2.1 Human tissue specimens and cell culture
Methods described in previous studies[16]. This study was approved by the Ethics Committee of The First Affiliated Hospital of Xi’an Jiaotong University, China.
2.2 Plasmid transfection
The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene. Cells were seeded into 6-well plates until 70%-90% confluency and transiently transfected with pcDNA3-flag-YTHDF2 or empty vector using the X-treme GENE HP DNA Transfection Reagent(Roche, Indianapolis, IN, USA) following the manufacturer’s protocol.
2.3 siRNA and transient transfection
Human YTHDF2 siRNA were purchased from GenePharma(Shanghai, China). YTHDF2 siRNA was transiently transfected 100nM per well using the X-treme GENE siRNA Transfection Reagent(Roche, Indianapolis, IN, USA) following the manufacturer’s protocol. RNA was extracted 48 hours later and protein was extracted 72 hours later for subsequent experiments.
2.4 miR transient transfection
Methods described in previous studies[16].
2.5 Quantitative real-time PCR (qRT-PCR)
Methods described in previous studies[16].
2.6 Western blot
Total proteins were extracted by RIPA lysis buffer(Roche, Indianapolis, IN, USA) and 1 mM PMSF on ice, proteins were separated by SDS-PAGE and then transmembrane. 5% skimmed milk was sealed at room temperature for 2 hours. First antibod was added to the membrane. TBST membrane was washed for 8 minutes and 5 times, and the corresponding second antibody (1:2000) was added, incubated for 2 hours, and TBST membrane was washed for 8 minutes and 5 times.
2.7 Luciferase reporter assay
Methods described in previous studies[16]
2.8 RNA m6A quantitative experiment
In this experiment, the total RNA content of m6A was determined by using the m6A RNA metrology Quantification Kit (ab185912, Abcam) of Abcam company. We measure m6A level following the manufacturer’s protocol. The absorbance of the measuring plate at 450 nm was measured by the enzyme scale instrument, and the RNA m6A content of each sample was calculated according to the standard curve. The formula is m6A% = [(sample OD-NC OD)/S] / [(PC OD-NC OD)/P]×100%, where S is the ng amount of sample RNA and P is the ng amount of positive control RNA.
2.9 Cell viability assay
Methods described in previous studies[18].
2.10 Transwell assay
Methods described in previous studies[16]
2.11 Cell apoptosis assay
Cell apoptosis analysis was performed using an Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (KeyGEN Biotech, Nanjing, China). Normal culture cells were selected in the logarithmic growth period, and the growth state was good for the experiment. After culture for 24 hours, the supernatant was introduced into EP tube, and cells were digested with trypsin without EDTA, then cell suspension was made and transferred to new EP tube. After centrifuging for 10 minutes at 1000rpm and 4 ℃, discard the supernatant; add 1ml of precooled PBS, gently blow to suspend the cells for 1000rpm, centrifuging for 10 minutes at 4℃, discard the supernatant; repeat step 3 and step 4 twice; re suspend the cells in 400ul × binding buffer; add 5ul annexin V-FITC to each sample to be tested, and add PI after mixing 5ul, mix well, react at room temperature for 15min, pay attention to avoid light, and try to get on the machine within 1h. The results were analyzed using the Cell-QuestTM Pro software (BD Biosciences, Bedford, MA, USA)
2.12 Statistical analysis
Data were presented as the means±SE and were analyzed using SPSS 22.0 software(Chicago, IL, USA). Statistical differences were tested by Chi-square test, two-tailed t-test, one-way ANOVA test or Fisher’s Exact test. Differences were considered significant at P<0.05(*) or highly significant at P<0.001 (**).