Ethics statement and human tissue samples
Twenty pairs of GC and adjacent tissues for real-time quantitative PCR were from the surgical patients in Xinqiao Hospital of Third Military Medical University (during 2016 and 2017)，and all resected specimens were confirmed by pathological examination. The informed consent was obtained for all patients. All clinical studies were approved by the Clinical Research Ethics Committee of Third Military Medical University, and were performed in accordance with the approved guidelines. Eighty pairs of GC and adjacent tissue microarray for immunostaining were purchased from SHANGHAI OUTDO BIOTECH CO., LTD (Shanghai, China).
MKN45, SGC7901, AGS, MGC803, BGC823 human gastric cancer cell lines and human gastric normal epithelial mucosa cell line (GES-1) were purchased from ATCC. All cells were cultured in RPMI-1640 or DMEM-HIGH GLUCOSE medium (Hyclone, USA) and supplemented with 10% fetal bovine serum (Hyclone, USA), 100IU/mL penicillin and 100µg/mL streptomycin (Invitrogen, USA). All cells were grown in a 37℃ humidified atmosphere containing 95% air and 5% CO2.
Preparation and infection of lentiviruses
Lentiviruses were purchased from Genechem Co., Ltd (Shanghai, China). Lentivirus which containing the full-length coding sequence (CDS) of TRPV1 (NM_080704) was designed to increase its expression in BGC823. Lentivirus-based shRNA was used to silence the expression of TRPV1 in MKN45. Sequences for TRPV1 shRNA and control were as follows: shRNA-1 (5’-GCATCTTCTACTTCAACTTCC-3’), shRNA-2 (5’-GGCCGACAACACGAAGTTTGT-3’) and control (5’-GTTCTCCGAACGTGTCACGT-3’). All the shRNA group that didn’t mark the number used shRNA-1. Cells were infected with lentiviruses according to the protocol of the manufacturer. Briefly, cells were attached in 24-well plates with amount of 1×105 cells/hole, lentiviruses were added into culture medium separately (the volume of lentiviruses was calculated with MOI 20), the medium were refreshed after 8 hours. Purinomycin was used to screen the stable cells after 72 hours of lentivirus infection.
Preparation and transfection of plasmids
The full length CDS of AMPK (NM_006251) was cloned into pcDNA3.1 to prepare overexpression plasmids; AMPK-siRNA sequence 5’-CTGCTTGATGCACACATGAAT-3’; CaMKKβ-siRNA sequence 5’- GTCAAGTTGGCCTACAATG-3’ were cloned into GV102 vector separately to prepare siRNA-knocked down plasmids. Transfection of plasmids into cells according to the protocol of [email protected] HD Transfection Reagent (Cat. No. E2311, Promega, USA). In brief, 1×105 cells were cultured in a hole of 24-well plates, 1.5 µL of transfection reagent and 0.5 µg of plasmids were mixed and added to 500 µL culture medium. The cells were incubated with the mixture for 72 hours.
RNA extraction and real-time quantitative PCR (qPCR)
Total RNA was extracted from each group by RNAiso Plus reagent (Cat. No. 9109, Takara, Japan). cDNA was synthesized using PrimeScript® RT-polymerase (Cat. No. R050A, Takara, Japan). Then, 50 ng of each cDNA was amplificated as a template, and qPCR was performed using a SteponePlus device (Art. No. 272008342, Life Technologies, USA) with a SYBR® Premix Ex TaqTM II kit (Cat. No. RR820A, Takara, Japan). All samples were run in triplicate, and β-actin was used as an internal control. Data were quantified using the 2−ΔΔCt relative quantitative method, normalized based on β-actin expression, and expressed as the ratio of TRPV1 to β-actin mRNA levels. The primers were designed as follows:
TRPV1: 5’-TGGTATTCTCCCTGGCCTTG-3’ (forward)
5’- CTTCCCGTCTTCAATCAGCG-3’ (reverse)
β-actin: 5’-GGCATCCACGAAACTACCTT-3’ (forward)
Tissue samples were paraffin embedded and cut into 5mm slices. After the treatment of dewaxing and rehydration, tissue samples were incubated with anti-TRPV1 (dilution 1:500, Cat. No. ab3487, Abcam, UK）overnight at 4℃ after blocked. TRPV1 was detected with HRP-conjugated anti-rabbit secondary antibody (dilution 1:1000, Cat. No. ZB-2301, ZSGB-BIO, China) and visualized with DAB. The negative control contained secondary antibody only. The staining results were observed by light microscopy, and IPP (Image Pro Plus 6.0) software was used to quantitatively score the tissue sections. Briefly, images of all tissue cores were acquired at the same time with a constant set of imaging parameters on the microscope and imaging software. The images were then subjected to optical density analysis by IPP software. The intensity range selection was based on histograms, with the intensity (I), saturation (S), and maximum hue (H) set at a range in which most of the brown diaminobenzidine color could be quantified. After defining the area of interest (AOI), the mean optical density of the selected area [integrated optical density (IOD)/unit area] was determined by the software and used to represent the immunoreactivity of the candidate protein within the tumor tissue. The acquired score of the optical density was normalized and subjected to IPP for analysis.
Cells of each group were attached to coverslips in 35 mm dishes and fixed in 4% polyformaldehyde for 15 minutes at room temperature. The coverslips were washed in PBS three times for 5 minutes. Cells were blocked in goat serum for 1 hour at room temperature, and then incubated with anti-TRPV1 antibody overnight at 4°C. After three washes with PBS, cells were incubated with Cy3 labeled anti-rabbit (diluted 1:1000, Cat. No. A0516, Beyotime, China) secondary antibody for 1 hour at room temperature. Finally, the nucleuses were stained with DAPI for 10 minutes. Images were captured on a confocal microscope (Leica SP5, Germany).
Western blot analysis
Whole-cell lysates were separated by SDS-PAGE on denaturing 10% or 12% gels and transferred to polyvinylidene fluoride membranes (Cat. No. ISEQ00010, Millipore, USA). The blots were blocked in 5% milk for 1 hour at room temperature and then separately incubated at 4°C overnight with the following specific primary antibodies: anti-TRPV1, anti-Ki67 (Cat. No. ab15580, Abcam, UK), anti-AMPK (Cat. No.5832, Cell Signaling Technology, USA), anti-phospho-AMPK (Cat. No.2535, Cell Signaling Technology, USA), anti-CaMKKβ (Cat. No. ab168818, Abcam, UK), anti-cyclin D1 (Cat. No. ab16663, Abcam, UK), anti-MMP2 (Cat. No.87809S, Cell Signaling Technology, USA), anti-β-catenin (Cat. No.8480, Cell Signaling Technology, USA), anti-phospho-β-catenin (Cat. No.9567, Cell Signaling Technology, USA), anti-AKT (Cat. No.9272, Cell Signaling Technology, USA), anti-phospho- AKT (Cat. No.4060, Cell Signaling Technology, USA), anti-ERK1/2 (Cat. No. ab184699, Abcam, UK), anti-phospho-ERK1/2 (Cat. No. ab214362, Abcam, UK), and anti-GAPDH (Cat. No. TA-08, ZSGB-BIO, China); all primary antibodies were diluted 1:1000. After rinsing, the blots were incubated in HRP-conjugated anti-rabbit or anti-mouse (diluted 1:1000, Cat. No. A0239 and A0216, Beyotime,China) secondary antibodies for 1 hour at room temperature. Enhanced chemiluminescence (Cat. No. 34094, Thermo, USA) was used to detect the immunoreactive bands. Human phospho-kinase array was used to detect changes in tumor-related signaling pathways (Cat. No. ARY003B, R&D Systems, USA). 2µM BAPTA-AM (Cas No. 126150-97-8, MedChemExpress, USA) was used to chelate intracellular calcium, cells were treated with BAPTA-AM for 2 hours. Each experiment was performed in triplicate and repeated three times. The gray value of the band measured by imageJ software for statistics.
Cell proliferation assay
Cell viability and proliferation were detected by CCK8 assay (Cat. No.C0038, Beyotime Biotechnology, China). Cells after cultured for 0, 24, 48 or 72 hours were added to 96-well plates (3000 cells/well) in triplicate and incubated in 100 µl of medium/CCK8 mixture (medium: CCK8, 9:1) at 1-2 hours before the endpoint of incubation. A Multiskan EX plate reader (Thermo Fisher Scientific, Germany) was used to quantify the viable cells by measuring the absorbance at 450 nm which can estimate relative cell numbers instead of counting cells. Experiments were repeated at least three times.
Cell cycle analysis
Cells were digested, centrifuged and washed twice with cold PBS, discarded the supernatant, and pre-cooled 70% ethanol was slowly added to the pellet, then cells were resuspended at 4 °C overnight. After centrifugation the next day, removed the ethanol, washed once with PBS, cells were incubated in a solution containing 0.2% Tween 20, 100 U/mL RNase, and 50 μg/mL propidium iodide for 20 minutes at 37°C. Cell cycle analysis was performed using flow cytometry in which samples were gated on live cells with an excitation wavelength of 488 nm and an emission wavelength of 620 nm. LMD files were further analyzed using ModFit LT (Verity Software House, Topsham, ME). Each experiment was performed in triplicate and repeated three times.
Fat plate clone formation test
Long-term survival of cells was assessed by the ability to form colonies. Cells were attached on 6-well plates with 3mL culture medium, each group of cells were calculated as 500 cells/hole. After 10-12 days, the cell culture medium was removed and cell clones were washed with PBS, then fixed with 4% polyformaldehyde. Counting the clone numbers after stained with crystal violet. (Relative clonogenicity = Clone numbers /Average clone numbers of control group). All experiments were repeated at least three times.
Transwell migration and invasion assays
Twenty-four-well transwell chambers (Corning, USA) were used for this assay. 5×104 cells were placed in each the upper chambers with 8-μm pores and cultured in 200 μL serum-free RPMI-1640. The lower chambers were filled with 500 μL completed RPMI-1640 medium. After 24 hours of incubation, cells that had migrated onto the lower surface were stained with crystal violet, and counted under a microscope (Olympus Corporation, Japan). The average value of three randomly selected fields was recorded as the number of migrated cells. Then, the upper surface of the polycarbonate filter was coated with 10% matrigel (Collaborative Biomedical, USA), and 1×105 cells were added to detect cell invasion. The other conditions were the same as those in the migration assay.
Attaching 1×104 cells on coverslips, cells were loaded with 5 μM Fura-2 AM (Cat. No. F1221, Invitrogen, USA) in physiological salt solution (PSS) at 37°C for 60 minutes and then washed in PSS or PSS with the inhibitor of TRPV1 SB-705498 50μM (Cas No.501951-42-4, MCE, USA). Then, cells on coverslips were mounted in a standard perfusion chamber on a Nikon microscope stage. The ratio of Fura-2 fluorescence with excitation at 340 or 380 nm (F340/380) was followed over time and captured with an intensified CCD camera (ICCD200) and a MetaFluor Imaging System (Universal Imaging, Downingtown, PA). PSS used in Ca2+ measurement contained the following: 140 mM Na+, 5 mM K+, 2 mM Ca2+, 147 mM Cl-, 10 mM HEPES, and 10 mM glucose, pH 7.4.
Tumor xenograft and peritoneal dissemination assay in nude mice
The animal use protocol was approved by the Third Military Medical University Committee on Investigations Involving Animal Subjects. All the animal care and experimental studies were conducted in accordance with the guidelines of the Animal Ethical Committee of Third Military Medical University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.8023, revised 1978). Animal studies were reported in compliance with the ARRIVE guidelines. 1×106 TRPV1-overexpression BGC823 cells and the negative control (NC) cells were injected into the armpits of seven 4-weeks old male nude mice respectively for tumor xenograft assay. TRPV1-overexpression BGC823 cells were injected into the right armpit and the NC cells were injected into the left side. The volumes of GC xenografts were assessed every 5 days. After 30 days of implantation, the mice were sacrificed and the tumor volume in mm3 was calculated by the formula1/2 (length× width2). For the peritoneal dissemination assay, 1×106 TRPV1-overexpression BGC823 cells and the NC cells were injected into the abdominal cavity of nude mice. Five weeks later, the mice were sacrificed, and the nodules were observed and counted.
Prism 8.0 software (GraphPad, San Diego, USA) was used to analyze the data. All data are shown as MEANS ± SD, the statistical significance between two groups was determined by Student’s t-test. The one-way ANOVA was used to compare three or more groups. Significant difference was expressed in the figures or figure legends as *P< 0.05. All the experiments were biologically repeated three times.