Patient tissues specimens collection
A total of 50 GC tissue samples in the cohort, which were formalin-fixed and paraffin-embedded, were recruited between January 2016 and December 2018 from patients who had underwent operation in Fujian Provincial Hospital. All samples were staged according to the criteria of the 7th Edition of the AJCC Cancer Staging Manual (Stomach, 2010). This study was approved by the Fujian Provincial Hospital Ethics Review Committee. Informed consent was written by each patient before the study.
GC cell lines (AGS, SNU-216, MKN45, AGS) and the immortalized human gastric epithelial cell line (GES-1) were purchased from the Cell Bank of the Chinese Academy of Sciences. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and antibiotics at 37 °C under 5% CO2.
Transfection vector construction
For the stable silencing of DLGAP1-AS2, lentivirus vector pLKD-U6-shRNA-EF1a-LUC-F2A-Puro-DLGAP1-AS2 (Obio Technology, shanghai, China) or control vectors were packaged and transfected into AGS cells. Cell were grown in 1 μg/ml puromycin (Invitrogen) to select the stable transfected cells. Moreover, for the overexpression of DLGAP1-AS2, the cDNA of DLGAP1-AS2 was PCR-amplified ad then sub-cloned into pcDNA 3.1 vector (Invitrogen, Carlsbad, Calif, USA). The efficiency of silencing or overexpression was subsequently quantified by qRT-PCR. The siRNAs and their controls (si-NC) were provided by GenePharma (Shanghai, China) and transfected using Lipofectamine 2000 (Invitrogen) when 70 % confluent growth.
Quantitative real-time PCR (qRT-PCR)
Total RNA from GC tissues or cell lines was extracted by TRIzol Reagent (Invitrogen, CA, USA). For the concentration and purity of RNA, RNA was detected with ultraviolet spectrophotometer using 260 nm and 280 nm. The real‐time PCR was performed using SYBR Premix Ex Taq (Takara) on the 7900 Real‐time PCR System. Reverse transcription was performed using PrimerScript RT Reagent Kit (TaKaRa, Kyoto, Japan). Fast SYBR green master mix (Life Technologies) applied to perform qRT-PCR. The relative expression was calculated using the 2−△△CT method. The β-actin acted as the internal mRNA control. The primer sequences were displayed in Table S1.
Western blot assay
The procedure details were conducted according to previous study. Proteins from transfected GC cells were extracted using RIPA buffer (Beyotime, China) and authenticated using BCA Kit (Pierce, Rockford, IL, USA). Protein was resolved using the SDS-PAGE Electrophoresis System and then transferred to PVDF membranes (Millipore, USA). Specific primary antibodies (anti-METTL3, 1:1000, ab195352, anti-c-MYC, 1:1000, ab32072) were incubated at 4 °C overnight. β-actin was the internal reference.
Methylated RNA immunoprecipitation (MeRIP) Sequencing and qPCR
All the specific manipulations were performed according to the protocol of Magna MeRIP™ m6A Kit (Merck Millipore). In brief, total RNAs were isolated from AGS cells and chemically fragmented into 100-300 nt. Fragmented fragments were incubated with m6A antibody for immunoprecipitation. The MeRIP was performed according to the manufacturer’s instructions. Samples were sequenced with the HiSeq PE150 platform. The enrichment of m6A containing mRNA was sent for quantitative RT-PCR. The RT-qPCR primers were listed in Table S1.
Quantitative analysis of glucose, lactate and ATP
The glucose uptake was quantificationally detected using Glucose Uptake Assay Kit (Fluorometric, Abcam, ab136956). The lactate production was quantificationally detected using Lactate Colorimetric/Fluorometric Assay Kit (BioVision, K607–100). The ATP generation was quantificationally detected using ATP Assay Kit (Beyotime, S0026).
Measurement of extracellular acidification rate (ECAR)
ECAR was analyzed using Seahorse XFe96 analyzer (Seahorse Bioscience, Agilent) as previously described. Briefly, knockdown and overexpression of DLGAP1-AS2 transfected cells (1×104 cells/well) seeded into 96-well XF cell culture microplate. Medium (pH 7.4) was sequentially added with 10 mM glucose, 1 mM glutamine, 50 mM 2-DG and 1 µM oligomycin. ECAR was shown in mpH/min and measured using an XF96 analyzer.
Actinomycin D assay
AGS cells were seeded in 6-well plates (2×105 cells/well). 24 hours later, cells were exposed to Actinomycin D (Act D, 2 μg/ml, Sigma) and harvested at indicated time point. The RNA remaining level was analyzed using qRT-PCR and normalized to mock group (0 hour).
RNA immunoprecipitation (RIP) assay
RIP assay was performed using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to manufacturer’s protocol. Specific antibodies (anti-YTHDF1, Abcam) was conjugated with magnetic beads and then dissolved in RIP buffer. Approximate 90% confluence, cells were lysed using complete RIP lysis buffer (100 μl) containing protease inhibitor and RNase Inhibitor (Millipore). Mouse anti-IgG antibody (Cell Signaling Technology, USA) acted as negative control. Then, total RNA was retrieved and the relative expression was detected by qRT-PCR analysis.
Probes were obtained from Genepharma (Shanghai, China). Fluorescence in situ hybridization (FISH) was performed using fluorescent in situ hybridization kit according to the manufacturer’s protocols (Genepharma). Cy3-labeled probe for DLGAP1-AS2 and FAM-labeled probe for c-Myc, YTHDF1 were used for localization of DLGAP1-AS2, c-Myc and YTHDF1 in GC cells. Nuclei was stained with 4,6-diamidino-2-phenylindole (DAPI) and images were obtained using confocal microscope (Olympus).
Animal models in vivo
Approximately 2×106 GC cells (AGS), transfected with sh-DLGAP1-AS2 or controls, were suspended in 100 μl PBS and then injected into flank of male BALB/c nude mice (5-week old). In the following observation, tumor size was recorded by vernier caliper. At the indicated time, mice were anesthetized and sacrificed for tumors neoplasm acquisition. All animal assays were performed in the light of institutional Fujian Provincial Hospital Animal Ethics Committee guidelines.
The data was expressed as mean ± S.D (standard deviation). All results were conducted using SPSS Statistics software (Armonk, NY, USA) or GraphPad prism 7.0 (La Jolla, CA, USA). Difference within groups was calculated using One-way ANOVA test, non-parametric Mann-Whitney test and student’s t-test. Overall survival rate was calculated using Kaplan-Meier analysis and log-rank test. *P ≤ 0.05 or **P ≤ 0.01 was considered statistically significant.