Cell Lines and Cell Culture
Human GC cell lines (AGS, BGC-823, MKN-45, MKN-28, and SGC-7901), a normal gastric epithelial cell line (GES-1), and model cells (HEK-293) were provided by the Biomedical Experiment Center of Xi’an Jiaotong University (China). The use of these cell lines was approved by the Ethics Committee of Yan’an University College of Medicine (China). Human GC cells were cultured in DMEM (PAA Laboratories, Pasching, Australia) containing 10% fetal bovine serum and RPMI1 640 medium (PAA Laboratories) at 37°C in a 5% CO2 incubator. The culture medium was changed once every 2–3 days. MKN-28 and SGC-7901 cells in the logarithmic growth phase were collected and subjected to the following experiments.
Cell Transfection
GC cells in the logarithmic growth phase were digested and inoculated onto a 6-well culture plate. After cells reached 60–80% confluence, the miR-335-5p-mimics and miR-335-5p-inhibitor (GenePharma, Shanghai, China) were added to the corresponding wells for further culture for 24–48 h.
Quantitative Real-Time PCR
RNA was extracted from GC cells using TRIzol. The cDNA was obtained by reverse-transcription using commercially available kits, according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the PrimeScriptTM RT Reagent Kit (Takara Bio, Kusatsu, Japan) and an iQ5 Optical real-time PCR System (Bio-Rad, Hercules, CA, USA). The following primer sequences were used for amplification: RT miR-335-5p, 5ʹ-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACacatttt-3ʹ; RT U6 5ʹ-CGCTTCACGAATTTGCGTGTCAT-3ʹ; miR-335-5p, forward 5ʹ-ATCCAGTGCGTGTCGTG-3ʹ and reverse 5ʹ-TGCTTCAAGAGCAATAACGA-3ʹ; U6, forward 5ʹ-GCTTCGGCAGCACATATACTAAAAT-3ʹ and reverse 5ʹ-CGCTTCACGAATTTGCGTGTCAT-3ʹ; MAPK10 forward 5ʹ-TTCTCAGGCACGGAATGG -3 and reverse 5ʹ-TAAGTTGCCATAGTGAAGATCTGAG -3ʹ; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5ʹ-TGAAGGTCGGAGTCAACGGATT-3ʹ and reverse 5ʹ-CCTGGAAGATGGTGATGGGATT-3ʹ. The 20-μL PCR system consisted of 10-μL of 2× RealStar Green Power Mixture, 1 μL of forward primer (10 μM), 1 μL of reverse-primer (10 μM), 2 μL of cDNA, and 6 μL of ddH2O. The amplification conditions were as follows: 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, and 72°C for 30 s, for a total of 40 cycles. Relative expression levels of the target genes were calculated using the 2−ΔCt method. GAPDH was used as an internal reference.
Western Blotting
Cells were lysed with precooled radio-immunoprecipitation assay lysis buffer supplemented with protease inhibitor (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min on ice. The supernatant was collected after centrifugation at 14,000 rpm at 4°C for 20 min. The protein concentration was determined using a bicinchoninic acid protein concentration determination kit (RTP7102; Real-Times Biotechnology Co., Ltd., Beijing, China). The samples (20 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. A GAPDH antibody was used as an internal reference. The membranes were washed with TBST and incubated with goat anti-mouse/rabbit antibody. Color development was performed using the chemiluminescence detection method, and images of protein bands were obtained for analyses. The primary and secondary antibodies were as follows: anti-GAPDH monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA; 5174S, diluted 1/1000), MAPK10 (Cell Signaling Technology; 2305S, diluted 1/1000), vimentin (Cell Signaling Technology; 5741S, diluted 1/1000), E-cadherin (ProteinTech, Rosemont, IL, USA; 20874-1-AP, diluted 1/1000), β-catenin (Cell Signaling Technology; 8480S, diluted 1/1000), CDK6 (Cell Signaling Technology; 13331S, diluted 1/1000), CDK4 (Cell Signaling Technology; 12790S, diluted 1/1000), CyclinD1 (Cell Signaling Technology; 55506S, diluted 1/1000), Bcl-2 (Cell Signaling Technology; 15071S, diluted 1/1000), Bax (ProteinTech; 50599-2-Ig, diluted 1/1000), goat anti-mouse immunoglobulin G (IgG) (ProteinTech; SA00001-1, diluted 1/3000), and goat anti-rabbit IgG (ProteinTech; SA00001-2, diluted 1/3000).
MTT Assay
Cell proliferation was assessed using the MTT Kit (Sigma, St Louis, MO, USA). Cells in the logarithmic growth phase were harvested and seeded on a 96-well plate. At 24, 48, and 72 h after seeding, 10 μL of MTT was added to each well and the cells were incubated for 4 h. Each well was supplemented with 150 μL of DMSO, and the optical density (OD) was recorded at 490 nm.
Colony Formation Detection
Transfected cells in the logarithmic growth phase were seeded onto a 6-well plate. After 2 weeks of culture, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. Images were obtained and cells were counted.
Flow Cytometry
Transfected cells in the logarithmic growth phase were inoculated onto a 6-well plate and cultured for 1 day. Cells were fixed in 70% ethanol for 24 h and treated with propidium iodide and RNase provided with the kit. The cell cycle distribution was detected by flow cytometry.
Dual Luciferase Reporter Assay
HEK-293 cells were divided into the miR-30a-3p and pmirGLO empty vectors, miR-335-5p and pmirGLO-MAPK10-WT (GenePharma), miR-335-5p, and pmirGLO- MAPK10-MuT (GenePharma) co-transfection groups, respectively. Untreated cells were used as controls. Wild-type and mutant MAPK10 were synthesized by GeneChem (Shanghai, China) as follows: wild-type MAPK10, up 5ʹ-cATTTAACTTCTAGTTGCTCTTGCc-3ʹ and down 5ʹ-tcgagGCAAGAGCAACTAGAAGTTAAATgagct-3ʹ; and mutant MAPK10, up 5ʹ-cATTTAACTTCTAGTTGATATCGCc-3ʹ and down 5ʹ-tcgagGCGATATCAACTAGAAGTTAAATgagct-3ʹ. Cells were inoculated onto 96-well plates and cultured for 24 h. Luciferase activity was detected using a microplate reader. Renilla luciferase was used as an internal reference.
Cell Invasion Assay
Transwell chambers (8-μm pore size; Millipore, Billerica, MA, USA) were coated with Matrigel (15 μg/filter; BD Biosciences, Franklin Lakes, NJ, USA). Cells (2.0 × 104) in serum-free medium were added to the upper chamber, and the bottom wells were filled with complete medium. The cells were allowed to cross the Matrigel-coated membrane for 48 h.
Wound-healing Assay
A wound-healing assay was performed to examine metastasis. Briefly, after cells reached 90% confluence in 12-well plates, a single scratch wound was generated with a 200-μL disposable pipette tip. The extent of wound closure was measured after 48 h.
Statistical Analysis
Results are shown as means ± SEM of at least three different experiments. The SPSS22.0 was used for statistical analyses. Bioinformatics analyses were performed using the ggstatsplot package in R. Experimental data were processed using GraphPad Prism7.0. Comparisons were conducted with the independent t-test. P < 0.05 was considered statistically significant.