A549, NCI-H460 and SPCA-1 human lung cancer cell lines obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GE Healthcare Life Sciences, Logan, UT, USA).

The A549 lung cancer cell line was plated onto 48-well plates. Following adherence of the cells for 8 h, sense hsa-miR-125a-3p and anti-sense hsa-miR-125a-3p was transfected into A549 cells. The cells were cultured for 1, 3, 5 and 7 days, respectively. Then, cell proliferation was evaluated by an MTT assay (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 10 µl of 5 mg/ml MTT was added into the medium. After 4 h, 150 µl dimethylsulfoxide (Sigma-Aldrich) was added for 15 min. The plates were then read on a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a test wavelength of 490 nm and a reference wavelength of 570 nm.

Total RNA was extracted from A549, NCI-H460 and SPCA-1 lung cancer cells and HBE normal cells by TRIzol reagent (Takara Bio Inc., Dalian, China), and cDNA was transcribed using the PrimeScript^® RT reagent kit (Takara Bio Inc.) according to the manufacturer's instructions. RT-qPCR was performed to evaluate hsa-miR-125a-3p expression on ABI 7500 system. The following primer sequences were used: Sense, 5′-ACA GGU GAG GUU CUU GGG AGCC-3′ and antisense, 5′-GGC UCC CAA GAA CCU CAC CUGU-3′ for hsa-miR-125a-3p; and sense, 5′-CTC GCT TCG GCA GCACA-3′ and antisense, 5′-AAC GCT TCA CGA ATT TGCGT-3′ for small nuclear RNA U6RNA, which was used as a reference gene. The primers were synthesized by Quanshijin Corporation (Beijing, China). PCR thermal cycling conditions were as follows: 40 cycles of 12 sec at 95°C and 1 min at 60°C using SYBR Green (Takara Bio Inc.).

The A549 and NCI-H460 lung cancer cell lysates were prepared and separated by SDS-PAGE (22–24). After transferring the protein from the gel to the membrane, the monoclonal mouse anti-human p53 (DO-2; sc-53394), MDM-2 (SMP14; sc-965) and β-actin (C-2; sc-8432) primary antibodies were used for incubation. The cells were then incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody. All of the antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The western blots were visualized usin Immobilon Western Chemiluminescent HRP Substrate (ECL) (EMD Millipore, Billerica, MA, USA).

The cell apoptosis rate was determined by flow cytometric analysis with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining (25,26). Briefly, 1×10⁵ lung cancer cells were plated into 6-well plates. After 6 h, the cells were transfected with sense hsa-miR-125a-3p, anti-sense miR-125a-3p and scrambled miRNA, respectively, which were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). After five days, cell apoptosis was detected by annexin V-FITC/PI (Abcam, Cambridge, MA, USA) dual staining analysis according to the protocols. Finally, the cells were detected and analyzed by flow cytometry on a FACSCalibur Cell Sorting system (BD Biosciences, Franklin Lakes, NJ, USA).

A549 cells were cultured in DMEM with 10% FBS and Transwell compartments (Corning Incorporated, New York, NY, USA) were prepared with 24-well format with an 8-µm pore size. The assay was conducted using methods described previously (27,28). Briefly, the lower compartment was filled with 2.6 ml DMEM with 0.5% FBS containing 40 µg/ml collagen I (Sigma-Aldrich). The Transwell insert was then added to the well by merging the bottom of the insert into the medium in the lower compartment. A549 cells (1×10⁵) were then added to the upper compartment. The cells were incubated in the Transwell plate at 37°C and 5% CO₂ for 8 h. Then, the insert was carefully removed. A549 cells on the lower side of the insert filter were quickly fixed in 5% glutaraldehyde for 10 min, and stained with 1% crystal violet in 2% ethanol for 20 min. Finally, excess crystal violet was removed by quickly merging the insert in ddH₂O for a few seconds and the number of cells on the lower side of the filter was counted under a microscope (CX22; Olympus, Tokyo, Japan).

Data were analyzed with Student's t-test using SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). The data are presented as the mean ± standard error of the mean. P<0.01 was considered to indicate a statistically significant difference.

The expression of has-miR-125a-3p was detected by RT-qPCR in a panel of three colorectal cancer cell lines. The HBE cell line was used as a control. As shown in Fig. 1, the results demonstrated that the mRNA levels of has-miR-125a-3p in the cancer cell lines were significantly lower than those in the HBE cells, suggesting that has-125a-3p may be a tumor suppressor gene. Notably, the expression of has-miR-125a-3p in NCI-H460 cells was lowest, and most significantly different compared with the control cells (P<0.01).

Hsa-miR-125a-3p was overexpressed in A549 lung cancer cells, and an MTT assay was used to analyze the cell proliferation. As shown in Fig. 2, the lung cancer cells were transfected with sense hsa-miR-125a-3p or anti-sense has-miR-125a-3p for 1, 3, 5 or 7 days, respectively, and the results showed that transfection of sense hsa-miR-125a-3p inhibited the growth of A549 cells. In addition, overexpression of anti-sense hsa-miR-125a-3p did not suppress the proliferation of lung cancer cells.

In order to detect the effects of has-miR-125a-3p on cell apoptosis in lung cancer cells, cell apoptosis rates were detected by annexin V-FITC and PI dual staining analysis. The NCI-H460 lung cancer cell line was transfected with sense has-miR-125a-3p and anti-sense has-miR-125a-3p, and cultured for 5 days. The lung cancer cells transfected with scrambled miRNA were used as negative controls. As shown in Fig. 3, the apoptosis rate of sense has-miR-125a-3p transfected NCI-H460 cells was significantly higher than that of the controls (P<0.01).

In order to detect the effect of hsa-miR-125a-3p on cell migration and invasion of lung cancer cells, a Transwell migration assay was performed. As shown in Fig. 4, the number of migrated A549 cells was 33.50±2.27 in the control group without any treatment. The number of migrated cells was 25.33±3.15, which was less than that in the control group (P<0.01). Additionally, transfection of anti-sense hsa-miR-125a-3p significantly promoted cell migration compared with transfection with sense has-miR-125a-3p (P<0.01). The data demonstrated that transfection of sense hsa-miR-125a-3p effectively inhibited the cell migration and invasion of A549 lung cancer cells.

To further investigate the mechanism of hsa-miR-125a-3p the regulation of apoptosis of lung cancer cells, western blot analysis was used to detect the expression of p53 and MDM-2. As shown in Fig. 5, transfection of sense miR-125a-3p increased the expression of p53 and inhibited the expression of MDM-2 in A549 cells. This is consistent with that observed in the NCI-H460 lung cancer cell line. Untreated cells and the cells transfected with scrambled miRNA were used as negative controls. These results suggested that hsa-miR-125a-3p could promote cell apoptosis by inhibiting the expression of MDM-2 to upregulate the expression of tumor suppressor p53.