MG-63 human osteosarcoma cells were brought from Shanghai Biotechnology Cell Experimental Center (Shanghai, China), and were cultured in the Dulbeco's modified Eagle's medium (DMEM) nutrient fluid, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin of 100 µg/ml final concentration (Beyotime Institute of Biotechnology, Jiangsu, China). The cells were placed in an incubator at 37°C, with 5% CO₂ and saturated humidity. Cells were left to grow to confluency of more than 80%, then adherent cells were digested with 0.25% trypsin and subcultured on a new flask. Every 2–3 days cells were subcultured, and maintained under the same conditions until ready for experiments. Cells for transfection experiments were selected when in the logarithmic phase.

We induced overexpression and knockdown expression of KISS-1 mRNA in cells of the osteosarcoma cell line MG-63 in vitro, and used the same type of cells as control by transfecting them with empty vector (GenePharma, Shanghai, China). The cDNA of KISS-1 from the osteosarcoma cells was amplified by RT-PCR, and the pcDNA3.1-vector (GenePharma) was used to make constructs for eukaryotic expression using standard molecular cloning techniques. Tranfections of the MG-63 cells with KISS-1 expression vector were done using Lipofectamine (both from GenePharma), and screened by G418 (1 mg/ml). MG-63 cells with stable high expression of KISS-1 were subcultured. Additionally, a number of small interfering RNAs (siRNAs) were designed to knock down the KISS-1 expression in the MG-63 cells. The most effective siRNA segment giving the best silencing of KISS-1 expression was used in a eukaryotic expression vector pSUPER-KISS-1-siRNA, and transfected using Lipofectamine (both from GenePharma) into the cells (screened by G418 at 1 mg/ml). The MG-63 cells with stable low expression of KISS-1 were subcultured and used in subsequent experiments. The control cell line was made by transfecting the empty pcDNA3.1-vector into MG-63 cells. KISS-1 expression was verified by RT-PCR method in all the resulting cell lines.

After 12, 24, 48 and 72 h of transfections, the cell proliferation was examined for each cuture flask with the method of MTT. Apoptosis was detected by flow cytometry, the mRNA levels of caspase-3, Bcl-2 and Bax (apoptosis markers) as well as the mRNA levels of LC3 and Beclin1 were detected by RT-PCR.

Cells were collected from the culture flasks when confluence reached 85% using trypsin digestion; 2,000 × g of the cell culture medium containing resuspended cells were centrifuged for 15 min, the cell pellet was saved and the supernatant discarded. Phosphate-buffered saline (PBS) was used to resuspend the pellet to a final concentration of 1×10⁶ cells/ml; 100 µl of the resuspended cells were placed on each well of a 96-well plate. The cells were incubated at 37°C with 5% CO₂ and saturated humidity for recovery and until attached. Then, 40 µl of MTT solution was added to each well. The cells were further cultured in the incubator for 4 h. In the following steps, the supernatants were removed, and 150 µl of DMSO were added to each well, shaking for 10 min. Finally, the absorbance (optical density, OD) at 490 nm wavelength was read in a microplate reader. The reference wavelength was 630 nm. The process was repeated three times to average the results.

The cells were collected and washed in PBS. The binding buffer in the apoptosis assays kit (ZSGB-BIO, Beijing, China) was used to make a cell suspension, with the cell density adjusted to 1×10⁶/ml; 100 µl of cell suspension were mixed with 5 µl of Annexin V-FITC, and allowed to react for 5 min at room temperature in the dark. Then, 10 µl of propidium iodide was added, the cells were left to react for 15 min at room temperature in the dark. Finally, 400 µl of 1X binding buffer were added, before FACSCalibur flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) within 1 h to detect the cell apoptosis.

The traditional TRIzol method (KeyGene Biotech, Nanjing, China) was used to extract RNA, and its concentration and purity were detected with a UV spectrophotometer. cDNA was synthesized using a Sensiscript RT kit. Primers were synthesized by Takara Bio, Inc. (Otsu, Japan), the sequences are shown in Table I.

The reaction system consisted of 2.5 µl 5X buffer + 1.5 µl MgCl₂ + 0.5 µl dNTP + 1 µl GAP-43 with sense and antisense primers + 0.3 µl Taq enzyme + 2 µl cDNA template and water to 25 µl. The reaction conditions were 95°C for 5 min, 95°C for 30 sec, 62°C for 30 sec, 72°C for 30 sec, and repeating the cycle 35 times, with a final at 72°C for 10 min extension. The dissociation curve was obtained and the 2^−ΔΔCt method was used to calculate the relative expression of mRNA.

SPSS 20.0 software (IBM, Armonk, NY, USA) was adopted for statistical analyses. All quantitative data were expressed as mean ± standard deviation (mean ± SD). The one-way analysis of variance (ANOVA) method was used for comparison among groups. The LSD-t method was used for comparison between two groups, and the repeated measure ANOVA was used for comparisons within one group. The differences were considered statistically significant at P<0.05.

The cell proliferation rates were evaluated for 72 h. The rates kept increasing for cells in the control and low expression groups, and decreased in the overexpression group. The rates in the low expression group were apparently higher than those in the control group. The differences had statistical significance (P<0.05) (Table II).

During the first 72 h after transfection, the cell apoptosis rates of the control and low expression groups did not change significantly, but that in the overexpression group increased. The rate of the overexpression group was significantly higher than that of the control group, and the low expression group had the lowest rates. The differences amongst groups had statistical significance (P<0.05) (Table III).

With time, the relative expression levels of caspase-3 and Bax mRNAs in the control and low expression groups remained the same, while the level of Bcl-2 increased. By contrast, in the overexpression group, the relative expression levels of caspase-3 and Bax mRNAs increased, and the level of Bcl-2 decreased. The levels of caspase-3 and Bax in the overexpression group were higher than those in the control group, with the low expression group showing the lowest level. The level of Bcl-2 in the overexpression group was lower than that in the control group, and the low expression group had the highest level. The differences had statistical significance (P<0.05) (Table IV).

With time, the relative expression levels of LC3 and Beclin1 mRNAs in the control and low expression group remained virtually unchange, while the level in the overexpression group increased. The expression in the overexpression group was higher than that of the control group, and the low expression group had the lowest levels. The differences had statistical significance (P<0.05) (Table V).