The MG63 and U-2 OS cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St Louis, MO, USA) and placed in a 5% CO₂ incubator. When 80% of cell fusion was completed, the cells were digested with 0.25% trypsin and subcultured in accordance with requirements in different Petri dishes until the cells grew to 70% confluence. In this experiment, rapamycin (RAPA; 100 nm), DDP (20 µM) and MTX (100 µM) (Sigma-Aldrich) were selected, and the cells were treated with the drugs for 48 h. Subsequent experiments could then be performed. The study was approved by the ethics committee of the Second Xiangya Hospital (Central South University, Changsha, China).

A target gene, chemically synthesized and linearly ligated into the AgeI lentiviral vector, was transformed into competent bacterial cells. Once the first colony was identified by polymerase chain reaction (PCR), the positive clones were sequenced and comparatively analyzed. The total volume of the reaction was 20 µl: 5 µl of sense oligonucleotide (200 µmol/l), 5 µl of antisense oligonucleotide (200 µmol/l), 2 µl of 10X annealing buffer and 8 µl of ddH₂O. A proper comparison was made to successfully clone a target plasmid, while purified viruses were measured by ELISA (KHB, Shanghai, China).

The level of miR-126 expression in the two osteosarcoma cell lines, MG-63 and U-2 OS, was detected by quantitative PCR following miR-126-overexpressing and -silencing lentiviral vector infection. Total RNA was isolated from using MirVana TM miRNA Isolation Kit (ABI, Roche, Branchburg, NJ, USA). Then the total RNA was transcribed into cDNA at 16°C for 30 min, 42°C for 30 min, 85°C for 30 min, and stored at 4°C. The reverse-transcription for GAPDH were conducted at 45°C for 1 h, then 70°C for 10 min, and saved at 4°C. The detection of miR-126 and GAPDH were conducted using the fluorescent dye SYBR Green. The sequences of the primers used for amplification were as follows: miR-126, F 5′-ACA CTC CAG CTG GGT CGT ACC GTG AGT AAT-3′ and R 5′-TGG TGT CGT GGA GGA GTC-3′; GAPDH, F 5′-GAA GGT CGG AGT CAA CGG ATT-3′ and R 5′-ATG GGT GGA ATC ATA TTG GAA-3′. The PCR reaction for miR-126 were 95°C for 5 min, 95°C for 15 sec, 60°C for 15 sec and a total 40 cycles were performed. GAPDH were conducted under 95°C for 5 min, 95°C for 15 sec, 60°C for 1 min and total of 50 cycles were performed. Fluorescence signals were collected at 85°C and GAPDH was used as an internal reference. After PCR was performed, the mRNA level was calculated by a comparative threshold cycle (Ct) method using the formula 2^−Δ∆Ct. miR-126 expression in the stably-transfected osteosarcoma cell lines was tested for using immunofluorescence analysis.

Culture medium containing 10% calf serum was used to prepare a single cell suspension, and ~10,000 cells per well were seeded in 96-well plates. The cells were cultured overnight until the cell density increased to 70%. The medium was replaced by 100 µl serum-free medium, cultured for 24 h and then synchronized. In the experiment, the cells were divided into different treatment groups, forming the control, RAPA (100 nM), DDP (20 µM) and MTX (100 µM) groups. A total of 10 µl MTT (5 mg/ml) was added to each well at different times (12, 24 and 48 h). The cells were incubated for 4 h and then the culture was terminated. With the culture supernatant in the net absorption hole, 150 µl dimethyl sulfoxide was added to each well. Optical density values at a 570-nm wavelength were measured with an automatic microplate reader (Dynatech MR4000; Dynatec Laboratories, Inc., El Paso, TX, USA).

A total of 2 ml (1×10⁶ cells/ml) of cells were seeded in 6-well plates, then treated with 100 nM of RAPA, 20 µM of DDP and 100 µM of MTX and the culture was stopped after 48 h. Following centrifugation for 5 min at 800 × g, the cells were collected through sedimentation, while the supernatant was discarded and washed twice with pre-cooled phosphate-buffered saline (PBS). Cold 75% ethanol was added overnight at a fixed temperature of 4°C. Following centrifugation at 1,500 × g for 5 min, the supernatant was discarded and the cells were washed with 3 ml of PBS once. In total, 400 µl ethidium bromide (50 µg/ml) and 100 µl RNase A (100 µg/ml) (Sigma-Aldrich) were added at 4°C in the dark for 30 min. A total of 20,000 cells were counted according to standard procedures through flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) and the treatment results were analyzed with ModFit (Verity Software House, Chula Vista, CA, USA), the DNA analysis software.

The cells in the logarithmic growth phase were digested and counted. The cell concentration was adjusted to 1×10⁵/ml and seeded in 6-well plates. Each well was provided with 2 ml cell solution and the cells were cultured in a 5% CO₂ incubator at 37°C. For the treatment groups, RAPA (100 nm), DDP (20 µM) or MTX (100 µM) were then added in the logarithmic growth phase. Meanwhile, a solvent control group was set, with two wells at the same concentration. Once the cell culture plate was cultured in the 5% CO₂ incubator for 48 h, the fluid culture was collected in a centrifuge tube where the cells were digested and collected at 4°C. The cells were centrifuged at a rate of 1,000 × g for 5 min. The supernatant was discarded and the cells were washed once with ice cold PBS. The cells were collected in a centrifuge tube at 4°C and centrifuged at 1,000 × g for 5 min. The supernatant was discarded, while the ice cold PBS cells were re-suspended, transferred to the Eppendorf tube at 4°C and centrifuged at 1,000 × g for 5 min. The supernatant was discarded again and 1 ml PBS was added. The re-suspended cells were pre-cooled and fixed with 70% ethanol at 4°C. The cells were centrifuged at 1,000 × g for 5 min and the ethanol solution was discarded. Next, the cells were washed twice with PBS and filtered through a 400-mesh sieve, prior to propidium iodide dye being added at 4°C in darkness for staining for 30 min. The proportion of cells in the G₀/G₁ phase, S phase and G₂ phase was then detected by flow cytometry.

SPSS software, version 19.0 (SPSS, Inc., Chicago, IL, USA) were conducted for data analysis. Continuous data were presented with mean ± standard deviation and examined with test of normality. Comparisons between two groups were conducted using Student's t-test and comparisons among multi groups were performed using homogeneity test of variance and one-way analysis of variance. Pairwise comparisons on mean values were determined using LSD t-test. P<0.05 was considered to indicate a statistically significant difference.

The titer of miR-126-overexpressing and -silencing recombinant lentivirus was 1.07×109 U/ml. Reverse transcription-PCR analysis showed that the miR-126 mRNA expression was increased in the stably-transected osteosarcoma cells overexpressing miR-126, while miR-126 mRNA expression was lowered in the osteosarcoma cells transfected with miR-126-silencing lentiviral vectors (data not shown).

The MTT assay showed that DDP and MTX exhibited inhibitory effects on the proliferation of the two osteosarcoma cell lines, MG63 and U-2 OS (both P<0.05), while RAPA, an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, did not significantly inhibit cell proliferation compared with the control (P>0.05). During miR-126 overexpression, cell proliferation was observed to be weaker, whereas cell proliferation became stronger as the expression of miR-126 was lowered in the DDP-treated cells at the same concentration (Fig. 1A and B).

Flow cytometry showed that the apoptotic rate of the osteosarcoma cells increased with the overexpression of miR-126, while the silencing of miR-126 expression interfered with the apoptotic rate. When DDP or MTX were applied, the apoptotic rate of the osteosarcoma cells was significantly promoted by miR-126 overexpression (Figs. 2 and 3; both P<0.05).

Although DDP or MTX have significant effects on the apoptotic rate in the miR-126-silenced osteosarcoma cells (both P<0.05), RAPA did not significantly promote the apoptosis of the osteosarcoma cells (Fig. 4; P>0.05).

The flow cytometry suggested that there would be a decrease in the proportion of osteosarcoma cells in the S phase as DDP, MTX and RAPA were added to the osteosarcoma cells (Figs. 5–7). DDP had the best inhibitory effect (both P<0.05), which was connected with changes in the miR-126 expression in the two osteosarcoma cell lines; the proportion of osteosarcoma cells in the S phase decreased with the increase of miR-126, while it increased as miR-126 was silenced (Fig. 5; both P<0.01).