Patient specimen collection
A total of 30 GC patients with no preoperative chemo- or radio- treatments were enrolled in this study. Samples were collected from patients who were diagnosed and underwent primary surgical resection in the Affiliated Cancer Hospital & Institute of Guangzhou Medical University (Guangzhou, China) during 2016-2017. After surgical, specimens were immediately frozen by liquid nitrogen and stored at -80 °C for further analysis. The present study was approved by the Ethics Committee of Affiliated Cancer Hospital & Institute of Guangzhou Medical University. Before all experiments were carried, the written informed consent was obtained from all patients.
Cell culture and hyperthermia treatments
The human gastric epithelial cell line, GES-1 and GC cell lines, HGC27, SGC-7901, BGC823, MKN-45 and AGS were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All cells were cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA), and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) at 37 °C in a humidified, 5% CO2 atmosphere. Cell culture medium was replenished every 3 days and when the cells reached 80~90% conﬂuence, cells were split at 1:5 for experiments. In a hyperthermia exposure group, cells in an incubator with preheated to 39˚C, 41˚C, 43˚C or 45˚C for 0.5, 1, 2 or 4 h. Cells from control groups were incubated at 37˚C for the same time periods. After hyperthermia exposure, cells were incubated at 37˚C for 8hours prior to experiments.
LncRNA, shRNA and miRNA transfections
For transfections, GC cells were seeded into 24-well plates at 5x104 density for 24 hours, transfection was performed using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, MA, USA) according to the manufacturer's protocols. Plasmid DNA for overexpression of lncRNA-MALAT1 was constructed according to previous reports (25) and transfected at 2 ug for 48 hours. The shRNA-MALAT1 or empty control was conducted from GenePharma (Shanghai, China). The miR-206 precursor, miR-206 inhibitors or negative control was purchased from RiboBio (Guangzhou, China) and transfected at 50 nM for 48 hours. The overexpression plasmid of GLS was purchased from Origen.com and transfected at 2 ug for 48 hours.
The Kaplan-Meier survival curves were drawn from the KM plot program (http://kmplot.com/analysis/) according to previous description(26). Prediction of the MALAT1-miR-206interaction was performed by starBase of ENCORI (http://starbase.sysu.edu.cn/) according to previous reports(27). The binding of miR-206 on 3’UTR of GLS was predicted from the TargetScan.org.
RNA extraction and qRT-PCR
Total RNAs were extracted using the Trizol reagent (Invitrogen, Carlsbad, MA, USA) according to manufacturer’s instructions. The quantities of RNA samples were measured using aND-1000 spectrophotometer (NanoDropTechnologies Inc., Wilmington, USA). For detection of lncRNA, one μg of total RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For detection of miRNAs, cDNA was synthesized using the TaqMan Advanced miRNA cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Quantitative PCR was performed using the SYBR Green method (Applied Biosystems Inc., Carlsbad, CA, USA). The thermal cycle was set as follows: 95°C for 1 min and 40 cycles at 95°C for 15 s, 58°C for 20 s, and 72°C for 20 s. Primers used for this study were listed as follows:
and Reverse: 5’-CGGAAGTAATTCAAGATCAAGAG-3’;
β-actin: Forward: 5’-CTGAGAGGGAAATCGTGCGT-3’
and Reverse: 5’-CCACAGGATTCCATACCCAAGA-3’;
miR-206: Forward: 5’-TGGAATGTAAGGAAGTGTGTGG-3’;
and Reverse 5’-ACACACTTCCTT ACATTCCATT-3’;
U6: Forward: 5’-CTCGCTTCGGCAGCACA-3’
and Reverse: 5’-AACGCTTCAGGAATTTGCGT-3’.
β-actin and U6 were used as an internal control for MALAT1 and miR-206, respectively. The Ct value was measured by the 2-ΔΔCt method. Experiments were performed in triplicate and repeated three times.
The wild-type MALAT1 or mutant MALAT1 and 3′-UTR of mutant or wild-type GLS were amplified and cloned into a pGL3 vector. Cells were co-transfected with the wild-type or mutant luciferase vectors and miR-206 or control miRNAs by Lipofectamine 2000 for 48 h. A dual-luciferase reporter gene assay was performed using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA). The relative luciferase activity was normalized to that of Renilla luciferase activity. Experiments were performed in triplicate and repeated three times.
Caspase-3 activity assay
Cells (5x104) were seeded in 24-well plates for 24 hours. After treatments, cells were collected and the activity of Caspases-3 was measured using a Caspase-3 Activity Assay Kit (Cell Signaling Tech. Danvers, MA, USA) according to the manufacturer’s protocol. Experiments were performed in triplicate and repeated at least three times.
Detection of glutamine metabolism and GLS activity
The glutamine uptake was measured using the Glutamine and Glutamate Determination Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The activity of GLS and GLS enzyme activity was measured by a Glutaminase Assay Kit (#E-133, Biomedical Research Service Center, Buffalo, NY, USA) according to the manufacturer’s instructions. Briefly, cells were homogenized and mixed with buffers from the kit. The mixture was incubated for 2 h at 37 °C, followed by incubating for another 1 h at 37 °C with new buffers from the kit. The optical density (OD) at 492 nm was determined by a microplate Spectro-photometer. Results were normalized to the cell numbers of each reaction. Experiments were performed and analyzed in triplicate.
Cell viability assay
The cell viability was determined by MTT assay (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. Cells (5×103) were cultured in 96-well plates and cultured for 24 hours. The next day, medium was refreshed cells were stained with 0.5 mg/ml MTT for 4 hours. Supernatant was discarded and cells were washed by PBS. Then 200 μl of dimethylsulfoxide (DMSO) was added for 2 hours at 37°C to dissolveprecipitates. The optical density (OD) of each well was measured at 490 nm using a spectrometer reader. Relative viability was calculated from the absorbance of hyperthermia treated cells/the absorbance of control cells. Experiments were performed in triplicate and repeated three times.
Total proteins were isolated from GC cells using the RIPA buffer (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA), with protease inhibitor cocktail (Bio-Rad, Hercules, CA, USA) according to the instructions of the manufacturer. Cells were washed with cold PBS and lysed withlysis buffer on ice. Samples were centrifuged at 10,000×g to collect the supernatant. Protein was quantified by Bradford assay (Biorad, Hercules, CA, USA). Equal amount of total protein (30 mg) of each sample was resolved on a 10% SDS-PAGE geland transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in TBST for 1 hour at room temperature. Membranes were washed with TBST and then incubated with primary antibodies at 1:1000 at 4oC for overnight. After complete washing by TBST, membranes were then incubated with horseradish peroxidase-linked secondary antibody at 1:3000 at room temperature for 1 hour, followed by visualization using Pierce ECL western blotting kit (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. β-actin was an internal control. Experiments were repeated three times.
Statistical analysis was performed using the Prism 6.0 software package (GraphPad Software, Inc.). Data are represented as mean ± standard deviation (SD). Student's t-test was used to compare difference between two groups and the one-way ANOVA was used to compare the continuous variables in multiple groups. Survival rate were analyzed by Kaplan–Meier method from the http://kmplot.com/analysis. Experiments were repeated three times. p<0.05 indicates statistical significance.