells (P<0.05; Fig. 3B).

To examine the functional consequences of DEPs in HBE cells and A549 cell lines, the Transwell assay was used to determine cell invasiveness and wound healing assays were used to detect cell motility in the cells treated with DEPs (100 µg/ml). Significant differences in the number of invasive cells were observed between the control and experimental groups (Fig. 4A). In addition, evident differences in the cellular migration remained between the treated cells and the control samples. A significant portion of HBE cells invaded the lower chamber (P<0.05), however this phenomenon was not observed in the A549 cells (P>0.05). Cell motility was suppressed by DEPs in the experimental group in HBE and A549 cells (P<0.05), but the effect was less prominent in A549 cells compared with HBE cells (Fig. 4B).