Clinical specimens
All clinical specimens of kidney cancer and adjacent tissues were sourced from Longhua District People’s Hospital of Shenzhen. They were obtained by surgical resection, stored, and transported in liquid nitrogen and stored in a freezer at -80 ℃.
Reagents
RPMI-1640 medium was purchased from Gibco Thermo Fisher Scientific (China). Fetal bovine serum (FBS) was purchased from Biological Industries (China). The primary antibody to PCK2 (#6924) was purchased from Cell Signaling Technology. The secondary antibody (catalog no. ab9485) was purchased from abcam. Anti-GADPH (#AFF389-50) was purchased from Affinity Biosciences.
Cell culture and transfection
Human RCC cell lines (OSRC-2, 786-O and A498) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai). They were cultured in RPMI-1640 medium supplemented with 10% FBS and incubated at 37℃ and 5% CO2. The lentivirus vector was purchased from Shanghai Gene Pharmaceutical Co., Ltd. (Shanghai, China). The lentivirus was used to construct stable transfected cell lines, and then, cells were harvested for PCR and western blotting experiments.
Reverse transcription quantitative (RT-q) PCR
Total RNA was extracted using a kit (Fastgene, Inc; Catalog no.220010). The extracted RNA was reverse transcribed into cDNA using the Prime kit with DNA Eraser (Takara Bio Inc.). The sequences of primers used were: PCK2 (forward): 5′-GAGCAACGGCTTCTGTCATTT-3′; PCK2 (reverse): 5′-TGCTTGACTCGTACCATGTCC-3′; GAPDH (forward): 5′-AAGAAGGTGGTGAAGCAGG-3′; GAPDH (reverse): 5′-GTCAAAGGTGGAGGAGTGG-3′.
Western blotting
Protein samples were lysed at 4 ℃ for 10 min with RIPA lysis buffer. Protein concentration was measured by the Coomassie brilliant blue method. The samples were mixed with 4× sample buffer and boiled for 30 min. The protein sample was loaded on a 10% SDS gel and electrophoresed for 2 h. Then, the separated protein was transferred to nitrocellulose membrane and blocked with 5% skim milk for 2 h at 24 ℃. Next, the film was cut according to the molecular mass of the target protein. Subsequently, the membrane was incubated with the primary antibody at 4℃ overnight. The next day, the secondary antibody (1:10,000) was incubated at 24 ℃ for 2 h. A scanning instrument (Amersham ImageQuant 800) was used to obtain protein bands. ImageJ was used for grayscale analysis.
Cell growth assay
To measure cell activity, the cells were counted and spread in the wells of a 96-well plate (5,000 cells per well). At 0, 24, 48, 72, 96 and 128 h, a cell counting kit-8 (CCK8) assay kit (Dojindo, China) was used to measure cell activity.
Cell EdU assay
Cell proliferation was detected using an EdU cell proliferation detection kit (Ribobio, Guangzhou, China). Cells were incubated with 50 μM EdU for 2 h before fixation, permeabilization, and EdU staining. The nuclei were stained with Hoechst at a concentration of 1 µg/mL for 30 min. The proportion of cells doped with EdU was observed with a fluorescence microscope.
Transwell migration and invasion experiment
The cells were digested with trypsin and resuspended in 500 µL RPMI-1640 medium, and the cell density was measured using a cell counter. To test invasion, the transwell membrane was coated with matrigel glue. A total of 2 × 104 cells were placed in the upper cavity of the transwell chamber (micropore diameter: 8 µm), and 750 µL RPMI-1640 medium supplemented with 10% FBS was added to the lower cavity, and then the cells were incubated. After 24 h, cells in the upper cavity that have not migrated or invaded were wiped off with cotton swabs, and the chamber was dried at room temperature. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with crystal violet for 10 min. After the filter membrane had dried, we observed the number of fine migrating and invading cells under a microscope.
Scratch experiment
The scratch experiment was performed with a culture insert tool. After the cells were digested, they were inoculated into the insert in the middle of the 24-well plate. After the cells filled the insert area, the insert could be removed with tweezers to produce a scratch with a width of 500 μm. Photos were taken over 24 h, picture data were collected, and the experimental results were analyzed.
High Performance Liquid Chromatography (HPLC)-tandem MS (MS/MS) analysis
HPLC analysis was performed using a UHPLC device (1290 Infinity LC, Agilent Technologies) coupled to a QTRAP (AB Sciex 5500). The mobile phase contained A=25 mM CH3COONH4+0.08% formic acid (FA) in water and B= 0.1% FA in acetonitrile. The samples were placed in an automatic sampler at 4 ℃, and the column temperature was kept constant at 40 ℃. The flow rate was set at 250 μL/min, and 2 µL of each sample was injected. The gradient was 90% B linearly reduced to 70% between minutes 0 to 12, then reduced to 50% for 6 min, reduced to 40% for 6 min, and then kept at 40% for 5 min. Then the B was increased to 90% in 30-30.1 min and kept for 30.1-37 min. Quality control samples were used to test and evaluate the stability and repeatability of this system, to set the standard mixture of amino acids metabolites, and to correct for the chromatographic retention time.
For MS/MS analysis, positive ion mode (ESI+) was used. The conditions were set as follows: source temperature, 500 ℃; ion Source Gas 1 (Gas1): 40; Ion Source Gas 2 (Gas2): 4; Curtain gas (CUR): 30; ionSapary Voltage Floating (ISVF), 5500 V; and MRM mode detection ion pair was adopted.
Statistical analysis
All data were analyzed using SPSS 23.0 (IBM Corp). The two groups were compared by a t-test. One-way ANOVA and Bonferroni’s test were used to evaluate the differences among multiple groups, and P < 0.05 was considered statistically significant.