Patients and diagnoses
Gastric cancer tissues were collected from 70 patients in the Department of General Surgery, First People’s Hospital of Yunnan, from November 2015 to March 2019. The patient cohort had an average age of 56.5 years (range: 26-80 years) and was comprised of 39 males and 31 females. Twenty-four patients had a tumour diameter > 4 cm. Patient data are listed in Table 1.
Dissected tissues were washed with saline immediately following isolation, frozen in liquid nitrogen, and stored at -80°C. All samples were classified as moderately differentiated tumours, according to the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) TNM staging system for gastric cancer (7th Edition) [15].
Total RNA extraction
Total RNA was extracted using the miRNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. RNA concentration and purity were calculated by the A260/A280 ratios, with all samples yielding purity values of 1.8-2.1.
Reverse transcription polymerase chain reaction (RT-PCR)
Complementary DNA (cDNA) synthesis was performed using the RT2 First Strand cDNA Synthesis Kit (Takara, Tokyo, Japan), and qPCR was performed using SYBR Green PCR Master Mix (Promega, Madison, WI, USA) on IQ5.0 equipment (PCR system; Bio-Rad, Hercules, CA, USA). The primer sequences used to target miR340 were as follows:
F: 5’-TTATAAAGCAATGAGACTGATT-3’,
R: 5’-TCAGTCTCATTGCTTTATAATT-3’.
Microarray hybridisation
Double stranded cDNA was initially labelled and hybridised to the microarray (Arraystar, Rockville, MD, USA). Following hybridisation and washing, slides were scanned with an Axon Gene Pix 4000B micro-array scanner (Molecular Devices, Sunnyvale, CA, USA).
Cell lines
SGC-7901 and BGC823 cells were isolated from gastric cancer patients and obtained from the American Type Culture Collection (ATCC). All culture conditions followed those described by the ATCC. KMB17 cells were purchased from the Kunming Medical Biology Institute，which were stored at the Department of Clinical Laboratory, First People’s Hospital of Yunnan.
Recombinant plasmid construction
Nucleic acids were isolated from KMB17 cells following 48 h culture using a phenol-chloroform extraction protocol. The miRNA-340 gene was amplified by PCR using primers that introduced 5’ BamHI and 3’ NotI restriction sites. PCR products were cloned into pCDNA3.1 to yield the recombinant interfering plasmid, pCDNA3.1-miR340 (Kunming Medical Biology Institute, Chinese Academy of Sciences).
Wound-healing assay
The wound-healing assay was used “to detect motility and migration changes in SGC-7901 and BGC823 cells, respectively. Approximately 3 × 105 cells were seeded in each well of a 6-well culture plate. Cells were transfected with pCDNA3.1-miR340, or empty vector control, and incubated for 12 h at 37°C. Cells were then scratched in the centre of each well using a P-20 pipette tip, washed three times in phosphate buffer saline (PBS), and cultured in serum-free medium for 0 h, 6 h, 12 h and 24 h under continual monitoring using an inverted microscope.
Migration assays
Cells (3 × 105 per well) were cultured for 12 h in 6-well cell culture plates containing trans-well inserts (8 μm pore size; Corning; New York, NY, USA) coated with fibronectin (10 µg/mL). Following the 12 h incubation, the culture medium was separated into two layers by the transwell insert, with the upper layer being serum-free and containing 0.1% BSA, while the bottom layer contained 20% foetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM). Following additional 24 h incubation, the bottom of the transwell insert was stained with Giemsa and the migration rates were calculated.
Annexin V/propidium iodide (PI) staining
Apoptosis was calculated by Annexin V/PI co-staining. Serum-free media containing L-mimosine (400 lM) was added to ensure G1 synchronisation. After 24 h incubation, media containing 10% FBS was added. Following an additional 12 h incubation, cells were fixed in 75% ethanol, re-suspended in 100 μL Annexin-V-Fluos labelling solution, 10 μL annexin reagent, and 10 μL PI solution in 150 μL incubation buffer (according to the manufacturer’s instructions; Roche, Basel, Switzerland). Samples were incubated in the dark at room temperature for 7 min followed by the addition of 100 μL incubation buffer. Fluorescence was observed by fluorescence-activated cell sorting (FACS) and the percentage of positive cells was determined based on fluorescence intensity using FAC Station Software (BD Biosciences, San Jose, CA, USA). An isotope control was included in the quadrant analysis. Experiments were performed in triplicate.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot
KMB17 and SGC-7901 cells were harvested and centrifuged at 1,000 g for 10 min (Thermo Fisher Scientific, Waltham, MA, USA). Cell pellets were re-suspended in lysis buffer (Beyotime, Jiangsu, China) followed by the addition of a proteinase inhibitor cocktail (1%) (Sigma-Aldrich, St. Louis, MO, USA), 25 mM NaF and 1 mM Na3VO4. The mixture was frozen to -80°C and thawed four times. The mixture was then centrifuged at 10,000 g for 30 min at 4°C (Thermo) and the supernatant containing RhoA was collected and separated using SDS-PAGE (8%). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and blocked for 2 h at 25°C using 5% BSA Tris-HCl and 0.05% Tween-20. Following blocking, the membrane was incubated with monoclonal anti-RhoA primary antibodies and rabbit anti-mouse monoclonal secondary antibodies. GADPH was used as the loading control and proteins were detected using an enhanced chemiluminescence system (ECL; BestBio, Shanghai, China).
Statistical analysis
Statistical analyses were performed using SPSS software (ver. 20.0; Chicago, IL, USA). Statistically significant differences were determined by one-way analysis of variance, while the chi-square test was used to calculate the significance of differences in detection rates between two groups. A P-value of < 0.05 was considered significant.