To examine METTL13 mRNA expression in GC, 48 pairs of gastric cancer samples were collected from diagnosed GC patients during surgical resection in Shanghai East Hospital. All the specimens were frozen in liquid nitrogen immediately after resection and then stored at − 80 °C fridge for subsequent experiments. In order to detect METTL13 expression at protein level in GC, a tissue microarray construction (TMA, Cat# HStmA180su11, containing 90 tumor tissues and 90 corresponding normal tissues) was purchased from Shanghai Outdo Biotech Co.,Itd and analyzed by two pathologists who are unknown about clinical information of patients after immunohistochemical staining. The staining results were classified as previously described (Wu et al. 2018). In brief, 0–100% was scored by positive cell percentage and 0–3 was scored on the basis of staining intensity. According to the product of percentage of positive cells and staining intensity: negative (0–0.5); slight positive (0.5–1.5); moderate positive (1.5–2.5); strong positive (≥ 2.5). Anti-METTL13 antibody (#ab186002, Abcam, Cambridge, MA, USA) was used at a dilution of 1:4000 for immunohistochemistry. The use of clinical samples and this study were approved by the Ethics Committee of Shanghai East Hospital, Tongji University School of Medicine. Written informed consent was obtained from each patient involved in this study.
Cell lines and cell culture
All the gastric cell lines (BGC823, AGS, MGC803, SGC7901, MKN28) were obtained from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which were maintained in DMEM (Dulbecco’s modified Eagle’s, Corning, Inc, Corning, NY, USA) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (M&C Gene Technology Ltd, Beijing, China). The cells were cultured in a humidified incubator with 5% CO2 at 37 °C.
RNA extraction and qRT-PCR assay
We used TRI Reagent to separate total RNAs from GC cells or human tissues (Sigma-Al-drich, MO, USA) referred to manufacturer’s instructions. First-strand cDNA were synthesized with PrimeScript RT reagent Kit, Takara, Kyoto, Japan. Analysis of qRT-PCR was performed on an ABI QuantStudio 6 Flex Real- time PCR system (Applied Biosystems, CA, USA) with TB Green Premix Ex Taq II (Takara, Kyoto, Japan). All primers used in the experiments are summarized in the Supplementary Table 1. 2−ΔΔCt method was utilized to assess relative fold change compared to the control samples. β-actin worked as the endogenous reference.
Transfection and lentivirus transduction
SiRNAs targeting METTL13, HN1L, eEF1A1 or eEF1A2 and control siRNA were synthesized by Gene Pharma (Shanghai, China). The target sequences are listed in the Supplementary Table 1. A full-length METTL13 or HN1L cDNA was cloned into pcDNA 3.1 mammalian expression vector respectively. EEF1A expression plasmids, pEnter-eEF1A1 and pEnter-eEF1A2 were purchased from Vigene Biosciences, Shandong, China. Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, US) was applied to transfect siRNAs or plasmids according to the manufacturer’s instructions. To establish the cells stably overexpressing METTL13, SGC7901 cells were transfected pcDNA3.1-METTL13 plasmid and pcDNA3.1 vector, respectively. Then cells were selected by G418 (900 μg/ml). Single colony was isolated and amplified. Western blot analysis was used to screen METTL13 overexpressed colonies. Lentiviral particles for silencing METTL13 (Lv-shMETTL13 and Lv-shNC based on siMETTL13-1 and si-NC sequence) were packaged by Genepharma (Shanghai, China). Stably infected cells were selected by puromycin (2 μg/ml) after transducing cells with lentiviral particles.
Western blot assay
Total proteins were extracted from cultured GC cells with RIPA buffer, and the supernatants were diluted in 5×SDS loading buffer after calculating protein concentration with a Pierce BCA protein assay kit (Takara, Japan). Western blot assay was carried out in accordance with the previous report (Yu et al. 2019). The signal bands were detected by Odyssey Infrared Imaging System (Li-COR Biosciences) based on manufacturer's instructions. The primary antibodies used in this study are as follows: METTL13 (#ab186002, Abcam), HN1L (#ab200571, Abcam), eEF1A (#2551, Cell Signaling Technology), β-actin (#81178, Santa Cruz Biotechnology).
Cellular proliferation assay
To evaluate GC cell viability, cell counting kit-8 assay was performed (CCK-8 reagent, Dojindo, Japan). In brief, 3000–5000 cells/well were seeded into 96-well plates in triplicates. 10 μl of CCK-8 solution was added into wells at scheduled time intervals (0 h, 24 h, 48 h, 72 h and 96 h). Optical density value at 450 nm was measured on an automated microplate reader (SpectraMax M5, Molecular Devices, USA) following 37 °C incubation for 1 h. For colony formation assay, 1500 GC cells were inoculated in 6-well plates per well. Two weeks later, colonies were fixed with 4% paraformaldehyde for 10 min. Then rinsed and stained with 0.5% crystal violet for 10 min at least. The number of colonies were calculated using Image J software (v1.52, National Institutes of Health Freeware, USA). The experiments were performed in triplicate and repeated at least three times independently.
Transwell migration assay
Modified 8 μm 24 well Boyden chambers (pore size; Costar; Corning, Inc.) were applied to evaluate cell migratory ability. In brief, 5 × 104 cells were seeded into upper chambers supplemented 400 μl of DMEM without FBS in triplicates. The bottom chambers were filled 800 μl DMEM containing 10% FBS. After cultured 24 h, cells on surface of bottom were stained with crystal violet and calculated under microscope. The experiments were repeated as least three times independently.
Wound healing assay
Cells stably overexpressing or knocking down METTL13 and corresponding control cells were collected and then planted into a 6-well plate in triplicates. A yellow plastic tip was used to scratch the cell monolayer (90% confluence). Then rinsed cells twice with 1× PBS and cultivated in serum-free DMEM medium for up to 48 h culture. The wound gap was photographed at 0 h, 24 h and 48 h under the light microscope (Nikon, Japan) with 200× magnification.
About 4–5 weeks old male BALB/c nude mice were provided by Sippr-BK laboratory animal corporation (Shanghai, China). To establish subcutaneous xenograft model, all mice were divided into two groups (n = 5 or n = 6 per group). 2 × 106 stably infected cells (BGC823-LV-shNC, BGC823-LV-shMETTL13) or stable cells lines (SGC7901-Vec, SGC7901-METTL13-15#) were collected and implanted into nude mice by subcutaneous injection at the site of the dorsal flank. Tumors were weighed and photographed 4 weeks later. For tumor metastasis assay in vivo, 1.5 × 106 cells were injected into each mouse by tail vein, respectively (n = 5 per group). After five weeks, mice were sacrificed and tumor nodules on lung surface were calculated. In addition, lung tissues were fixed with 4% paraformaldehyde and hematoxylin and eosin (H&E) staining was performed. All animal handling and experimental procedures used in this study were approved by the Ethics Committee of Shanghai East Hospital.
The public data in this study were obtained from Co-expedia database (http://www.coexpedia.org/) (Yang et al. 2017) and Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/) based on The Cancer Genome Atlas (TCGA) database (Tang et al. 2017). Genes co-expressed with METTL13 in gastric cancer were examined using Co-expedia database. Through GEPIA online tools (http://gepia.cancer-pku.cn/), we analyzed the correlation between HN1L and METTL13 expression in gastric cancer.
Statistical analysis for three independent experiments was analyzed by GraphPad Prism 8. The quantitative data was represented as mean ± SD. Student t test and one-way ANOVA were performed to calculate the difference comparison. The χ2 test was performed to analyze the relationships between METTL13 expression and a series of clinicopathological characteristics. P < 0.05 was considered to be significant.