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  • 4μ8C br Materials and methods br Tissue samples and cell lin

    2020-08-30


    2. Materials and methods
    2.1. Tissue samples and cell lines
    All GC tissues (160 pairs of GC tissues and para-carcinoma tissues used for immunohistochemistry and 84 pairs used for quantitative real-time polymerase chain reaction) included in the present study were obtained from patients who were admitted to the Affiliated Hospital (Group) of Putian University in China and provided written informed consent before surgery. The tissues were collected using a protocol approved by the Ethics Committee of the Affiliated Hospital (Group) of Putian University based on the Declaration of Helsinki. Four human GC cell lines (MKN28, SGC7901, AGS and HGC27) were obtained from the Chinese Academy of Sciences, Shanghai, China. All cell lines were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA).
    2.2. Immunohistochemistry (IHC)
    2.3. Quantitative real-time polymerase chain reaction (qRT-PCR)
    Total RNA was extracted from the GC tissues (50 mg) using the TRIzol reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer's instructions. One microgram of RNA was reverse-transcribed in a final volume of 20 μl under standard conditions using the RevertAid First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). PCR was performed with SYBR Green Master Mix (Roche, Basel, Switzerland) using an ABI 7500 Real-Time PCR System (Applied Biosystems, Life Technologies). The data were analysed with the 2− Ct method. The forward primer for CDH17 was 5′-GGA CAG AGAAGC CGG AAG TC-3′, and the reverse primer was 5′-GAA CAAGCC CGT GTA GTC CTT-3′. GAPDH was used as control; the forward primer for GAPDH was 5′-AGGGCTGCTTTTAACTCTGGT-3′, and the reverse primer was 5′-TCTCGCTCCTGGAAGATGGTG-3′. 
    2.4. Small interfering RNAs (siRNAs) and transfection
    Two siRNA sequences for hamster CDH17 (siCDH17 #1 and siCDH17 #2) were employed, 5′-CCAAGAACCGAGUCAAAUUTT-3′ and 5′-CCACGUUUCUCCAGUCAAATT-3′, and the control sequence was 5′-UUCUCCGAACGUG UCACGU-3′. All the siRNAs were obtained from GenePharma (Shanghai, China). The pcDNA3.1(−)/CDH17 plasmid was preserved in our laboratory. The siRNA sequences and plasmids were transfected with Lipofectamine 2000 (Life Technologies) into AGS 4μ8C according to the manufacturer's recommended protocol.
    2.5. Cell proliferation assay
    Cell proliferation was measured using the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan). Briefly, the cells were seeded into 96-well plates (2.5 × 103 cells/well) and cultured at 37 °C. Then, 10 μl of CCK-8 solution was added to each well, and the plates were incubated for 2 h at 37 °C. The proliferation rate was measured using a scanning multiwell spectrophotometer at 450 nm. Each of the conditions was repeated in triplicate.
    Cells (5 × 105) were seeded in six-well plates and allowed to form a monolayer at 80–90% confluence. The cell monolayer was then scrat-ched using a 10-μl pipette tip. After the detached cells were removed by washing, the cells were incubated at 37 °C. The initial scratch area and the remigration of cells to the wound were photographed for 36 h under serum starvation conditions. Each set of conditions was repeated in triplicate.
    2.7. Cell invasion assay
    Cell invasion assays were performed using Matrigel-coated upper chambers (BD Bioscience). Briefly, cells (2 × 105) suspended in 200 μl of serum-free medium were seeded into the upper chambers. Then, 1 ml of the medium containing 10% serum as a chemo-attractant was added to the lower chamber. After incubation for 24 h at 37 °C, the cells on the upper sides of the filter were gently removed with cotton swabs. The cells that had migrated or invaded the lower surfaces of the filter were fixed in 4% paraformaldehyde for 30 min and stained with 1% crystal violet for another 30 min. Each set of conditions was repeated in tri-plicate.
    2.8. Western blotting
    Western blotting analysis was performed as previously described (Liu et al., 2016). Anti-human CDH17 was obtained from Abcam (Abcam, Cambridge, UK), and an anti-human phospho-inhibitor of nuclear factor kappa-B (IκB) α, anti-human NF-κB (p65), and anti-human IκBα were purchased from Cell Signalling Technology (Danvers, MA, USA).
    The concentration of MMPs in the cell culture medium was de-termined using the commercial Human MMPs Quantikine ELISA Kit (R &D Systems, Minneapolis, MN, USA). The absorbance was measured at 450 nm and corrected using the reading at 540 nm obtained using a Benchmark microplate reader (Bio-Rad, Model 680, Hercules, CA, USA).
    2.10. Statistical analyses
    The results are presented as the means ± SDs and were analysed using SPSS software (version 17.0). An unpaired student's t-test was