Clinical tissue specimens
Twelve paired fresh GC tissue specimens and adjacent normal tissue specimens from patients were obtained from the First Affiliated Hospital of Soochow University (Suzhou, China) between May 2017 and August 2018. All tissue specimens were snap-frozen in liquid nitrogen. This study was approved by the Institutional Review Board of the First Affiliated Hospital of Soochow University. Written informed consent was obtained from each patient. Basic information of clinical tissue specimens are provided in Supplementary Table 1.
Cell culture
Human GC lines AGS, SGC-7901, and MKN-45, normal gastric mucosa cell GES-1, Jurkat, and THP-1 cells were purchased from the Chinese Academy of Science Cell Bank (Shanghai, China). All cells were cultured in RPMI-1640 (Biological Industries, BeitHaemek, Israel) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 1% Penicillin -streptomycin (Beyotime, Shanghai, China, #C0222) in a humidified incubator with 5% CO2 at 37°C.
Cell transfection
Two small interfering RNAs (siRNA) targeting human H19 (siRNA-1, and siRNA-2), were obtained from RiboBio Life Science Co., Ltd. (Guangzhou, China). Negative control (NC), miR-519d-3p mimic and miR-519d-3p inhibitor were purchased from Genepharma Co., Ltd. (Shanghai, China). pReceiver-Lv105-LDHA (LDHA) plasmid and pReceiver-Lv105-negative control plasmid were purchased from Gene Copoeia (Guangzhou, China). SGC-7901 and MKN-45 cells were transfected with siRNA reagents or plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Lentiviral infection
Lentivirus vectors carrying human H19 shRNA and negative control lentivirus vectors were manufactured by Genepharma Co., Ltd. (Shanghai, China). For lentivirus infection, SGC-7901 and MKN-45 cells were infected with lentiviral particles at a multiplicity of infection (MOI) of 25. The efficiency of infection was confirmed by counting GFP-expressing cells under a fluorescence microscope.
RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA from tissue samples or cells was extracted using TRIzol reagent (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. For gene expression analysis, a total of 1 µg RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara, Shiga, Japan, #RR036A). RT-qPCR reactions were performed on a CFX96 TouchTM real-time PCR system (Bio-Rad, CA, USA) using AceQ universal SYBR qPCR Master Mix (Vazyme, Nanjing, China, #Q511) according to the manufacturer’s instructions. For miRNA expression analysis, 1 µg of total RNA was used for first-strand DNA synthesis using a miRNA 1st Strand cDNA Synthesis Kit (Vazyme, #MR101-02) and RT-qPCR was conducted using a miRNA universal SYBR qPCR Master Mix kit (Vazyme, #MQ101-02). Sample and reference genes were analyzed in triplicates. Individual gene expression was normalized to β-actin, while miRNA expression was normalized to small nuclear RNA U6. The primer sequences for RT-qPCR are provided in Supplementary Table 2.
Western Blot analysis
Cells were lysed with RIPA buffer (Beyotime, #P0013D) supplemented with protease inhibitors and phosphatase inhibitors (Beyotime, #P1045). Protein concentrations were examined with an Enhanced BCA Protein Assay Kit (Beyotime, #P0010). Total protein (30 μg) was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, #P0012AC) and transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life science, Germany). The membranes were blocked with 5% BSA (Fcmacs, Nanjing, China, #FMS-WB021) for 1.5 h and then incubated with the rabbit anti-human/rabbit LDHA (Beyotime, AF1660), mouse anti-human/mouse β-actin (CST, #3700) at 4 °C overnight. On the following day, the membranes were incubated with the corresponding HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Beyotime) for 1 h at room temperature. The protein bands were visualized with ECL reagents (NCM Biotech, Suzhou, China, #10100) in a Chemi DocTM MP Imaging System (Bio-Rad). The band density was analyzed using ImageJ software.
Glucose consumption and lactate production assay
The glucose and lactate levels were measured using a glucose assay kit (robio, Shanghai, China, #361510) and a lactate assay kit (Jiancheng, Nanjing, China, #A019-2-1) following the manufacturer’s protocols.
CCK-8 assay
The cell proliferation was assessed at 0, 24, 48 and 72 hours using Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan, #CK04). Briefly, Cells were plated into 96-well plates at a density of 3´103 cells per well. Then, 10ul CCK8 solution was added to each well and cells were incubated for another 2 h at 37°C. The optical density (OD) at 450nm was measured. Each group contained 5 replicates and all experiments were performed in triplicate.
Colony formation assay
Cells were seeded into 6-well plates at 1´103 cells per well and cultured in a humidified incubator with 5% CO2 at 37°C for 10-14 days. The cell colonies were fixed in methanol for 30 min and then stained with 1% crystal violet (Sigma) for 30 min. Then, the colonies were imaged and counted.
Cytoplasm and nuclear localization
Cytoplasm and nuclear were separated from the cells using MinuteTM Cytoplasmic and Nuclear Extraction Kit (inventbiotech, USA, #SC-003) according to the manufacturer's instructions.
Luciferase reporter assay
PmirGLO vectors containing the wild type (WT) or mutant (MUT) H19 or LDHA 3’UTR were obtained from Realgene Biotech (Nanjing, China). Luciferase reporter assay was performed following the previous protocols [26]. Briefly, MKN-45 cells were cultured in a 12-well plate and co-transfected with WT or MUT luciferase plasmids and miR-519d-3p mimic or control miRNA using Lipofectamine 2000. 24h after transfection, the cells were lysed with Passive Lysis Buffer (Promega, Madison, USA, #E1910), and the luciferase activity was measured with a Dual Luciferase Reporter Assay System (Promega, #E1910) according to the manufacturer's instructions.
Flow cytometry assay
For intracellular staining of IFN-γ, γδT cells were treated with CM or CM+lactate (Sanjia, Suzhou, China, #SL017769) for 24h, and stimulated with Cell Activation Cocktail with Brefeldin A (Biolegend, USA, #423304) for 10h. The cells were stained with PC7-conjugated antibody against CD3 (Biolegend) and FITC-conjugated antibody against γδT (Biolegend) at 4°C for 20 minutes. Then the cells were fixed and permeabilized with a Fixation/Permeabilization Solution Kit (BD Biosciences, #554714) according to the manufacturer’s instructions, and stained with PE-conjugated antibody against IFN-γ (Biolegend) allowing analysis by a flow cytometry analyzer (Beckman Coulter).
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of human IL-2 in the supernatants of Jurkat cells were examined with ELISA Kit (NeoBioscience, Shenzhen, China, #EHC003.96) according to the manufacturer's instructions.
Tumor-associated macrophages (TAMs) induction
THP-1 cells (1.5×105 cells/ml) were treated with 200 ng/ml phorbol myristate acetate (PMA, Solarbio Science & Technology Co., Ltd, Beijing, China.) for 24 h and polarized into macrophages [27]. To induce TAMs, THP-1 macrophages were cultured with CM of GC cells for a further 24h prior to harvesting.
Xenograft tumor
Six-eight-weeks-old female BALB/c athymic nude mice were purchased from the Shanghai Laboratory Animal Center. All animal experiments were approved by the Institutional Animal Care and Use Committee of Soochow University (Suzhou, China). All the mice were randomly divided into two groups (n=4 each group). 5´106 MKN-45 cells with or without H19 knockdown were subcutaneously injected into the layer of the right flank of the mice. Tumor volume (mm3) was calculated according to the following equation: V (mm3) = S2(mm2) × L(mm)/2, where S and L are the smallest and the largest perpendicular tumor diameters, respectively [28]. After 21 days, mice were sacrificed and the xenograft tumor tissues were removed, photographed and weighted.
Conditioned medium
Cells (3×105) were seeded in 6-well plates and cultured overnight. The culture medium was changed to fresh medium in each well. After 24 h, the conditioned medium (CM) was collected.
γδT cell separation and culture
γδT cells were isolated from human peripheral blood mononuclear cells (PBMCs) and cultured as our previously described [29].
Statistical analysis
All data were presented as mean ± SD. Student’s t-test was used for comparisons between two groups. For survival curves, Kaplan-Meier method with log-rank or Gehan-Breslow-Wilcoxon test was used. Significant differences were displayed as follows: *p<0.05, **p<0.01, ***p<0.001 and ns. not significant.