Cell Lines and Cell Culture
Human GC cell lines GES-1、AGS、BGC-823、 MKN-45、MKN-28 and SGC-7901, and the HEK-293 model cells, were provided by the Biomedical Experiment Center of Xi’an Jiaotong University (China). The use of these cell lines was approved by Ethics Committee of Yan’an University College of Medicine(China). The human GC cells were cultured in the DMEM medium (PAA Laboratories, Pasching, Australia) containing 10% fetal bovine serum (FBS) and the 1640 medium (PAA Laboratories), in a 37°C, 5%CO2 incubator. The culture medium was changed once every 2–3 d. The MKN-28 and SGC-7901 cells in the logarithmic growth phase were collected and subjected to the following experiments.
Cell Transfection
The GC cells in the logarithmic growth was digested and inoculated onto the 6-well culture plate. When 60–80% confluence was reached, the desired transfected fragments (miR-335-5p -mimics and miR-335-5p -inhibitor were purchased from GenePharma, Shanghai, China) were mixed and added into 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 with the reverse-transcription using the commercially available kits, according to the manufacturer’s instructions. Quantitative real-time PCR was performed with the PrimeScriptTM RT Reagent kit (Takara Bio, Japan) on the iQ5 Optical real-time PCR System machine (Bio-Rad, 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 -3ʹ 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 2×RealStar Green Power Mixture, 1-μL Forward-primer (10 μM), 1-μL Reverse-primer (10 μM), 2-μL cDNA and 6-μL 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 totally 40 cycles. Relative expression levels of the target genes were calculated with the 2−ΔCt method. GAPDH was used as internal reference.
Western Blot Analysis
Cells were harvested and lysed with lysis. Total protein concentration was determined with the BCA method. The protein samples were separated with 7.5–12.5% SDS-PAGE, and electronically transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with anti-β-actin, anti- MAPK10 primary antibody (Sanying Biological Co., Ltd., Wuhan, Hubei, China), at 4°C overnight. The membrane was then incubated with tris buffer solution with tween (TBST) -conjugated secondary antibody (Sanying Biological Co., Ltd.) at room temperature for 1 h. Color development was performed with the chemiluminescence detection method, and protein bands were imaged and analyzed with the Q550CW software (Leica, Heidelberg, Germany). β-actin was used as internal reference.
MTT Assay
Cell proliferation was assessed with the MTT kit (Sigma, St Louis, MO, USA). The cells in the logarithmic growth phase were harvested and seeded onto the 96-well plate. At 24 h, 48 h, and 72 h after seeding, respectively, 10 μL MTT was added into each well to incubate the cells for 4 h. The well was added with 150 μL DMSO, and t optical density (OD) was recorded at 490 nm.
Cloning Formation Detection
The transfected cells in the logarithmic growth phase were seeded onto the 6-well plate. After 2 w of culture, the cells were fixed with 4% paraformaldehyde, and stained with crystal violet. The stained cells were then observed, photographed, and counted.
Flow Cytometry
The transfected cells in the logarithmic growth phase were inoculated onto the 6-well plate, and cultured for 1 d. Cells were fixed in 70% ethanol for 24 h, which were then treated with PI and RNase within the kit. Cell cycle distribution was detected by flow cytometry.
Dual Luciferase Reporter Assay
The HEK-293 cells were divided into the miR-30a-3p and pmirGLO empty vector, miR-335-5p and pmirGLO- MAPK10-WT (GenePharma), miR-335-5p and pmirGLO- MAPK10-MuT (GenePharma) co-transfection groups, respectively. Cells without treatment were used as control. The MAPK10 wild-type and mutant fragments 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. These cells were inoculated onto the 96-well plates, and cultured for 24 h. The luciferase activity was detected by the microplate reader. Renilla was used as internal reference.
Cell invasion assay
The 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 plated into the upper chamber, and the bottom wells were filled with complete medium. The cells were allowed to invade across the Matrigel-coated membrane for 48 h.
Wound-healing assay
Awound-healing assay was performed to examine the capacity for cell metastasis. Briefly, once the cells had grown to 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 48 h after wounding.
Statistical Analysis
The SPSS22.0 software was used for statistical analysis. Bioinformatics analysis was performed by the R language ggstatsplot package. Experimental data were processed by the GraphPad Prism7.0 software. Comparison was conducted with the Independent t-test. P<0.05 was considered as statistically significant.