1Reagents and specimens
DMEM medium, 2.5 g/L trypsin, fetal bovine serum, penicillin and streptomycin double antibody solution, and tetramethylazozolium salt (MTT) were purchased from sigma company. CA No.: C11995, 03-050-1A 10270-106, 03-034-1A, A056. The BCA kit was purchased from biyuntian company；LipofectamineTM 3000 was purchased from thermo, USA, and GLS siRNA was purchased from Guangzhou Ruibo biological company, vinculin, GLS1, β- Actin, CDK4, celeaved-Caspase3, FOXO1 and EGR1 antibodies were purchased from Abcam company. CB-839 was purchased from Selleck company in Shanghai.Source of specimen: the specimens of 12 cases of osteosarcoma tissue and para cancerous tissue used in the study were from amputated patients in the Department of bone oncology, the First Affiliated Hospital of Sun Yat-Sen University (approval No.:  060), and all patients were diagnosed by postoperative pathology. All patients signed informed consent.
2 Main Methods
2.1Tissue protein acquisition
After amputation, osteosarcoma and its distal normal muscle tissue far away from the reaction area were obtained. The tissue was grinded in a precooled grinder for 30 s, and the tissue was fully broken to obtain its protein, which was stored in a refrigerator at - 80 ℃.
2.2Cell culture and siRNA transfection of GLS1
Osteosarcoma cell lines U2OS, U2OS / MTX300, MNNG / HOS, HOS, 143B, SJSA-1 were purchased from the American ATCC cell bank. They were cultured in DMEM medium containing 10 g/L fetal bovine serum, 1% penicillin and streptomycin at 37 ℃ in a 5% CO2 incubator for 2-3 days. According to the instructions of LipofectamineTM 3000, the siRNA bacterial solution of GLS1 was transfected into 50% of normal cells, namely, siGLS1-nc, siGLS1-1, and siGLS1-2. After 8 hours of transfection, the normal medium was changed. After 48 hours of culture, the cells were lysed for the detection of GLS1 protein expression and subsequent function test.
Table 1 Synthetic sequence of siRNA
2.3 Western blot
After 48 hours of transfection, the cells were collected and placed in a precooled EP tube. 0.3 ml RIPA lysis buffer was added into the EP tube, and then placed in a 4 ℃ shaker for 30 min, followed by 15000×g. After centrifugation for 60 min, the supernatant was the total protein. According to the kit instructions of Biyuntian, the cytoplasmic proteins were extracted. SDS electrophoresis was carried out and then transferred to PVDF membrane. 5% skimmed milk powder was sealed at room temperature for 1 h, and 1:1000 primary antibody was incubated overnight at 4 ℃. The 1:5000 second antibody was incubated at room temperature for 1 h, and then the chemiluminescent agent was added for detection. The X-ray film was exposed in the dark room, and the conventional development and fixation were carried out. Image J software was used to analyze the relative expression level of target protein. The experiment was repeated three times and the average value was calculated.
2.4 MMT assay
The cells in logarithmic phase were digested and counted, and then the 96 well plate was laid, 3000 cells per well, 100ul system. For four consecutive days, 50ul MTT (1:4 diluted MTT mixture) was added at a fixed time every day. The cells were cultured in 37 ℃ and 5% CO2 incubator for 4 hours. The upper liquid was absorbed, 200ul DMSO was added, and the absorbance (D) was detected at 490nm wavelength. The experiment was repeated three times, Calculate the average.
2.5Cell cloning formation assay
After the cells were digested and counted in logarithmic phase, the plates were laid in 6-well plates with 900 cells per well and 2ml DMEM. The medium was changed once every 4 days. After 2 weeks, the medium was removed. The plates were fixed with 4% paraformaldehyde for 30min and stained with 0.05% crystal violet for 30min. Then the whole plates were photographed and counted. The experiment was repeated three times and the average value was calculated.
2.6 Transwell assay
After the logarithmic phase cells were digested and counted, 8 × 104 cells were added into each upper chamber, and the culture system was 100 μL DMEM medium containing 1% FBS, adding 600 μml DMEM medium containing 10% FBS in the lower chamber was placed in 37 ℃ and 5% CO2 incubator for 24 hours. A proper number of cells were observed to penetrate into the lower layer to complete the experiment. The chamber was taken out, fixed with methanol for 30 minutes, stained with crystal violet for 30 minutes, and then photographed. Five random fields were recorded under 20 times magnification for data analysis. The experiment was repeated three times and the average value was calculated.
2.7 Analysis of gene expression by quantitative real-time PCR
（1）RNA extraction：When the cell density was about 60%, CB-839 (8μM) After 48 hours, discard the medium, add 2 ml PBS, wash for 3 times, and add 700 μml PBS buffer solution, standing at 37 ℃ for 5 min, blowing different parts of the lysate in the dish to make it fully fused. Use the precooled centrifuge at the speed of 15000 R / min for centrifugation for 5 min, collect the liquid at the bottom, and add 700 μ L 70% ethanol, centrifuge at 10000 R / min for 1 min, then discard the liquid in 2 ml collecting tube and add 500 μml RNA wave buffer I, centrifuged at 10000 R / min for 1 min. Continue to replace the new 2 ml collecting tube and add 500 μml. After centrifugation at 10000 R / min for 1 min, a new 2 ml collecting tube was replaced and 500 μml RNA was added. After centrifugation at 10000 R / min for 1 min, a new 2 ml collecting tube was finally replaced. Centrifugation at 15000 R / min for 2 min, the remaining liquid in the red column was dried, and the Hi bind matrix was dried. The red column was moved into a 1.5 ml collecting tube again, and 40 μml was added. The RNA solution was collected by centrifugation at the speed of 15000 R / min for 2 min. the RNA concentration in the cells was detected by microplate reader. The RNA solution was stored in an ultra-low temperature refrigerator at - 80 ℃ for freezing to prevent degradation.
（2）mRNA Reverse transcription：The genomic DNA removal reaction solution system was prepared with pre cooled ice box, and the experiment was carried out according to the operation process of TaKaRa reverse transcription reagent.
（3）mRNA Real Time PCR: The experiment was carried out according to the operation procedure of TaKaRa q-PCR reagent, with 0.8 for each group μL's forward primer, 0.8 μL's reverse primer, 0.4 μL ROX reference dye II, 2 μL of cDNA template, 10 μL TB Green Premix Ex Taq II and 6 μL DEPC enzyme free water to make 20 μL. The total system was added to the q-PCR plate, covered with a film, and centrifuged at 3000 R / min for 3 min in a precooled high-speed centrifuge. In the first stage, the reaction parameters were set at 95 ℃ for 30 s, the second stage was set at 95 ℃ for denaturation for 5 s, and the next stage was annealed at 60 ℃ for 34 s for 40 cycles. In the third stage, the reaction parameters were set at 95 ℃ for 15 s, 60 ℃ for 1 min, 95 ℃ for 15 s, about 1 h to complete the whole quantitative amplification experiment, According to the experimental data, CT value is selected for further statistics and drawing.
3 Statistical analysis
GraphPad PRISM 8.0 was used for mapping and SPSS 21.0 software（(IBM Corporation, Armonk, NY)） was used for statistical analysis. The experimental data were calculated by means ± Standard deviation (mean ± SD). T-test was used for comparison between the two groups, and one-way ANOVA was used for comparison between multiple groups. P < 0.05 was considered as statistically significant.