2.1 Human tissue specimens and cell culture
SKOV3 was obtained from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China), 3AO was from the Shandong Academy of Medical Sciences (Jinan, China). Cells were maintained in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum at 37°C under a humidified 5% CO2 atmosphere. Human ovarian cancer tissue samples and normal ovarian tissue samples were collected from patients at The First Affiliated Hospital of Xi’an Jiaotong University, PR China. 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(Boston, MA, USA). 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
miR-145 mimic and negative control were purchased from Ribo-Bio Co. Ltd. (Guangzhou, China). SKOV3 and 3AO cells were transiently transfected with 60 nM miR-145 mimic or negative control using the X-treme GENE siRNA Transfection Reagent (Roche, Indianapolis, IN, USA) following the manufacturer’s protocol.
2.5 Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For mRNA detection, first-strand cDNA was synthesized using a RevertAid first strand cDNA synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Quantitative real-time PCR was performed using a SYBR Premix Ex Taq™ II kit (Takara, Dalian, China) on a CFX96 real-time PCR system (Bio- Rad, Hercules, CA, USA).
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, and then incubated overnight at 4°C with rabbit anti-human YTHDF2(1:1000, Cell Signaling Technology, Danvers, MA, USA). 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
Cells were co-transfected with pRL-TK vector (20 ng), wild-type (WT-3’ UTR) or mutant (MUT-3’ UTR) reporter vectors (180 ng), along with miR-145 mimic or negative control at a final concentration of 20 nM using the X-treme GENE siRNA Transfection Reagent. 24h after transfection, the relative firefly luciferase activity (normalized to Renilla luciferase activity) was measured using a dual-luciferase reporter gene assay system (Promega, Madison, WI, USA), and results were depicted as the percentage change over the respective control.
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 measured 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
Cells in logarithmic growth phase were inoculated into 96-well plates with 5000 holes per hole, 100μL of culture medium was added into each hole and incubated overnight in a 37℃，5% CO2 incubator, then add berberine for 48 hours. 10μL CCK8(7Sea, Shanghai, China) was added to each pore and incubated at 37℃ for 4 hours. The absorbance value of each pore OD 450 was determined by enzyme labeling(PerkinElmer, Waltham, MA, USA).
2.10 Transwell assay
Cells were trypsinized and counted. A total of 1×105 cells in 100 μl serum-free medium were added into millicells (Millipore Co., Bedford, MA, USA). 500 μl of 1640 medium containing 20 % newborn bovine serum was added to the bottom chambers as the chemotactic factor. After incubation for 24 at 37 °C. Migratory cells were counted and averaged from images of five random fields (original magnification ×200) captured using an inverted light microscope. Each cell count was performed by three researchers.
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 400μl × binding buffer; add 5μl annexin V-FITC to each sample to be tested, and add PI after mixing 5μl, 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 (**).