The 95D and 16HBE cells were obtained from the Chinese Academy of Sciences Type Culture Collection Cell Library Committee (Shanghai, China). These cells were cultured in RPMI-1640 cell culture medium supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare Life Sciences, Little Chalfont, UK), 100 units/ml penicillin and 100 units/ml streptomycin sulfate in a humidified 5% CO₂ incubator at 37°C. The total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), resolved in nuclease-free water and preserved at −80°C.

The total RNA was purified to remove any mRNA prior to sequencing. A GeneChip miRNA 2.0 array (Affymetrix, Santa Clara, CA, USA) was used to analyze the miRNA expression profile and one-way analysis of variance (ANOVA) was used to identify any significant differences among the groups. P<0.05 was considered to indicate a statistically significant difference. A fold change >2 was used as the standard in identifying the differentially expressed miRs.

The prediction of target candidates of miR-125b was performed using miRGen targets on three authority target predicting software programs, TargetScan 5.1 (http://www.targetscan.org/), Pictar (http:/pictar.mdc-berlin.de/) and miRanda (http://www.microrna.org/microrna/home.do). The miR-125b targets were then entered into the DAVID Bioinformatics Resources 6.7 software to perform Gene Ontology (GO) annotation. In addition, DIANA-miRPath software (http://diana.cslab.ece.ntua.gr/pathways/) was used to perform online gene enrichment analysis of the miR-125b putative targets, which compared the set of miR-125b targets to all known Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.kegg.jp/) pathways. Subsequently, the information containing the numbers and P-values, in the form ‘In (P-value)’, of the enrichment genes in the specific pathways were examined. The interaction and reaction networks between the genes were also analyzed according to the pathway data provided by the KEGG pathway database.

The transfections were performed using siPORT NeoFX transfection reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. The cells were trypsinized at the logarithmic growth period and were resuspended in normal growth medium at 1×10⁵ cells/ml. The siPORT NeoFX transfection reagent was diluted (1:30) in Opti-MEM I (Invitrogen Life Technologies) and incubated for 10 min at room temperature. Subsequently, anti-miR-125b (Invitrogen Life Technologies) and anti-negative control (Anti-miR™ Negative Control #1, AM17010; Invitrogen Life Technologies) were diluted in Opti-MEM I. The transfection reagents were mixed and incubated at room temperature for 10 min. The transfection complexes were dispensed into the empty wells of a six-well culture plate. The cells were added to the transfection complexes and were incubated at 37°C for 24 h. Following transfection, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to examine the expression levels in the miR-125b-transfected cells and to confirm the transfection efficiency. RT-qPCR analyses for miR-125b were performed using TaqMan microRNA assays (Applied Biosystems Life Technologies, Foster City, CA, USA).All reagents, primers and probes were obtained from Applied Biosystems. The 15 μl RT reaction consisted of 7 μl Master mix (100 mM dNTPs (with dTTP) 0.15 μl; MultiScribe™ Reverse Transcriptase, 50 U/μl 1.00 μl; 10X Reverse Transcription buffer 1.50 μl; RNase Inhibitor, 20 U/μl 0.19 μl; Nuclease-free water 4.16 μl), 3 μl stem-loop RT primer (ID 000449), and 5 μl RNA sample. The reactions were incubated on ice for 5 min and followed by 1°C for 30 min, 42°C for 30 min, 85°C for 5 min and held in 4°C. The 20 μl PCR reaction included product from RT reaction (minimum 1:15 dilution) 1.33 μl; TaqMan MicroRNA assay (20X) 1.00 μl (ID 000449); TaqMan 2X Universal PCR Master mix, No AmpErase UNGa 10.00 μl; Nuclease-free water 7.67 μl. Amplification was performed using the Step One Plus Detection System (Applied Biosystems), and initiated at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec, 60°C for 1 min and annealing and extension at 60°C for 1 min.

An MTT assay was used to determine the cell proliferation. The cells (1×10⁴) were seeded into each well of a 96-well plate. The cells were transfected in triplicate wells with 50 nM anti-miR-125b or anti-miR negative control and were cultured in complete culture medium for 48 h. The culture supernatants were then removed and 200 μl MTT solution (0.5 mg/ml; Amresco LLC, Solon, OH, USA) was added to each well, followed by incubation for 4 h at 37°C. The supernatants were then removed and 150 μl dimethyl sulfoxide (Sigma-Aldrich) was added into each well and agitated to dissolve the formazan crystals. The optical density (OD) was measured at 570 nm in an automatic enzyme standard microtiter plate reader (MRX; Dynex Technologies, Chantilly, VA, USA). The experiment was repeated three times.

The cells were analyzed for annexin V binding and propidium iodide (PI) incorporation to distinguish between apoptotic and necrotic cells. Cells (3×10⁵) were seeded into each well of a 96-well plate and transfected in triplicate wells with 50 nM anti-miR-125b or anti-miR negative control, followed by culture in complete culture medium for 48 h. The cells were harvested, washed twice in ice-cold PBS, resuspended in 500 μl binding buffer and stained for 30 min with Annexin V-fluorescein isothiocyanate and PI. The stained cells were analyzed using a flow cytometer (TY4124; BD Technologies, Durham, NC, USA) at an excitation wavelength of 488 nm and emission wavelength of 525 nm for fluorescein isothiocyanate fluorescence and 610 nm for PI fluorescence. The experiment was repeated three times.

The cells (3×10⁵) were seeded into each well of a 96-well plate and transfected in triplicate wells with 50 nM anti-miR-125b or anti-miR negative control, followed by culture in complete culture medium for 48 h. The cells were harvested, washed in ice-cold PBS and fixed with 70% ice-cold ethanol for 24 h. Subsequently, the cells were resuspended in 100 μl RNase and cultured for 30 min at 37°C. The suspension was mixed with 400 μl PI for 30 min in the dark and the stained cells were analyzed using a flow cytometer at an excitation wavelength of 488 nm and 610 nm for PI fluorescence. The experiment was repeated three times.

Matrigel was thawed at 4°C overnight and then diluted (5 mg/ml) in serum-free cold RPMI-1640 (Corning Inc., Corning, NY, USA) at a dilution of 1:8 and was transferred into the upper chamber of a 24-well Transwell system (100 μl in each well). The Transwell was incubated at 37°C for ~2–3 h for gelling. Subsequently, 100 μl cell serum-free RMPI-1640 suspension containing 1×10⁴ anti-miR-125b-transfected cells were added to the Matrigel. The lower chamber of the Transwell was filled with 600 μl RPMI-1640 containing 10% FBS. The 24-well plate was incubated at 37°C for 20–24 h. The cells which did not invade and remained in the upper chamber of the Transwell were removed by washing with PBS. Subsequently, 4% paraformaldehyde was used to immobilize the cells that had invaded the lower chamber and hematoxylin and eosin staining was performed. The cells in the lower chamber of the Transwell were counted under a light microscope (magnification, ×400; BX41; Olympus Corp., Tokyo, Japan). The experiment was repeated three times.

Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation. Analysis of variance and Dunnet’s test were performed to compare the differences between the exposure group and the control group. P<0.05 was considered to indicate a statistically significant difference.

Gene chip analysis revealed that 45 miRs were significantly differently expressed in the 95D cells compared with the 16HBE cells, of which 30 were upregulated and 15 were downregulated. The fold-changes in the dysregulated miRs are shown in Fig. 1. Confirmation of the expression of miR-125b by RT-qPCR supported the results of the gene chip analysis (data not shown).

Analysis was performed using miRGen Target software, which involved the prediction of possible targets of miR-125b by TargetScan, Pictar and miRanda. A total of 72 genes were identified as possible putative targets of miR-125b (data not shown). The GO terms of the biological process were further analyzed, revealing 21 associated functional annotations. The results demonstrated that terms associated with the function of phosphorylation, including the phosphate metabolic process and protein amino acid phosphorylation, were annotated by several gene targets of miR-125b. Stress-associated terms were also annotated by several targets. Of note, among the 21 terms, the term ‘regulation of MAP kinase kinase kinase (MAP3K) cascade’ was found (data not shown). In the KEGG pathway analysis, the transforming growth factor (TGF)-β signaling pathway scored highest (5.85). The Wnt signaling pathway, closely associated with the development and progression of cancer, also scored highly in the miR-125b enrichment pathway analysis (Table I). To further examine the impact of miR-125b on important pathways, pathway graphs, indicating the regulatory sites and interactions, were analyzed. In the TGF-β KEGG pathway graph, several genes at key sites were found to be potentially regulated by miR-125b. ACVR1C is involved in activating the TGF-β receptor and transmitting the signal to SMAD2, which affects cell apoptosis and cell cycle by activating a series of transcription factors (Fig. 2A). In the Wnt signaling KEGG pathway graph, DVL3, a possible target gene of miR-125b, regulates the mitogen-activated protein kinase (MAPK)-c-Jun N-terminal kinase (JNK) apoptotic process. APC and PPP2R1B affect the adherence and junctions between cells by regulating β-actin and are associated with migration and invasion (Fig. 2B).

Following anti-miR-125b transfection, miR-125b levels were significantly inhibited to 3% of those in the negative control (Fig. 3).

Under light microscopy, no significant differences were observed in the morphology or density of the anti-miR-125b-transfected 95D cells compared with those of the cells in the negative control group. The MTT assay, used to examine the effect of miR-125b downregulation on the viability of the 95D cells, demonstrated no significant differences in the OD values among the groups (P>0.05; Table II).

PI and Annexin V were used to stain the 95D cells and the apoptotic rate was determined using flow cytometry. No significant difference was observed between the blank and the negative control groups (P>0.05). The apoptotic rate of the cells in the positive group was significantly higher than that of cells in the other groups (P<0.05). Of note, the apoptotic rates of the cells with downregulated miR-125b were significantly higher compared with those in the negative control group (P<0.05; Fig. 4 and Table III).

The cell cycle of anti-miR-125b-transfected 95D cells was assessed using flow cytometry. The results demonstrated that downregulation of miR-125b in the 95D cells induced G1/S phase arrest compared with the negative group (P<0.05; Fig. 5 and Table IV).

A Transwell assay was used to evaluate the impact of downregulated miR-125b on the invasive ability of 95D cells. No significant change was observed in the number of invaded cells between the negative control group and the blank group (P>0.05). However, the number of invaded cells in the anti-miR-125b-transfected group was significantly lower compared with that in the negative control group (P<0.05; Fig. 6).