Cell lines and culture
Authenticated GC cell lines, SGC-7901 and MKN-28, were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco’s modified Eagle medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin under a humidified atmosphere containing 5% CO2 at 37°C.
Cell transfection
SGC-7901 and MKN-28 cells (2 × 105/6-well plate) were cultured to 50%–70% confluence. Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) was used to transfect the cells with 50 nM saRNA targeting retinoblastoma (Rb) or negative control saRNA (dsControl). The mock control was treated with Lipofectamine 2000 alone.
RNA extraction and quantitative reverse-transcription PCR
Total RNA from tissues or cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed using the RT-PCR kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Quantitative PCR was conducted using the SYBR Green Premix reagent (Tiangen) and ABI Prism 7000 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The sequences of the primers used in this study are as follows: forward, 5'- CTC TCG TCA GGC TTG AGT TTG-3' and reverse, 5'- GAC ATC TCA TCT AGG TCA ACT GC -3' for Rb; and forward, 5'-GGA GCG AGA TCC CTC CAA AAT-3' and reverse, 5'-GGC TGT TGT CAT ACT TCT CAT GG-3' for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR data were analyzed using the 2−DDCt method, and expression was normalized to that of GAPDH.
Western blotting
Total cellular proteins were extracted using 1% NP-40 and 1 mM phenylmethylsulfonyl fluoride and were quantified using the bicinchoninic acid assay (Sigma-Aldrich, St. Louis, MO, USA). The proteins were then separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. After sequential incubations with primary and secondary antibodies, the bands were visualized by enhanced chemiluminescence using the ImmobilonTM Western Kit (Millipore, Burlington, MA, USA). GAPDH served as a loading control.
Cell proliferation assay
The proliferation of the GC cell lines SGC-7901 and MKN-28 was assessed using the Cell Counting Kit-8 (CCK8: Beyotime Biotechnology, Shanghai, China). All procedures were performed according to the manufacturer’s instructions.
Cell migration and invasion assays
After 72 h, the cells were harvested following treatment with the Rb-targeting saRNA, dsControl, mock transfection, or saRNA/short-hairpin RNA combinations. Cell invasion and migration were measured via Transwell assays using chambers with membranes containing 8 μm pores (Corning, Corning, NY, USA). The cells were suspended in 100 μL serum-free medium and seeded in the upper chamber, and the lower chamber was filled with 500 μL complete media. After incubation at 37°C for 24 h, the Transwell membranes were fixed and stained with 0.5% Crystal Violet/methanol solution for 30 min. The cells that did not migrate were removed using cotton swabs, and the membranes were photographed using a digital camera. In the cell invasion assay, the Transwell membranes were coated with Matrigel (BD Biosciences, San Jose, CA, USA), and the cell incubation period was 48 h.
In vivo model of tumor growth
All animal experiments were approved by the Animal Care and Ethics Committee of Shandong Provincial Hospital affiliated to Shandong University and conducted in the Animal Center of Shandong Provincial Hospital affiliated to Shandong University. Animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press). The Rb-targeting saRNA and dsControl saRNA were transfected into GC cells, and the mock control group consisted of GC cells treated with the transfection reagent only. Nude mice were randomly divided into three groups (n = 6) consisting of mice injected with the following: (1) GC cells transfected with the Rb-targeting saRNA (saRNA group), (2) GC cells transfected with the dsControl saRNA (dsControl group), and (3) mock transfected GC cells (mock group). The cells (5 × 106) were subcutaneously injected into the right side of nude mice. After 21 days, the mice were sacrificed, and solid tumors were isolated for further analysis.
Statistical analysis
All experimental results are expressed as the mean ± standard deviation, and one-way analysis of variance followed by the Tukey–Kramer test for multiple comparisons were conducted using Prism statistical software (GraphPad, San Diego, CA, USA). Differences with a value of P < 0.05 were considered statistically significant.