2.1.Clinical samples. Human primary GC and corresponding normal gastric tissue were collected from the Department of Gastroenterology at the Xingtai People’s Hospital. All patients were GC patients from March 2015 to August 2020 and underwent partial gastrectomy. The research protocol was approved by the Ethics Committee of the Xingtai People’s Hospital, and written consent was obtained from each patient.
2.2. Cell lines and transfection. The normal gastric mucosa epithelial cell line (GES-1) and GC cell lines (AGS, HGC-27, BGC-823, and MGC-803) were purchased from Procell (Wuhan, China). The above-mentioned cells were cultured in RPMI-1640 (Gibco, NY, USA) supplemented with 10% fetal bovine serum (Clark Bio, USA) and 1% penicillin/streptomycin (Gibco, NY, USA). The cells were cultured in a humidified atmosphere of 95% air and 5% CO2. Cell transfection was performed as described in the previous  by studyusing Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The miR-150-5p, miR-205-5p, and miR-30b-3p mimics, mimic-NC, miR-30b-3p inhibitor, inhibitor-NC, si-circCOL12A1, and si-NC were purchased from GenePharma Co., Ltd (Shanghai, China). si-circCOL12A1-F: CAUUUAAAUAUAGCUGUAGTT; si-circCOL12A1-R: CUACAGCUAUAUUUAAAUGTT; the circCOL12A1 overexpression vector, and luciferase reporter vector were constructed by Biocaring Biotechnology Co., Ltd (Shijiazhuang, China) and confirmed by Sanger sequencing.
2.3.RNA isolation and qRT-PCR. The RNAeasy Mini Elute Kit (QIAGEN) was used for total RNA isolation, and the NanoDrop 2000 system was used to measure RNA concentration and quality. Complementary DNA was synthesized using the M-MLV First Strand Kit (Life Technologies) with random hexamer primers and diluted cDNA 5- to 10-fold according to the concentration. MicroRNA reverse transcription was conducted using the miScripIIRT kit (QIAGEN GmbH, D-40724 Hilden, GERMANY) following the manufacturer’s protocol. Platinum SYBR Green qPCR Super Mix UDG kit (Invitrogen) was used for quantitative real-time PCR (qRT-PCR) analysis along with the ABI 7500 FAST System (Life Technologies). GAPDH was used as an internal reference gene for standardization. The 2−ΔΔCt formula was used to calculate relative gene expression as described previously . The primers used were as follows: COL12A1-F: CAAGCTCATTGTAGTCGACATCAGCC; COL12A1-R: GTCTTGACTTGGGAGAGCGGC; circCOL12A1-F: CCTGTAGGAGGTGGCTGC; circCOL12A1-R: GGTCTTTCCCCAGCTACTCAGC; miR-29a-5p: GCGACTGATTTCTTTTGGTGTTC; miR-30b-3p: GGCCTGGGAGGTGGATGTTTAC; miR-133a-3p: TTTGGTCCCCTTCAACCAGC; miR-193b-3p: AACTGGCCCTCAAAGTCCCG; miR-200c-3p: TAATACTGCCGGGTAATGATG; miR-338-3p: CCAGCATCAGTGATTTTGTTG; miR-432-3p: CTGGATGGCTCCTCCATGTC; miR-605-3p: GGCAGAAGGCACTATGAGATTTAG; miR-579-3p: GGCTTCATTTGGTATAAACCGCG; miR-877-5p: GTAGAGGAGATGGCGCAGG; miR-1178-5p: GGCAGGGTCAGCTGAGCATG.
Microarray was used to detect the circRNAs expression as previously described. Total RNAs from five GC tissues and corresponding normal gastric tissues were extracted and digested with RNase R (Epicentre, Inc., Madison, WI, USA) to remove linear RNAs and thus enrich circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were purified using an RNeasy Mini Kit (Qiagen, Venlo, Netherlands). The concentration and specific activity of the labeled cRNAs (pmol Cy3/µg cRNA) were measured using a NanoDrop ND-1000 (Thermo Scientific, Waltham, MA, USA). One microgram of each labeled cRNA was fragmented by adding 5 µl of 10× Blocking Agent and 1 µl of 25× Fragmentation Buffer; after heating the mixture at 60°C for 30 min, 25 µl of 2× Hybridization Buffer was added to dilute the labeled cRNA. Next, 50 µl of hybridization solution was dispensed into the gasket slide and assembled to the circRNA expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent hybridization oven (Agilent Technologies, Santa Clara, CA, USA). The hybridized arrays were washed, fixed, and scanned using the Agilent Scanner G2505C. Agilent Feature Extraction software (version 184.108.40.206) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies).
2.4. Western blot analysis. Western blot analysis of cells and tissues was conducted as previously described[22, 23]. RIPA lysis buffer was used to extract proteins from cultured cells and frozen tissues. A modified Bradford method was used for protein quantitation, and an equal amount of protein was loaded onto the gel. Proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% milk for 2 h and incubated with primary antibody overnight at 4°C. The following antibodies were used: ZEB1 (1:1000, ab245283), E-cadherin (1:1000, 20874-1-AP), vimentin (Vim; 1:1000, 10366-1-AP), CDK6 (1:1000, 14052-1-AP), MMP2 (1:1000, 10373-2-AP), and β-actin (1:1000, sc-47778). The HRP-labeled secondary antibody (1:10000, Rockland) was incubated with the membranes for 1 h at room temperature. Immobilon™ Western chemiluminescence HRP substrate (Millipore) was used to develop the membrane and detected by ECL (enhanced chemiluminescence) Fuazon Fx (Vilber Lourmat). FusionCapt Advance Fx5 software (Vilber Lourmat) was used to capture and process the images. All experiments were repeated three times.
2.5. Target prediction. To identify potential miRNAs of circCOL12A1 as methods of Z. Yang et al. 2021, we used the miRanda (www.microrna.org), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html), and RNA22 (https://cm.jefferson.edu/rna22/Interactive/). For predicting the target gene of miRNA, we used Targetscan (http://www.targetscan.org).
2.6. Luciferase assay. For the luciferase assay we followed the methods of Z. Yang et al. 2021 . The HGC-27 cells were seeded into a 12-well plate. For the circCOL12A1-miRNA luciferase assay, HGC-27 cells were co-transfected with miRNA mimics or NC mimics and the circCOL12A1-luciferase reporter gene or empty vector. For the miR-30b-3p-ZEB1 luciferase assay, HGC-27 cells were transfected with the miRNA-194-3p mimic or NC mimic and ZEB1-3’UTR luciferase reporter (wt or mut). A dual Glo luciferase assay system (Promega, Madison, WI) (LB955, Berthold Technologies) with flash and luminescence was used to measure luciferase activity.
2.7. Oligo pull-down. HGC-27 cells were incubated with a biotin (bio)-labeled oligonucleotide probe for circCOL12A1 (Bio-5′-CTCTCACTGAAACAGAAATGTTCCGAATAAC, GenePharma Co., Shanghai) at 37°C for 4 h. M-280 Streptavidin Dynabeads (Life Technologies) were added per 100 pmol of biotin-DNA oligonucleotide, and the mixture was rotated at 37°C for 30 min. The magnetic beads were captured using a magnet (Life Technologies) and washed five times. Each experiment was repeated three times as previous description .
2.8. Transwell assay. The transwell assay was performed as previous description. A Transwell chamber with an 8 µm pore size (Costar, Massachusetts) was used to detect the migration ability of HGC-27 cells. In short, 3 × 104 cells/well of HGC-27 cells was seeded into the upper compartment with a serum-free medium. Medium containing 10% FBS was added to the lower compartment. After culturing at 37°C and 5% CO2 for 24 h, the cells were treated as indicated. The cells with a higher migration ability on the upper side of the chamber migrated to the lower chamber. The number of migrating cells was counted in three randomly selected areas by microscopy.
2.9. MTT assay. Cell viability was measured using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, HGC-27 and AGS cells were seeded into 96-well plates. Cells were transfected with the indicated vector for 24 h; a 20 µL MTT reagent (5 mg/mL; Sigma-Aldrich) was added to each well and incubated for 3 to 4 h. A microplate reader (Thermo Fisher, USA) was used to measure the absorbance.
2.10. Colony formation assay. For the colony formation assay we followed the methods of Z. Yang et al. 2021, 100 cultured cells/well were seeded into six-well plates, cultured for 1 week, and were then fixed in glacial acetic acid/methanol solution. Then, 0.5% crystal violet was used to stain the colonies. Colony numbers were counted under a microscope.
2.11. ChIP assay. The chromatin immunoprecipitation (ChIP) assay was conducted as previously described . Briefly, Caki-1 cells were treated with formaldehyde. The cross-linked chromatin was prepared and sonicated to an average size of 400–600 bp. The samples were diluted 10-fold and then precleared with protein A-agarose/salmon sperm DNA for 30 min at 4°C. The DNA fragments were immunoprecipitated overnight at 4°C with anti-MAZ or anti-IgG antibodies. After reversing the crosslinks, MAZ occupancy on the MAP2K2 promoter was examined. The results were determined via qRT-PCR. The ChIP primer sequences were as follows: MAP2K2-prom-F1: GTGGTAAGGCAAGCGAGGGCG; MAP2K2-prom-R1: AGGGGAGGGGCGGCCACAAG; MAP2K2-prom-F2: GGTTCTCTCAGCCCCAGCCTG; MAP2K2-prom-R2: GGCGCCCTCGCTTGCCTTAC; MAP2K2-prom-F3: CCATCCTGGCTAACACGGTG; and MAP2K2-prom-R3: GGAGTGCAGTGGTGCGATCTC.
2.12. Statistical analysis. Data were presented as the mean ± SEM. A Student’s t test was used to analyze differences between the two groups. Spearman’s correlation analysis was used to determine correlation coefficients. P < 0.05 was considered statistically significant. GraphPad Prism 7.0 software was used for the analyses (GraphPad Software).