Cell culture
HEK293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). SGC-7901 cells and HUVECs were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China). HEK293Tnd SGC-7901 cells were cultured in DMEM medium (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Thermo Fisher Scientific). GSCs were maintained in serum-free DMEM/F12 medium (Thermo Fisher Scientific) containing 2% B27 supplement (Thermo Fisher Scientific), 20 ng/ml epidermal growth factor (Sino Biological Co., ltd., Beijing, China), 1% N2 supplement (Thermo Fisher Scientific), 10 ng/ml fibroblast growth factor-2 (Thermo Fisher Scientific) and 1% penicillin-streptomycin solution (Thermo Fisher Scientific). Human umbilical vein endothelial cells (HUVECs) were cultured in complete endothelial cell medium (ECM) (Sciencell Research laboratories, Carlsbad, CA, USA). All the cells were cultured in a 5% CO2 incubator at 37°C.
Sortingnd determination of GSC subpopulations by flow cytometry
CD24 and CD44 have been identified as the biomarkers of gastric cancer stem cells [24, 25]. In this text, CD24+CD44+ GSCs were sorted from SGC-7901 cells using a flow cytometry. Briefly, cells were incubated on ice for 30 min with primary antibodies against CD24 (cat. no. ab202073, Abcam, Cambridge, UK) and CD44 (cat. no. ab6124, Abcam). Next, iFluor 488 goat anti-rabbit IgG (cat. no.16800, AAT Bioquest lnc., Sunnyvale, CA, USA) and iFluor 555 goat anti-mouse IgG (cat. no.16739, AAT Bioquest lnc.) were added into cells post PBS buffer wash. After 30 min of dark incubation, cells were washed twice with PBS and re-suspended in PBS solution containing 2% FBS. Finally, GSC subpopulations were sorted or analyzed using the flow cytometry (BD Biosciences, San Diego, CA, USA) post filtering.
Reagents and plasmid construction
The 5-fluorouracil (5-FU) drug was purchased from MedChemExpress lnc. (Monmouth Junction, NJ, USA). The full-length fragment of WT1-AS, WT, XBP1, or TP53as subcloned into pLenti6.3⁄V5-DEST lentiviral vectorcat. no. V53306, Thermo Fisher Scientific) to generate corresponding lentiviral overexpression plasmid (WT1-AS (+), WT(+), XBP1(+), or TP53(+). Also, 3 pairs of oligonucleotides targeting WT1-AS or XBP1 were constructed into pLenti6 BLOCK-iT-DEST vector (cat. no. K494400, Thermo Fisher Scientific) to generate WT1-AS(-) or XBP1(-) knockdown plasmids. The overexpression primer sequence and knockdown oligonucleotides were presented in Table 1.
Lentiviral package
Lentiviral overexpression or knockdown plasmid was transfected into HEK293T cells along with packaging plasmid mix (Novobio Biotechnology Co., ltd., Shanghai, China) using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Cell supernatants containing lentiviral particles were collected at 60 h post transfection. After centrifugalized (1000 ×g, 10 min, 4˚C) and filtered with 0.45 µm filters, lentiviral supernatants were concentrated in 1 ml DMEM medium by supercentrifugation (50000 ×g, 2h, 4˚C). After subpackage, lentiviral concentration solutions were stored at -80 ˚C. The titers of lentiviruses (108 transducing units/ml) were determined by double dilution method.
Established of cell lines stably transduced with lentiviral particles
GSCs at the logarithmic phase were seeded into 6 well plates and cultured in complete medium containing lentiviral particles (multiplicity of infection=0) and 8 µg/ml polybrene. At 72 h after transfection, cells were screened withµ for 3 weeks. Overexpression or knockdown effect of target genes was detected by RT-qPCR assay.
RT-qPCR assay
Total RNA was isolated from GSCs using the Trizol reagent (Thermo Fisher Scientific) according to the instructions of manufacturer. RNA was reversely transcribed into cDNA first strand using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) and oligo dT/random primers under the reaction conditions: 25˚C for 5 min, 50˚C for 60 min, and 70 ˚C for 15 min. Next, cDNA was amplified and quantified using SYBR Green I fluorescent dyThermo Fisher Scientific), Platinum Taq DNA Polymerase (Thermo Fisher Scientific) and specific quantitative primers. The reaction procedures were: 95 ˚C for 2 min, 40 cycles of 95 ˚C for 10s, 60 ˚C for 30s, and 70 ˚C for 45s. The quantitative primer sequences were presented in Table 1. β-actin functioned as the housekeeping gene and relative expression levels of lncRNAs and mRNAs were calculated using the 2-ΔΔCt method.
CCK-8 assay
Cell viability was examined using the Cell Counting Kit-8 (CCK-8) kit (Beyotime Biotechnology, Shanghai, China) according to the protocols of manufacturer. Briefly, CCK-8 solution was co-incubated with treated cells at the indicated time points. After 2 h of incubation at 37°C, the optical density (OD) values were measured at 450 nm using a MK3 microplate reader (Thermo Fisher Scientific).
Soft agar colony formation assay
Cell proliferative potential was examined by soft agar colony formation assay. Firstly, the mixture solution (3 ml) containing equal volume of 1.2% agarose and 2× complete medium supplemented with 2×antibiotics and 20%FBS were added into the 6 cm culture dishes to prepare the base agar layer. Next, cells at the logarithmic phase (1000 cells/well) and the equal-volume mixtures (3 ml) of 0.7% agarose and 2× complete medium containing 2×antibiotics and 20%FBS were added into the upper layer. Next, the culture dishes were maintained for 14 days in a 5% CO2 incubator at 37°C. Finally, the number of colonies was counted under a microscope.
Cell cycle detection
For cell cycle determination, cells were fixed overnight at 4˚C with pre-cold 70% ethanol. After low-speed centrifugation (500 ×g, 10 min, 4˚C), cells were incubated for 30 min with the staining solution containing Propidium Iodide (PI, 50 µg/ml, Sigma-Aldrich), RNase A (100 µg/ml) and 0.2% Triton X-100 in the dark. Next, cell cycle distribution patterns were measured using a flow cytometry (BD Biosciences).
Cell apoptotic rate detection
Cell apoptotic percentage was determined using an Annexin V-PE/7-AAD Apoptosis Detection kit (Nan Jing KeyGen Biotech Co., Ltd., Nanjing, China) following the instructions of manufacturer. Briefly, cells were re-suspended in Binding Buffer and then stained with Annexin V-PE and 7-AAD solutions for 15 min at room temperature under a dark environment. Finally, cell apoptotic patterns were analyzed using a flow cytometry (BD Biosciences).
Transwell migration assay
Cells (2×104 cells/well) suspended in serum-free medium were seeded into the upper chamber of Transwell plate (8µm pore size; Costar Corning, Corning, NY, USA) and complete medium containing 10% FBS was added into the low chamber of plate. The plates were cultured in a 5%CO2 incubator for 24 h at 37 ˚C. Next, cells on the top surfaces of the Transwell filters were removed. Cells on the low surfaces were fixed with 4% paraformaldehyde for 30 min and stained with 1% crystal violet solution (Sigma-Aldrich, lnc., St Louis, MO, USA) for 10 min. Finally, cells were imaged and counted using a microscope.
Wound healing assay
Cells were seeded into 24-well plates and cultured overnight at 37°C. When cells grew to the full confluency, a straight scratch wound was created using a sterile 10μl pipette tip. After washed three times with PBS solution, cells were maintained in serum-free medium at 37°C in a 5% CO2 incubator. The wound images were captured at 0 h and 24 h post scratching using a microscope (CKX31, Olympus, Tokyo, Japan). Finally, the migratory distance of cells under the same filed were measured and statistically analyzed.
Western blot analysis
Cell lysates were prepared using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) supplemented with protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins (40μg/sample) were separated through SDS-PAGE and then transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). After blocked with 5% skim milk, the membranes were incubated with primary antibody against vimentin (cat. no. bs-0756R), TWIST (cat. no. bs-2441R), CTNNB1 (beta catenin, cat. no. bs-20599R), MMP2 (cat. no. 10373-2-AP), E-cadherin (cat. no. CDH1, bs-10009R), N-cadherin (cat. no. CDH2, ab18203) and GAPDH (cat. no. bs-0755R) for 12 h at 4˚C. Next, the membranes were probed with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (cat. no. D110058, Sangon Biotech Co., Ltd., Shanghai, China) for 1 h at room temperature. Finally, protein bands were developed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific). All primary antibodies were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China).
In vitro angiogenesis assay
HUVECs were plated in 24-well platesre-coated with Matrigel (Costar Corning) and pre-treatedith CFDA-SE (MedChemExpress, 5 µM) for 10 min at room temperature. Next, HUVECs were cultured in condition medium containing 50% ECM complete medium (Sciencell) and 50% GSC cell supernatants (volume ratio=1:1). Six hours later, cells were imaged using Olympus IX51 fluorescence microscope (Olympus, Tokyo, Japan) and angiogenesis patterns (tube lengths) were analyzed using an Olympus Cellsens 1.5 software (Olympus).
In vitro 3D culture
Cells at the logarithmic growth phase were collected and re-suspended in the complete medium containing 2.5% Matrigel (volume ratio, Costar Corning) at a density of 104 cells/ml. Next, cells (200 µl) were added into 96-well plates pre-coated with agarose. After low-speed centrifugation (1000 ×g, 10 min, 4˚C), cells were cultured at 37°C in a 5% CO2 incubator. Medium was replaced with fresh medium every other day. Next, these 3D multiple cell tumor spheres were imaged using a microscope after 7 days.
Promoter luciferase reporter assay
The luciferase reporter containing WT1-AS promoter sequences (-1K~+200) were constructed and corresponding WT1-AS reporter lentiviruses were generated by Novobio Biotechnology Co., ltd. GSC cells stably transduced with XBP1(+), XBP1(-), NC lentiviruses were also infected with the WT1-AS reporter lentiviruses, followed by the detection of luciferase activities.
In vivo mouse xenograft experiments
All animal experiments were performed with the approval of Experimental Animal Center of the Affiliated Hospital of Jining Medical University. A total of 45 athymic BALB/c mice (male, 6-8 weeks old) were purchased from Shanghai Laboratory Animal Research Center (Shanghai, China) and raised under the standard conditions. Mice were randomly divided into 9 groups: WT1-AS(+) (subcutaneous), WT1-AS(-) (subcutaneous), NC (subcutaneous), WT1-AS(+) (peritoneal), WT1-AS(-) (peritoneal), NC (peritoneal), WT1-AS(+) (tail vein), WT1-AS(-) (tail vein), and NC (tail vein) groups. Each group contains 5 mice.
For subcutaneous xenograft experiments, GSCs (106 cells) stably transduced with WT1-AS(+), WT1-AS(-), or control (NC) lentiviruses were injected into the subcutaneous tissues of mice in corresponding groups, respectively. Tissue volume was measuredt 4 weeks after injection and calculated with the formula: Volume=0.5×length×(width)2. Tumors were resected at weeks post injection.
For peritoneal xenograft experiments, GSCs 106 cells) transduced with WT1-AS(+), WT1-AS(-), or control (NC) lentiviruses were intraperitoneally injected into the corresponding mice. The volumes of ascetic fluid were measured. Tumors were resected and weighed at weeks upon injection.
For lung tumor metastasis models, GSCs106 cells) stably transduced with WT1-AS(+), WT1-AS(-), or control lentiviruses were administrated into corresponding mice via the tail vein. Lung tissues were obtained at
Partial tumor tissues were fixed with 10% formalin, embedded with paraffin and cut into 5 μm sections, followed by hematoxylin-eosin (HE) staining and Ki-67 immunohistochemistry (IHC), and cell subpopulation analysis. HE analysis was carried out using the Hematoxylin and Eosin Staining Kit (Beyotime) according to the manufacturer’s instructions. Briefly, tumor sections were deparaffinized with xylene, rehydrated with different concentrations of ethanol, and stained with hematoxylin and eosin. After dehydrated, permeabilized and sealed, the sections were imaged under a microscope. For IHC analysis, tumor sections were deparaffinized and rehydrated. After the removal of endogenous peroxidase and treatment of antigen retrieval, sections were blocked with 10% normal goat serum for 30 min at room temperature and incubated overnight at 4˚C with primary antibody against Ki67 and then probed with horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature. Next, the sections were incubated with 3,3’-diaminobenzidine (DAB) solution and counterstained with hematoxylin. After the routine treatment of dehydration, clearing and mounting, the sections were imaged. Cell subpopulation analysis was performed with the experimental procedures similar with cell sorting except cell sorting procedures.
RNA-seq
RNA was isolated from GSCs stably transduced with lentiviruses using Trizol Reagent (Thermo Fisher Scientific), followed by the measurement of RNA concentration, purity and integrality. After quality control, RNA was purified, fragmented, and transformed into cDNA library. Next, cDNA library was enriched, quantified by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and sequenced using the Illumina HiSeq 2500 instrument (Illumina).
Raw data obtained after RNA-seq were processed into clean data by removing the sequences with 3’ adapters or reads with the average quality score < Q20. Next, filtered clean data were aligned to the reference genome (Homo_sapiens.GRCh38.dna.primary_assembly.fa) using the HISAT2 software (http://ccb.jhu.edu/software/hisat2/index.shtml). Read counts were normalized using the FPKM (fragments per kilo bases per million fragments) method. Gene were regarded as differentially expressed when they satisfied the conditions: |log2FoldChange| > 1 and P value <0.05. Cluster analysis for differentially expressed genes were performed using the long distance hierarchical clustering method (Complete Linkage) and Euclidean distance metric (R language: Pheatmap software package) according the expression levels of the same genes in different samples and expression patterns of different genes in the same sample. Expression trends of genes in the same cluster were similar. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed using KAAS software. The top 5 KEGG pathways were picked out according to the P values (the 5 minimum P values).
Clinical samples
GC tumors and adjacent normal tissues were collected from 3 cases of GC patientsnderwent surgery sections during May 2017 to July 2018. Our study was conducted with the approval of the Medical Ethics Committee of the Affiliated Hospital of Jining Medical University, and the written informed consents from all participants.
Statistics Analysis
Data (in vitro and in vivo experiments) analysis was performed using GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA) with the results presenting as mean ± standard deviation. Difference between groups was analyzed using paired or unpaired T test. Difference among groups was analyzed using two-way ANOVA along with Sidak post-hoc test or one-way ANOVA along with Tukey’s post-hoc test. P < 0.05 was regarded a significant difference.