The human gastric cancer cell lines (BGC-823, SGC7901) and human gastric epithelial cell (GES-1) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, NY, USA) in a humidified air at 5% CO2 atmosphere at 37℃.
RNA extraction and real-time quantitative PCR (RT-qPCR)
RNA was isolated by Trizol (Invitrogen, CA, USA) according to the manufacturer’s protocols. RNAs were reverse transcribed to cDNA by employing PrimeScript RT Master Mix (Takara, Dalian, China) following the manufacturer’s protocol. PCR amplification was conducted with the SYBR Premix Ex TaqTM Kit (Takara, Dalian, China). GAPDH and U6 were used to normalize the expression. The relative expression was calculated using the 2−ΔΔCt method.
Oligonucleotides and transfection
The plasmid carrying the CREB1 CDS domain was used to overexpress the CREB1 in gastric cancer cells and a pcDNA (pcDNA3.1) vector was used as a negative control (GenePharma, Guangzhou, China). The cDNA encoding CREB1 CDS domain was amplified by PCR, and then subcloned into the pcDNA3.1 vector (Invitrogen, CA, USA) to obtain the pcDNA-CREB1. The miR-450 mimic and the negative control (NC) were synthesized by GenePharma (Guangzhou, China). The transfection was performed by using Lipofectamine 3000 Reagent (Life Technologies, Carlsbad, CA, USA).
Cell proliferation assay
Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to examine gastric cancer cell proliferation. In brief, BGC-823 or SGC7901 cells (1 × 104/well) were seeded in 96-well plates and seeded for 0 h, 24 h, 48 h and 72 h, respectively. Then BGC-823 or SGC7901 cells were incubated with 10 µL CCK-8 solution for 4 h at 37℃. The optical density (OD) was recorded using a microplate at 490 nm (Multiskan MK3, Thermo Scientific, USA). For colony formation assay, BGC-823 and SGC7901 cells (2 × 104 cells/well) were seeded in 24-well plates. After incubation for 12 days, the cells were immobilized with paraformaldehyde for 30 min, and stained with 10% crystal violet for another 30 min. Colonies were counted and photographed with a light microscope (Olympus, Tokyo, Japan).
Cell apoptosis assay
Flow cytometry was conducted to investigate the gastric cancer cell apoptosis. Cells (1 × 104 cells/well) were seeded in six-well plates and culture for 48 h. Thereafter, cells were washed using PBS, treated with 5 µL of annexin V-FITC and 5 µL of PI in the dark for 15 min at room temperature. Then apoptosis cells were detected through a FACScalibur Flow Cytometry (BD Biosciences, CA, USA).
Cell migration and invasion assays
Wound healing and transwell chamber (5-µm pore size, Costar, Cambridge, MA, USA) assays were conducted to examine gastric cancer cell migration and invasion abilities. For wound healing assay, cells were seeded into six-well plates. The supernatant fluid was removed when BGC-823 or SGC7901 cells were highly confluent (> 90%). Scratches were made using a sterile pipette tip, with the scratch width remaining the same. After continuous culture for 48 h, the width of the scratch was photographed and recorded under a microscope (⋅ 100). For invasion assay, BGC-823 or SGC7901 cells were estimated by transwell assays. In short, transfected BGC-823 or SGC7901 cells were added to the upper chamber loaded with matrigel (Corning, Cambridge, MA) and the bottom of the chamber were supplemented with complete medium containing 1% FBS. 48 h later, cells on the surface of membranes were wiped out. Invaded cells were fixed in 10% formaldehyde, dyed with 0.1% crystal violet and then counted with a light microscope (Olympus, Tokyo, Japan).
Hematoxylin-eosin staining (HE) assay
Tumor slices were stained with hematoxylin for 5 min, then rinsed for 1 min, and returned to blue by 1% ammonia (30 s). Afterwards, slices were flushed with running water (1 min). Furthermore, slices were stained by 0.5% H&E (for 1 min), rinsed (for 30 s), made into transparent, and finally mounted with neutral gum.
Immunohistochemical (IHC) assay
Tumor sample slides were deparaffinized (xylene), rehydrated (ethanol), and incubated with H2O2 at 37 °C for 10 min. Tumor sections were incubated with antibody against Ki67 (ab15580; 1: 1000; Abcam, USA) at 4 °C overnight., followed by incubation with the biotin-conjugated goat anti-rabbit immunoglobulin G secondary antibody (ab6721; 1: 1000; Abcam, USA) at 37 °C for another 30 min. DAB was used as chromogenic agent. Images were obtained using a microscope (× 200).
The slices were washed by PBS and then immobilized for 30 min with 4% paraformaldehyde. After washed with PBS once, the slices were added with 0.1% Triton X-100 for 2 min, and then washed with PBS once. Afterwards, 3% H2O2 was used for incubation (5 min). Then the slides was rinsed, and maintained with 50 µL TUNEL at 37 °C overnight. After rinsed by PBS again, the TUNEL reaction was visualized by chromogenic staining with DAB (Sigma-Aldrich). Light microscope was applied to observe the slides and the TUNEL-positive BGC-823 or SGC7901 cells were calculated in Image J software.
Dual luciferase reporter assay
A putative 3’-untranslated regions (3’-UTR) of CREB1 was mutated using mutagenesis kit (Promega, USA). Wild type and mutant sequences were amplified and inserted into the vector to construct luciferase reporter plasmids according to the manufacturer’s instructions (Promega, USA). The luciferase activities were detected with the dual luciferase reporter kit (Promega, USA). Luciferase activity was measured by dual-luciferase reporter assay system (Promega) and presented as firefly luciferase intensity normalized to Renilla luciferase activity.
Western blotting analysis
Total proteins in tissues or cells were dissolved with RIPA lysate buffer (Beyotime Inc, Shanghai, China). The protein was quantified using BCA protein assay Kit (Thermo Scientific, CA, USA). The protein samples were separated by 12% polyacrylamide gel, which were transferred to the PVDF membrane, sealed with 5% skim milk powder. The membrane was incubated with the primary antibody at 4℃overnight and then incubated with HRP coupled secondary antibody (Santa Cruz Inc, CA, USA) at room temperature for 1 h. The protein signal was detected by ECL detection reagents (Thermo Scientific, CA, USA) and GAPDH worked as the internal reference.
Xenograft tumors in nude mice
Female nude mice (6-week-old, 18–22 g) were provided by Nanjing Medical University and housed under germ free conditions. Animal care and use were carried out according to the ethical guidelines by the First Affiliated Hospital of USTC Animal Care and Use Committee. Nude mice were maintained in a 12 h light/12 dark cycle in a temperature- and humidity-controlled environment. To detect the effect of miR-450 on tumor growth in vivo, BGC-823 cells (1 × 106 cells) were injected subcutaneously into the right axilla of the nude mice. Following a 30-day period, nude mice were sacrificed, and neoplasms were isolated for further analyses. Note that the tumor volumes were recorded every week and calculated with the formula: Volume = 0.5 × length × wide2.
All data are presented as the mean ± SD. Each bar expressed the mean ± SD of three independent experiments. Statistical significance between two or multiple groups was analyzed by t-test or one-way ANOVA using SPSS 17.0 (SPSS Inc, Chicago, IL, USA). Experiments were repeated three times independently. Statistical significance was assumed when P < 0.05.