Human GC tissue preparation
In the Department of Oncological Surgery, First Affiliated Hospital, Xi’an Jiaotong University, China, we collected seventy GC tissue specimens and matched adjacent normal tissue samples from sufferer who were diagnosed, between August 2017 and November 2018. We gained the consent form from patients before tissue collection. The obtained tissue specimens were promptly refrigerated and stockpiled at -70 °C. The Ethics Committee of The First Affiliated Hospital of Xi’an Jiaotong University authorized the researches.
Human normal gastric epithelial cell line (GES-1) and GC cell lines (BGC-823, MKN-45 and AGS) were obtained from the Cell Bank (Shanghai, China). The Cell Bank Cells have authenticated these cell lines. These cells were maintained in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) including 10% fetal bovine serum (Gibco), streptomycin (100 µg/mL) and penicillin (100 U/mL), and were cultured at 37 °C in a thermotank under 5% CO2 and 95% air.
Hsa-miR-559 vector construction
Hsa-miR-559 precursor expression vector (called after miR-559) and control empty vector (called after control) were constructed with chemosynthetic oligonucleotides and incorporated into the pcDNA6.2-GW/EmGFPmiR plasmid on the basis of the manufacturer’s instructions. Full-length human TRIM14 gene DNA was cloned into the pCMV2-GV146 vector (Genechem Co. Ltd, Shanghai, China). Transfection was fulfiled through using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions.
Anti-miR-559/TRIM14 siRNA synthesis and transfection
MiR-559 inhibitors (named anti-miR-559), interfering RNA oligonucleotides, were were synthesized by Gene Pharma (Shanghai, China). The sequence of anti-miR-559 was 5′- UUUUGGUGCAUAUUUACUUUA-3′. The useless siRNA was used as a control (named anti-miR-Control), and its sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′. The miR-559 inhibitors were transfected into human GC MKN-45 and BGC-823 cells with Lipofectamine 2000. Small interfering RNAs (siRNAs) were used to silence or knock down the human TRIM14 gene. TRIM14 siRNA and negative control siRNA (named NC-siRNA) were designed and synthesized by GenePharma. The siRNAs were transfected with Lipofectamine 2000 and diluted to 70 nM for use in future experiment.
Dual-luciferase reporter assay
The binding site for miR-559 in the 3′-UTR of TRIM14 was constructed with synthetic oligonucleotides (Beijing AuGCT DNA-SYN Biotechnology Company, China) and inserted to the pmirGLO Dual-Luciferase expression vector (called after TRIM14-WT). The mutated 3′-UTR sequences of TRIM14 were also cloned and named TRIM14-MT. The pre-miR-559 plasmids and reporter plasmids (WT or MT) were cotransfected into HEK293T cells. The cells were collected and examined 24 hours after transfection. We measured the reporter activity with the Dual-Luciferase Assay System (Promega, Madison, USA).
Quantitative real-time PCR (qRT-PCR)
RNA was extracted from GC tissue specimens and cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The SYBR Premix Ex Taq II Kit (Takara, China) was used to measure miR-559 expression and TRIM14 mRNA expression. qRT-PCR was performed. The data were normalized to RNU6B (U6) or GAPDH gene expression. The primer sequences contained the miR-559 reverse-transcription primer (5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTTTTGGT-3′), miR-559 PCR forward primer (5′-ATCCAGTGCGTGTCGTG-3′), miR-559 PCR reverse primer (5′-TGCTTAAAGTAAATATGC-3′), U6 reverse-transcription primer (5′-CGCTTCACGAATTTGCGTGTCAT-3′), U6 PCR forward primer (5′-GCTTCGGCAGCACATATACTAAAAT-3′), U6 PCR reverse primer (5′-CGCTTCACGAATTTGCGTGTCAT-3′), TRIM14 PCR forward primer (5′-GCAGAAACTCAGCCAAGAA-3′), TRIM14 PCR reverse primer (5′-CTTGACTCTGCATTAGCCT-3′), GAPDH forward, 5′-GCCACATCGCTCAGACAC-3′; GAPDH reverse, 5′-GCCCAATACGACCAAATCC-3′. The 2−ΔΔCt method was used in the qRT-PCR analysis.
Cell proliferation assay
Human GC MKN-45 and BGC-823 cells were seeded into three 96-well plates (4-parallel wells/group) and cultured for 24 hours. Then, the GC cells were transfected with Control, miR-559, anti-miR-Control, anti-miR-559, NC-siRNA (70 nM), TRIM14 siRNA (70 nM), vector control or TRIM14 expression vector for 24, 48 or 72 h, respectively. Cell proliferation was examined using MTT assays (Sigma, St Louis, MO, USA) on a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 492 nm.
Cell counting assay
To measure cell proliferation, 2.0 × 105 cells were plated in 6-well plates (3-parallel wells/group) and cultivated for 24 hours. MKN-45 and BGC-823 cells were transfected separately with control, miR-559, anti-miR-Control, anti-miR-559, NC-siRNA (70 nM), TRIM14 siRNA (70 nM), vector control and TRIM14 overexpression vector. The quantity of cells was counted at 24, 48 and 72 hours after transfection through using a Countess automated cell counter (Life Technologies Corp., Carlsbad, USA).
Cell cycle analysis
MKN-45 and BGC-823 cells were cultured in 24-well plates (3-parallel wells/group) and transfected for 24 hours. Next, the cells were collected for analysis by trypsinization and fixed in 70% ice-cold ethanol at 4 °C. The fixed cells were washed by using PBS and stained with 50 µg/ml propidium iodide and 50 µg/ml RNase A (DNase-free) for 10–15 min at room temperature. Finally, cell cycle distribution was detected by using fluorescence-activated cell sorting (BD Biosciences, USA), and the different cell cycle populations were analyzed with ModFit software (Bio-Rad Laboratories, Hercules, CA, USA).
MKN-45 and BGC-823 cells were seeded into 12-well plates (3-parallel wells/group) and transfected for 48 hours. The cells were harvested and washed with PBS. We assessed cell apoptosis with an Annexin-V FITC Apoptosis Kit (Invitrogen, Thermo Fisher Scientific, USA) on the basis of the manufacturer's instructions. A flow cytometer (BD Biosciences, USA) was used to measure apoptotic cells. The changes of apoptosis were analyzed by using the ModFit software.
Western blot was performed according to standard methods. Human GC tissue specimens and cultivated cells were lysed using RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA) and centrifuged at 12,000 g. Next, we detected the protein concentration with the bicinchoninic acid (BCA) assay. The extracted protein was divided by 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). The nitrocellulose membranes were treated for 30 min in blocking solution including 5% nonfat dry milk and incubated with primary antibodies at 4 °C. The primary antibodies were including mouse monoclonal anti-TRIM14 (1:1,000, Cell Signaling Technology, USA), mouse monoclonal anti-AKT (1:1,000, Cell Signaling Technology, USA), mouse monoclonal anti-cyclin D1 (1:1,000, Santa Cruz, CA, USA), mouse monoclonal anti-p-AKT (1:1,000, Cell Signaling Technology, USA), mouse monoclonal anti-CDK2 (1:1,000, Santa Cruz, CA, USA), rabbit monoclonal anti-Bcl-2 (1:1,000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bax (1:1,000, Santa Cruz, CA, USA), and mouse monoclonalanti-GAPDH (1:5,000, Santa Cruz, CA, USA). The membranes were subsequently treated by using ECL reagent (Pierce, Rockford, IL, USA) for chemiluminescence detection. The blots were scanned and recorded, and the band densities were analyzed using PDQuest software.
All experiments were fulfilled minimally in triplicate independently. The data were analyzed with SPSS 25.0 software (SPSS, Inc., Chicago, IL, USA). The data were represented as the means ± SEM from at least 3 experiments. One-way ANOVA and Student's t-test were used to analyze the statistical significance of differences between groups. Correlation analysis between miR-559 and TRIM14 in human GC tissues was performed with Pearson's correlation analysis. Values of p < 0.05 were deemed to indicate statistically significant differences.