2.1 Data availability
The mRNA expression data and methylation data were obtained from the Gene Expression Omnibus (GEO) database with the login numbers GSE33335 and GSE30601.
2.2 Tissue sample collection and follow-up
The tissues used for immunohistochemistry (IHC) were composed of 83 pairs of tumor and adjacent non-tumor tissues that were collected from patients with GC who underwent curative gastrectomy at Tianjin Medical University Cancer Hospital (Tianjin, China) between January 2004 and August 2007. All patients underwent standard follow-up after curative surgery. The median follow-up time was 26 months (range: 3–70 months). The tissue used for the MassARRAY methylation test consisted of 97 cancerous tissues and 6 paracancer tissues from patients undergoing radical gastrectomy between January 2003 and July 2007 at Tianjin Medical University Cancer Hospital (Tianjin, China), of which 97 cancerous tissues were closely observed. The median follow-up time was 18 months (range: 2–85 months). The clinical data included the patient’s age, sex, endoscopy result, chest X-ray, B-mode ultrasound, and surgical methods. Between August and November 2019, we collected 30 pairs of GC and adjacent non-tumor tissues for RNA extraction. The study was conducted with patient consent. The experimental study protocol and the use of clinical data were approved by the Ethics Committee of the Cancer Institute of Tianjin Medical University and the Cancer Hospital of Tianjin Medical University (Tianjin).
2.3 Cell culture and transfection
The cell lines of MKN45, NCI-N87, BGC-823, MGC-803, SGC-7901, SNU-1, KATO-III, HGC-27, and GES-1 were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone) and 1% antibiotics (penicillin/streptomycin) (Gibco). AGS cells were cultured in F12k (Gibco) with 10% FBS and 1% antibiotics. The HEK293T cell line was cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco) with 10% FBS and 1% antibiotics. Cells were maintained at 37℃ with a 5% CO2 atmosphere. The expression level of ZSCAN18 was upregulated by a plasmid (pLVX-IRES-Puro-ZSCAN18), and an empty vector was transfected into the cells (pLVX-IRES-Puro-vector). Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to verify the transfection efficiency.
We prepared 83 paraffin-embedded primary gastric tumor tissues and adjacent non-tumor tissues. The ZSCAN18 antibody concentration was 1:150. The staining intensity of ZSCAN18 was estimated by three independent pathologists without knowledge of the clinical data. The staining intensity was divided into 0 (negative), 1 (weak), 2 (medium), and 3 (strong) ranks. Zero and 1 were considered negative; 2 and 3, positive.
2.5 Western blot
The western blot experiment was conducted in accordance with standard procedures. The primary antibodies used for this research were the ZSCAN18 antibody (TA505326, OriGene), VINCULIN (ab219649, Abcam), AURORA (ab52973, Abcam), GEMINI (ab195047, Abcam), and LAMIN-B1 (660951-1-AP, Proteintech).
2.6 Cell viability assay
Cell viability was detected using a Cell Counting Kit 8 (CCK-8) reagent. After the cell count, cells were placed in a 96-well culture plate; the AGS cell line had 1 × 103 cells per well, and the NCI-N87 cell line had 3 × 103 cells per well. Testing started 2 h after the addition of 10 µl of the CCK-8 reagent. AGS was tested once a day, and NCI-N87 was tested once every other day.
2.7 Colony formation assay
The colony-forming method was used to detect the proliferation of GC cells. We conducted experiments on AGS and NCI-N87 cell lines using 1000 AGS cells per well and 8000 NCI-N87 cells per well. After the cell count, the cells were inoculated into 6-well plates and incubated at 37℃ for 12 to 14 days. Cell fluid was changed periodically until a visible clone formed.
2.8 Animal experiment
Ten 4-week-old female BALB/C nude mice (Vital River) were purchased to construct a subcutaneous tumor xenograft. A total of 3 × 106 overexpressed ZSCAN18 and control NCI-N87 cells were injected into the lateral dorsal subcutaneous flank of nude mice. Xenograft tumor formation was measured every 4 days. Three weeks after injection, the nude mice were euthanized by cervical-dislocation sacrifice after intraperitoneal injection of 2% pentobarbital sodium (0.5 mL), and the tumor volume (V) was measured by the formula: V = 1/2 × length × (width)2. Tumor tissues were excised for immunohistochemistry.
2.9 5-Aza-2′-deoxycytidine treatment
AGS and NCI-N87 cells were laid 12 h in advance, and the cell density was approximately 30% on the next day. Cells were incubated with a concentration of 2 µM of 5-aza-2′-deoxycytidine (DAC, Sigma, St. Louis, MO). The liquid was changed every 24 h. The treatment lasted for 96 h.
2.10 RNA sequencing and analysis
NCI-N87 cells were stably transfected with PLVX-IRES-Puro-ZSCAN18 or empty vector (PLVX-IRES-Puro). RNA from total samples was isolated and purified using TRIzol (Invitrogen, CA, USA). Then, NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) was used to control the quantity and purity of total RNA. The captured mRNA was fragmented at 94℃ for 5 to 7 min using a magnesium ion fragmentation kit (NEBNext® RNA Fragmentation Module, article no. E6150S, USA). cDNA was synthesized from the segmented RNA using Invitrogen SuperScript™ II Reverse Transcriptase (article no. 1896649, CA, USA). Illumina Novaseq™ 6000 (LC Bio Technology Co, Ltd, Hangzhou, China) was used for double-terminal sequencing according to the standard operation, and the sequencing mode was PE150. Cutadapt (https://cutadapt.readthedocs.io/en/stable, version for cutadapt 1.9) was used to remove the joint plane raw data processing software. A significant difference was analyzed between samples, and the multiple of the difference (fold change [FC] > 2 or [FC] < 0.5 with a p value < 0.05) defined differentially expressed genes (DEGs), which were analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment using R packs.
2.11 MassARRAY analysis of the methylation level of ZSCAN18
Instructions to extract cells or tissues using the DNA extraction kit (BioTeKe Corporation) were observed. The DNA samples to be tested were processed using a commercial NaHSO3 kit (ZYMO). The gene fragments to be detected were enriched and amplified by PCR reaction, and the product length was 200 to 700 bp. The PCR product was treated with shrimp alkaline phosphatase to remove free deoxyribonucleoside-5′-triphosphates (dNTPs) in the system. A transcriptase digestion reaction was followed by resin purification. The purified products of the resin were transported to a 384-well SpectroCHIP® bioarray (Agena, Inc) using an Agena NanodispenserRS1000 dot sampling instrument (Agena, Inc). EpiTYPER™ software provided advanced and convenient quantitative analysis of DNA methylation.
2.12 Statistical analysis
IBM SPSS Statistics (version 24.0; Armonk, NY, USA) and GraphPad Prism 8 software (GraphPad Software Inc, La Jolla, CA, USA) were used to analyze the data for this study. The t-test was used to compare statistical differences between the two groups. The chi-squared test and Fisher’s exact test were used to analyze the relationship between methylation and various clinicopathological parameters. Overall survival was determined by log-rank test and the Kaplan-Meier method. The Spearman correlation test was used to analyze the correlation between gene expression and the methylation level. A receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of methylation sites in cancerous and adjacent tissues. In univariate analysis, survival differences were estimated using the Kaplan-Meier method (log-rank test), and independent prognostic factors were subsequently identified in Cox proportional risk regression models for multivariate analysis. p < 0.05 was considered statistically significant.