RNA-seq
RNA-seq was performed with SETDB1 stable knockdown cell line SW620 by Biomarker Technology Corporation (Beijing, China) as previous described with log2|fold change| ≥ 1.5 and P-value < 0.05 [9].
Survival Analysis
The Human Protein Atlas (HPA, http://www.proteinatlas.org) is a database that maps tissues, and organs using information on proteins, transcriptomes, and system biology. The HPA pathology database's patient records for CRC patients were used for the survival analysis.
Cell Culture And Transfection
Human CRC cell lines used in this study were SW620, HCT116 and SW480. The origin and culture conditions were described before [9]. Using Lipofectamine 2000, the cells were transfected with ADORA2A siRNA or ADORA2A overexpressed plastic after the cell density reached around 50%. As a negative control, cells were transfected with nonsense siRNA or plastic (NC). ADORA2A siRNA was designed by Ruibo (Guangzhou, China) and overexpressed plastic was obtained from OBiO Technology (Shanghai, China).
Real‑time Quantitative Pcr
Total RNA was extracted from cells after 24 h of transfection using RNA-simple total RNA kit (Tian gen, China), and then reverse transcribed to cDNA by Prime Script RT reagent Kit with gDNA Eraser (TaKaRa, Japan). TB Green Premix Ex Taq™ (TaKaRa, Japan) was used to express ADORA2A mRNA. After normalization to β-actin, the relative quantification of ADORA2A was calculated using the2−∆∆Ct technique. Primers used were as follows: ADORA2A sense: 5’-TCGCCATTGACCGCTACATTGC-3’, antisense:5’-AAACGACAGCACCCAGCAGATG-3’,β-actin sense:5’CATGTACGTTGCTATCCAGGC-3’, antisense:5’CTCCTTAATGTCACGCACGAT-3’.
Western Blot And Antibody
For cells that had been transfected for 48 hours, ice-cold RIPA buffer (Beyotime, China) containing protease inhibitors and phosphatase inhibitors was used to lyse the cells. (Beyotime, China). The primary antibodies used in this study were as follow: anti-ADORA2A (1:2000, Abcam); anti-Bcl-2 (1:1000, Bimake); anti-Bax (1:1000, Bimake); anti-caspase3 (1:1000, Proteintech); anti-caspase9 (1:1000, Proteintech); anti-p-PI3K (1:1000, Proteintech); anti-PI3K (1:1000, Bimake); anti-p-AKT (1:2000, Cell Signaling Tech- nology); anti-AKT (1:1000, Immuno Way).
Cck8 Assay
CCK8 assay was performed with CCK-8 kit (Beyotime, China). 2000 ~ 2500 cells were transfected for 24 hours were placed into a 96-well plate. 100µl spent medium was replaced with an equivalent volume of new medium containing 10µl of CCK8 after the cells had been cultivated. The cells were then incubated for 1 h at 37°C. Using a microplate spectrophotometer, the OD value at 450 nm was measured to calculate the rate of cell growth.
Colony Formation Assay
In order to see cell clones, 800 cells were put into a 6 well culture plate and grown for two weeks at 37°C in 5% CO2. The clones were stained for 10 minutes with 1% crystal violet staining solution after being fixed with 4% methanol. An optical microscope was used to assess the number of generated clones.
Wound Healing Assay
Approximately 5×105 cells were transfected and put onto a 6 well culture plate after 48 hours. When the cell density is around 90%, cell scratches are conducted using a 10µl pipet tip, and the cell debris is entirely removed using RPMI 1640 media. Wells were filled with complete media containing 10% fetal bovine serum and cells were incubated at 37℃ in 5% CO2. At 0 hours, 24 hours, and 48 hours, respectively, the microscope camera captured the reduced distance of cell scratches.
Transwell Invasion Assay
Cells (1×106) that transfected for 24 h, were seeded in the upper chamber of transwell chamber (Corning, USA). 45ul of mixed Matrigel (Serum-free medium: Matrigel = 9:1) were added to the transwell chamber (Corning, USA). Transfection after 48 hours, the upper chambers of the wells contained 200uL of cell suspension, whereas the bottom chambers received 600ul of RPMI1640 medium with 30% fetal bovine serum. Before being preserved in methanol for 30 minutes and stained for 5 minutes with a 1% crystal violet staining solution, the chambers were cultured for 48 hours. Using a microscope camera, cells that migrated to the bottom surface of the membrane were counted and recorded.
Apoptosis Detection
Knockdown cell apoptosis was analyzed using Annexin V-FITC Apoptosis Detection kit (Dojindo, Japan) and overexpress cell apoptosis was analyzed using Annexin V-Alexa Fluor 647/PI Apoptosis Detection kit (Yeasen Biotechnology, China). After transfected for 24h, cells were harvested and operated according to the instructions. Statistical analysis was performed using Flowjo software (Tree Star, Ashland).
Statistical analysis
The experimental data were analyzed using GraphPad Prism 9.0 software, and statistical analyses were carried out using SPSS 26.0. The mean and standard deviation were used to express the measured data (SD). The two groups were compared using the Student's t-test, and three or more groups were compared using a one-way ANOVA. Statistical significance was determined to be P<0.05, and the significance shown in * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.