The human NSCLC cell line A549 was purchased from the American Type Culture Collection (Manassas, VA, USA). RPMI-1640 medium, fetal bovine serum (FBS), PBS, bovine serum albumin (BSA), dimethylsulfoxide and Cell Counting Kit-8 (CCK-8) were purchased from Beijing Transgen Biotech Co., Ltd. (Beijing, China). Antibodies against matrix metalloproteinase (MMP)-2, MMP-9, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, poly (ADP-ribose) polymerase (PARP), β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Lipofectamine^® 2000 and Opti-MEM were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). AnnexinV/fluorescein isothiocyanate (FITC) kit and Matrigel invasion chambers were purchased from BD Biosciences (San Jose, CA, USA). The Transwell invasion chambers were purchased from Costar (Corning Life Sciences, Cambridge, MA, USA). Crystal violet staining solution was purchased from Beyotime Institute of Biotechnology (Haimen, China). Scrambled sequence and miR-145 mimic were purchased from Biotend (Shanghai, China).

The A549 cells were grown in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, and were cultured at 37°C in a humidified atmosphere containing 5% CO₂. All cells used in the present study were subjected to <20 passages.

In the exponential phase, A549 cells were seeded at a density of 5×10⁵ cells/well in a 6-well plate for 24 h before transfection. Lipofectamine 2000 was used for the transfection of scrambled sequence: Forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′; or miR-145 mimic, forward, 5′-GUCCAGUUUUCCCAGGAAUCCCU-3′ and reverse, 5′-GGAUUCCUGGGAAAACUGGACUU-3′, according to the manufacturer's protocol. A final concentration of 100 nM miR-145 mimics and 100 nM negative control miRNA was used for transfection. At 24 or 48 h, the transfected cells were collected and used in the subsequent experiments.

Cell proliferation was detected using the CCK-8 assay. Briefly, A549 cells were transfected with scrambled sequence or miR-145 mimic. Transfected and non-transfected cells were incubated in 96-well plates at a density of 4×10³ cells/well. At 24, 48 and 72 h post-transfection, CCK-8 solution was added (10 µl/well) and cells were incubated at 37°C for 2 h. Absorbance at 450 nm was measured using a Universal Microplate Reader EL800 (Bio-Tek instruments, Inc., Vermont, MA, USA).

For the invasion assays, 5×10⁴ cells were plated in a 8.0 µm pore size Matrigel invasion chamber without serum and the lower chamber contained RPMI-1640 supplemented with 20% FBS that acted as a chemoattractant. At 24 h, the non-invading cells were removed with cotton swabs. The invasive cells located on the lower side of the chamber were fixed in 4% formaldehydeat room temperature for 15 min, stained with 0.2% crystal violet staining solution at room temperature for 30 min and counted under a phase-contrast microscope (Olympus Corporation, Tokyo, Japan) in three random fields (magnification, ×100).

The migration of the A549 cells was evaluated using wound-healing assays. Cells (5×10⁵ cells cells/well) from the three groups (CON, non-transfected control; NC, non-specific negative control; and MIMIC, miR-145 mimic) seeded in a 6-well culture plate to form a confluent monolayer and were then wounded using 100 µl pipette tips. The scratch wounds were observed, and images were captured at 0 and 24 h, under a phase-contrast microscope (magnification, ×40).

At 48 h post-transfection, A549 cells from the three groups (CON, non-transfected control; NC, non-specific negative control; and MIMIC, miR-145 mimic) were seeded in 6-well plates and fixed with 4% paraformaldehyde for 10 min at 4°C. Subsequently, cells were washed three times with ice-cold PBS and stained with 10 mg/l Hoechst 33258 for 10 min at 25°C in the dark. Nuclei were observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) (magnification, ×200).

The cell apoptosis assays were performed using an Annexin-V/FITC kit. Cell apoptosis was detected using flow cytometry at 48 h after transfection. The transfected cells were collected and washed with ice-cold PBS prior to being stained with Annexin-V/FITC and propidium iodide (PI) solution for 15 min in the dark. The proportion of the apoptotic cells was determined using a flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software (version 10; Tree Star, Inc., Ashland, OR, USA). All experiments were performed three times.

At 48 h post-transfection, cells from the three groups (CON, non-transfected control; NC, non-specific negative control; and MIMIC, miR-145 mimic) were collected and lysed in radioimmunoprecipitation assay buffer containing phenylmethanesulfonyl fluoride and phosphatase inhibitor cocktail (Applygen Technologies Inc., Beijing, China). Each sample was centrifuged at 17,105.6 × g for 10 min at 4°C to remove cell debris and the supernatant was collected for immune blotting. Protein concentrations were calculated using BSA. Equal quantities of proteins (40 µg) were loaded and separated by SDS-PAGE (10% gels) for 2 h at 100 V under reducing conditions. The proteins were transferred onto polyvinylidene difluoride membranes in a Tris-glycine transfer buffer. Subsequent to blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 2 h, the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies were: Anti-Bax (cat. no. 2774), anti-Bcl-2 (cat. no. 2872), anti-caspase-3 (cat. no. 9662), anti-PARP (cat. no. 9542), anti-MMP-2 (cat. no. 4022), anti-MMP-9 (cat. no. 3852) and β-actin (cat. no. 8457; dilution of all, 1:1,000). The membranes were washed with Tris-buffered saline with Tween-20 (TBST) three times and incubated with secondary HRP-conjugated antibodies (Anti-rabbit IgG; cat. no. 7074; dilution, 1:5,000) at 25°C for 2 h. Membranes were washed with TBST three more times and immune reactive protein bands were detected by an enhanced chemiluminescence kit (Beijing Trans gen Biotech Co., Ltd., Beijing, China). Image Quant TL 7.0 software (GE Healthcare, Chicago, IL, USA) was employed to quantify protein expression levels.

Data were analyzed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation. Statistical analysis was performed using independent two-sample t-tests, or by one-way analysis of variance (ANOVA) with Tukey's post-hoc test for ≥3 groups. P<0.05 was considered to indicate a statistically significant difference.

A549 cells were transfected with scrambled sequence or miR-145 mimic, and their proliferative ability was assessed using a CCK-8 assay. At 24, 48 and 72 h post-transfection, the optical density values of the miR-145 mimic group, the non-transfected group, and non-specific negative control were 0.335±0.012, 0.426±0.002 and 0.349±0.048; 0.704±0.008, 0.928±0.070 and 0.903±0.032; 1.558±0.049, 2.028±0.209 and 1.987±0.155, respectively (Fig. 1A). Therefore, ectopic miR-145 expression significantly decreased the proliferation of A549 cells (P<0.05).

Boyden chamber Transwell assays were employed to investigate the effect of miR-145 expression on the invasive ability of A549 cells. Typical micrographs of the Transwell filters are presented in Fig. 1B. The invasive cell count (Fig. 1B and C) indicated that invasive capacity was significantly decreased in the miR-145 mimic group compared with that in the non-transfected group (P<0.001). The invasive cell count of the non-transfected group compared with the non-specific negative control demonstrated no significant difference (P>0.05). Additionally, the migratory ability of A549 cells in response to miR-145 treatment was evaluated using a wound healing assays, which identified that treatment with an miR-145 mimic decreased the percentage of wound closure compared with the non-transfected group, 24 h post-transfection (P<0.01; Fig. 1D and E). No difference in wound closure was observed between the non-specific negative control and the non-transfected group (P>0.05; Fig. 1E).

MMP-2 and MMP-9 expression is associated with tumor invasion and metastasis (15–17). Therefore, the present study investigated the expression levels of MMP-2 and MMP-9 in response to miR-145 treatment in A549 cells using western blot analysis (Fig. 2A). MMP-2 and MMP-9 expression was significantly decreased in the miR-145 mimic group compared with that in the non-transfected group (P<0.05; Fig. 2B). There was no significant difference between the non-transfected group and the non-specific negative control in the expression of MMP-2 and MMP-9 (P>0.05; Fig. 2B). These results indicate that increased miR-145 expression inhibits the invasion and migration of A549 cells possibly by repressing the expression of MMP-2 and MMP-9.

Apoptotic cells were detected in A549 cells using the fluorescent DNA-binding dye Hoechst 33258, which stains the nuclei of normal cells blue and those of apoptotic cells bright blue or even white (Fig. 2C). The results indicated that the number of apoptotic cells was increased in the miR-145 mimic group compared with that in the non-transfected group (P<0.001; Fig. 2D), there was no significant difference in the number of apoptotic cells between the non-transfected group and the non-specific negative control (P>0.05). Next, flow cytometric analysis using Annexin V-FITC/PI double staining depicted apoptosis in A549 cells (Fig. 3A). Cell apoptosis of ectopic miR-145 expression was increased and the apoptotic index was significantly higher when compared with the non-transfected group. The results indicated that ectopic miR-145 expression induced apoptosis of A549 cells (P<0.001; Fig. 3B). There was no significant difference between the non-transfected group and the non-specific negative control (P>0.05). Collectively, these results indicate that deregulation of miR-145 expression promoted the apoptosis of A549 cells.

In order to elucidate the potential molecular mechanism underlying the effect of miR-145 on apoptosis of A549 cells, the expression of Bax, Bcl-2, procaspase-3 and PARP was examined using western blot analysis. miR-145 treatment increased the expression of Bax, but led to a marked decrease in the expression level of Bcl-2. Thus, the Bax/Bcl-2 ratio was increased in the miR-145 mimic group compared with the non-transfected group (P<0.01; Fig. 4A and B). Furthermore, miR-145 over expression promoted the cleavage of procaspase-3 and PARP (P<0.05; Fig. 4C and D). No difference in the Bax/Bcl-2 ratio, or the cleavage of procaspase-3 or PARP were observed between the non-transfected group and the non-specific negative control group (P>0.05).