Specimen collection

All GC tumor tissue specimens and non-tumor tissue samples were obtained from GC patients at Sun Yat-Sen University Cancer Center between March 2013 and May 2015. All tissues were frozen in -80℃ liquid nitrogen immediately after surgery. This study was supported and managed by the Ethics Committee of Sun Yat-Sen Memorial Hospital and Affiliated Cancer Hospital & Institute of Guangzhou Medical University.

Cell culture

Human normal gastric epithelial cell line (GES1), gastric cancer cell lines (AGS, BGC823, MGC803, SGC7901, MKN45, MKN74, NCI-N87, NUGC4 and HGC27) were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China). All cells were cultured in RPMI-1640 medium (NUGC4 in DMEM) supplemented with 10% or 20% (HCG27 only) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in 5% CO2.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from GC cell lines and samples using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The precipitated RNA was resuspended in 30 µl of RNase-free water. Then, 2 µg RNA was reverse-transcribed into cDNA using a reverse transcription kit (Promega, Madison, USA).

Expression levels of miR-183 were measured by qRT-PCR, which was carried out with SYBR® Green master mix kit (Promega Corporation). All experiments were performed in triplicate. Relative fold expression changes were calculated using the comparative CT method (2-ΔΔCT), and the endogenous control, U48, was used for normalization of miRNA expression and GAPDH was for mRNA expression. U48 (HmiRQP9001 and P01011A, GeneCopoeia), miR-183-5p (HmiRQP0244 and P01011A, GeneCopoeia).

Wound healing assay

Cells were plated on six-well plates and incubated at 37℃ overnight. A standardized scratch was made on the cell layer by a 200 µL micropipette tip when cells reached 90–100% confluence. Images were captured with an inverted microscope at 0, 24, 48, 72 and 96 h. The scratched wound distances were measured using Image J software.

Transwell assay

Migration assays were performed in 24-transwells. Briefly, cells were seeded in the upper chambers and starved overnight. The lower chamber was supplemented with 750µL RPMI-1640 medium containing 10% FBS. After 24-hour migration, filter was fixed with methanol and stained with crystal violet. The supernatant cells were scraped off gently with cotton swab and lower cells were imaged and counted.

CCK-8 assay

The cell counting kit-8 (CCK-8, Dojindo, Japan) was performed to measure the half-maximal (50%) inhibitory concentration (IC50) values of GC cells exposed to different drug concentrations. GC cells were seeded into 96-well plates. 24 h later, the cells were treated with different concentrations of paclitaxel (Taxol) ranging from 0 to 1000 ug/ml for 48 h. Then the CCK-8 solution (10µL) diluted by fresh RPMI-1640 medium was added to each well and incubated at 37 °C. Finally, the OD values were detected at the wavelength of 450 nm using a microplate reader.

Western blot

Cells were lysed in RIPA buffer and protein concentration of the supernatant was determined by Pierce™ BCA Protein Assay Kit (Thermo Scientific, 23227). 30 µg protein were separated by SDS-PAGE on 10% gel and transferred to PVDF membranes. PVDF membranes were overnight incubated with primary antibodies at 4 °C. Washed with Tris-Buffered Saline and Tween 20 (TBST) 3 times, the membranes were incubated with appropriate secondary antibodies at room temperature for 2 h. After washing, the chemiluminescence image system Tanon 2000 (Tanon, China) was utilized to visualize the immunoreactive bands on the membranes.

Tumor formation and thermochemotherapy treatment in vivo

All animal experiments were conducted in accordance with research ethics and were accepted by the Guangzhou Medical University Laboratory Animal Center Committee approvement. Female BALB/c nude mice (4–5 weeks old, weighing 12–14 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China), kept in SPF mice house. HGC27 miR-183-5p mimics cell suspension was injected subcutaneously into mice with 1 × 10^6. Intraperitoneal injection of PTX (15 mg/kg) or water bath hyperthermia starting from the 6th day, and the cycle was once every 6 days, a total of 3 times. The mice were killed at the end of 24 days experiment period and subcutaneous xenograft tumors were separated for qRT-RCR test.

Dual luciferase reporter assay

HEK293T cells were seeded in 96-well plates and transiently co-transfected with psiCHECK-PPP2CA plus hsa-miR-183-5p, psiCHECK-PPP2CA plus NC, psiCHECK-PPP2CA-mut plus hsa-miR-183-5p and psiCHECK-PPP2CA-mut plus NC. 48 h after co-transfection, luciferase reporter assay (Promega) was performed according to the manufacture’s specifications.

Statistical analysis

All experiments were independently repeated three times. The results are expressed as means ± standard deviation. All data analysis was performed by SPSS statistical software (SPSS 16.0). The differences in variables between groups were determined by Student’s t-test. Cox regression analysis was utilized for evaluated clinicopathological features and miR-183-5p expression. Survival curves were estimated by Kaplan Meier curves and log-rank tests. A value of p less than 0.05 was considered statistically significant.