The following study was approved by the Ethics Committee of Wuhan University (Wuhan, China). Primers specific to human miR-196b (gene ID 442920) were designed as follows: 5′-CGGTTAACCCCTTCCTTGACGCATTTG-'3 (sense) and 5′-CGACTCGAGAACCTAACCCTACCTGCTGTGA-3′ (anti-sense). The primers were synchronized by Takara Biotechnology Co., Ltd. (Dalian, China) by introducing HpaI and XhoI restriction enzyme sites at the terminals. The normal male peripheral blood genomic DNA template was used to amplify specific fragments by polymerase chain reaction (PCR). Then the PCR product and pLL-3.7 plasmid (Takara Biotechnology Co., Ltd.) were digested with HpaI and XhoI and ligated overnight using T4 ligase at 16°C. The ligated product was transferred to E.coli STBL-3 competent cells (Takara Biotechnology Co., Ltd.) and selected for positive clones by plating on an ampicillin+LB plate. pLL-miR-196 plasmid was purified from the positive clones. Using Lipofectamine® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10 mg pLL-miR-196b and 5 mg pCMV-VSV-G were delivered to HEK293FT cells (Cell Bank of the Chinese Academy of Science, Beijing, China). Prior to transfection, the cells were seeded on a six-well plate at a density of 2×10⁶ cells per well and co-cultured with modified RPMI-1640 medium containing 2 ml 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) until confluence reached 70–90%. Virus-containing cell culture fluid was collected at 48 h post-transfection, centrifugated at 3000 × g for 15 min at 4°C, filtered through a 0.45-µm filter, and maintained at −80°C. MKN28 gastric cancer cells (Cell Bank of the Chinese Academy of Science) were incubated on a six-well plate at a density of 1×10⁶ per well with modified RPMI-1640 medium supplemented with 2 ml 10% FBS. After 24 h, virus-containing supernatant was delivered to the cells to serve as the miR-196b group. For the control group, the same procedure was carried out, with the exception that the virus-containing supernatant was replaced with 100 µl phosphate-buffered saline (PBS). The transduced cells demonstrated green fluorescence under the fluorescence microscope. The expression of miR-196b was measured using quantitative PCR (qPCR).

When cells were in the exponential phase of growth, TRIzol reagent (Thermo Fisher Scientific, Inc.) was used to isolate total RNA. mRNA reverse transcription was generated using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc.) following the instructions. The reaction system was as follows: 2 µl 5X PrimeScript buffer, 2 µl total RNA (0.25 µg/µl), 1 µl primers (10 pM), 0.5 µl PrimerScript RT enzyme mix, and 6.5 µl RNase-free dH₂O. The reaction parameters were 37°C for 15 min, followed by 85°C for 5 sec and 4°C for the hold. Quantitative analysis was performed using a Thunderbird SYBR® qPCR mix kit (Toyobo Co., Ltd., Osaka, Japan). The reaction system was as follows: 12.5 µl 2X qPCR mix, 2.0 µl of each 2.5 µM primer, 2.0 µl reverse transcription products, and 8.5 µl ddH₂O. The amplification parameters were 40 cycles of 95°C for 3 sec, 95°C for 5 sec, 60°C for 30 sec, and 68°C for 45 sec. For the qPCR primers, see Table I.

Cells were lysed for total protein extraction using RIPA buffer (250 µl, Beyotime Institute of Biotechnology, Shanghai, China) for 24 h after being incubated on a six-well plate. The expression of miR-196a, PI3K, AKT and mTOR was measured using a western blot kit (Biosdec, Wuhan, China). Rat anti-human miR-196a, PI3K, AKT and mTOR antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Dallas, TX, USA).

Successively, 50 µg total protein was separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a 0.45 µm polyvinylidene fluoride membrane and incubated overnight at 4°C with rat anti-human monoclonal antibody (1:3000 dilution). Following decoloration, horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at a 1:3000 dilution was added for 30 min at room temperature and unbound antibody was washed away. Proteins were visualized by enhanced chemiluminescence detection reagents (Thermo Fisher Scientific, Inc.) and exposed to X-ray film after raffinate clearance. Exposure conditions and developing and fixing were based on the light intensity. Developed films were processed with BanScan software (Glyko Inc., Novato, CA, USA) to determine optical densities.

Cells were seeded on a 96-well plate at a density of 2000 cells per well. When the cells reached 80% confluence, the culture medium was removed before adding 20 µl MTT reaction solution (5 mg/ml, Beyotime Institute of Biotechnology) to each well. Once the cells had been incubated in the dark for 4 h at 37°C, the supernatant was removed and 150 µl DMSO (Beyotime Institute of Biotechnology) was added to each well and agitated for 10 min at room temperature. Optical density was measured at 490 nm using the enzyme-linked immunosorbent assay.

Cells were seeded on a six-well plate at a density of 1×10⁶ per well and allowed to incubate until cell adhesion using the conventional culture technique. The culture medium was removed, then cells were suspended, centrifuged and then fixed with precooled 75% ethanol overnight at −20°C. Cells were centrifuged to remove supernatant and washed twice with PBS. A total of 450 µl PBS was added to each well to resuspend cells. Subsequently, 50 µl propidium iodide (0.5 mg/ml, Beyotime Institute of Biotechnology) was added, agitated and the cells were maintained in a water bath at 37°C for 30 min. Cells were again centrifuged to remove the supernatant and PBS was used to resuspend the cells. The cell cycle was determined by flow cytometer (BD-FACSCalibur, BD Biosciences, San Jose, CA, USA).

Soft agar (5%) was added to the medium at a 1:9 ratio and mixed well before being placed on dishes and cooled at room temperature. Exponentially growing cell suspension containing 1×10³ cells/ml was prepared. Cell suspension (1.5 ml) was then added to an equal volume of 0.5% soft agar, agitated and incubated on a dish at 37°C under 5% CO₂ for 2 to 3 weeks. The formation of colonies was calculated using the formula: Colony formation rate (%) = (number of colonies/number of cells incubated) × 100.

Cell invasion ability was tested using a Transwell chamber model (Chemicon; EMD Millipore, Billerica, MA, USA). Cell suspension was adjusted to a concentration of 1×10⁵ cells/ml. Then, 50 µl cell suspension was placed in the upper chamber. After 24 h, cells that had migrated to the lower chamber were fixed with 10% formalin and stained with Giemsa to quantitate the number of transmigrated cells under an inverted microscope. The transmigration rate was the number of cells transmigrated over the total number of cells.

Six 5-week-old BALB/c nude mice purchased from Wuhan University Center for Animal Experiments, China, were randomly assigned to the miR-196a group and control group, with three mice per group. In the miR-196a group, 1×10⁵ miR-196a-transfected MKN28 cells were suspended in a serum-free RPMI-1640 medium (0.1 ml), and administered by subcutaneous injection into the back of the nude mice. Three days later, another injection with the same number of cells at the same concentration was administered. In the control group, the mice underwent the same treatment as the miR-196a group with the exception that the MKN28 cells were not miR-196a-transfected. Animals were sacrificed 4 weeks after tumor formation and tumor weight was determined.

Data were expressed as the means ± standard deviation and were processed with the paired t-test using SPSS 16.0 software (SPSS, Inc., Chicago, Illinois, USA). P<0.05 was considered to indicate a statistically significant difference.

MKN28 cells exhibited green fluorescence when transfected with lentivirus-mediated miR-196b. In addition, the expression of miR-196b RNA was ~20-fold higher than that in the control group (Fig. 1A). The results of the MTT assay revealed that miR-196b activation enhanced the proliferative ability of MKN28 cells compared with that of the control group (P<0.01; Fig. 1B). We assessed the effect of miR-196b on the cycle of gastric cancer cells by flow cytometry and observed that gastric cancer cells in the G0/G1 phase decreased, while those in the S and G2 phase of the cell cycle increased compared with the control group (P<0.05; Fig. 1C).

Colony-forming cell assay revealed that the cell cloning efficiency increased to (78±12)% following miR-196b activation compared with (32±8)% in the control group (P<0.01; Fig. 2A). Transwell invasion assay revealed (48±16)% cell migration following miR-196b activation, which was far higher than the (12±4)% observed in the control group (P<0.01; Fig. 2B).

The results of the tumorigenecity test revealed increased tumor growth following miR-196b transfection (Fig. 3A). Four weeks after injection of miR-196b-transfected gastric cancer cells into nude mice, the tumor weight was significantly higher than that of the control group (P<0.01; Fig. 3B).

Western blot analysis revealed that the levels of PI3K/AKT/mTOR protein in gastric cancer cells were 0.38±0.06, 0.46±0.08 and 0.22±0.06 in the miR-196b transfection group, which was far higher than the levels 0.18±0.03, 0.16±0.04 and 0.09±0.03 observed in the negative control group (P<0.01; Fig. 4A). qPCR analysis demonstrated significant upregulation of PI3K/AKT/mTOR mRNA in gastric cancer cells following miR-196b transfection (Fig. 4B).