Tissue specimens and cell culture
161 cases of GC and matched adjacent normal gastric tissue samples were collected from Nanfang Hospital, Southern Medical University. No chemotherapy or radiation therapy was administered before surgery. Prior to patient consent and approval were obtained from the Institutional Research Ethics Committee. GC cell lines (GES-1, SGC7901, AGS, BGC803, MGC803, MGC823, BGC823, MKN28, MKN45) were obtained from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China). These cells were cultured in PPMI-1640 medium containing 10% fetal bovine serum (Invitrogen Life Technology).
Overexpression and knockdown cell lines
The WTX whole CDS region lentivirus expression vector was synthesized by Invitrogen Company (Shanghai, China). WTX shRNAs fragments were synthesized by Genechem company (ShangHai, China), verified and established lentivirus expression vector. MiR-20a-5p mimics and inhibitor lentivirus were constructed by GenePhama (Shanghai, china). Target cells (2×105) were infected with 1×106 lentivirus transducing units in the presence of 1µg/ml polybrene.
Cell proliferation, colony formation, migration and wound healing assays
Cell proliferation was assessed by the cell counting kit-8 (CCK8) assay. Briefly, 2000 cells were seeded to each well of 96-well plates in triplicate. After incubation for indicated time, 5μl CCK-8 buffer (DOJINDO, Japan) was added to each well and incubated for 1 h at 37 °C. The absorbance was measured at 450 nm using Microplate Autoreader (Bio-Rad, Hercules, CA, USA).
For colony formation assay, Cells were plated on 6-well plates (200 cells/well) and cultured until the visualization of cell colonies appeared, which takes approximately 2 weeks. The colonies were fixed and stained with hematoxylin. The colony which contained more than 50 cells were counted for further statistical analysis.
For cell migration assay, 1×105 cells were seeded onto Transwell chambers (BD Biosciences, San Jose, CA, USA) under the same treatment conditions and incubated for 48–72h. Cells that had migrated through the filter were stained with hematoxylin and counted in five fields per insert for further statistical analysis.
For wound healing assays, the pretreated sub-line cells were seeded into 6-well plated and scratched with a pipette tip in 24h, after the clearance of wounded floating cells with PBS, the cells were continuously cultured for another 48h and took photos in every 6h.
Immunohistochemistry (IHC), In situ hybridization (ISH) and scoring
The paraffin-embedded sections were stepwise dewaxed from xylene, gradient ethanol to water, and performed IHC staining according to protocol. The following primary antibodies were used: WTX (diluted 1:200, Abcam), Ki-67 (diluted 1:500, Abcam), p-AKT and p-mTOR (diluted 1:200, Cell Signaling Technology).
ISH assay were performed under RNase-free conditions. The miR-20-5p probe and ISH kit were purchased from Boster Company (Wuhan, China). The slides were stepwise dewaxed, gradient ethanol to water, and refixed as protocol. Then they were incubated in 50 μg/ml Proteinase K, prehybridise for 4 h at 60 °C, 1.5 μg/ml probe incubated 18h at 60°C in series; following 2×SSC, 1×SSC, and 0.1×SSC strict rinsing; blocking, anti-digoxin biotin, SABC-POD, streptavidin-HRP, DAB substation, and hematoxylin counterstain in sequence to detect the positive signal.
The results were scored as previously described and scored as a sum of the staining intensity and percentage of positive tumor cells[12]. Briefly, the staining intensity scaled with 0-3. Percentage of positive cells staining was scored as 0-4. The final staining was summed by intensity and percentage as 0-12. And then adapted to 4-point IRS: 0-1(−), 2–3(+), 4-8(+ +), and 9-12 as (+ + +). Finally, we set (+) as WTX expression cut off point: (−) as negative; (+) ~ (+ + +) as positive. The clinical pathologic meanings of the IHC data were analyzed by Chi-square analysis. IHC staining and statistics were performed in a blind manner.
Immunofluorescence (IF)
Cells were cultured on dishes and fixed with 4% paraformaldehyde at 4°C for 15 min. After blocked with normal goat serum for 30 min, cells were incubated with primary antibodies against WTX (1:200) and PI3K (1:200) at 4°C overnight, and then incubated with Alexa Fluor 488-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech) and 594-conjugated goat anti-rabbit IgG(H+L) (Proteintech) for 2h at room temperature. DAPI was used to position nuclear. The stained cells were mounted with 80% glycerol/PBS for subsequent examination by a laser-scanning confocal microscope (FV1000, Olympus, Japan) using FV10-ASW 4.0 viewer software (Olympus).
Immunoblotting
The proteins were eluted by denaturation buffer; separated by SDS-PAGE gel, transferred to a PVDF membrane (Pierce), followed by milk blocking, then and primary antibodies (WTX, diluted 1:500, Abcam; PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K, 4E-BP1, diluted 1:1000, Cell Signaling Technology; GAPDH, diluted 1:2000, Proteintech) and secondary antibodies incubating. Finally, the proteins were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences Europe, Germany) according to the manufacturer’s instructions.
RNA extraction and quantity real-time PCR (qRT-PCR).
Total RNA was extracted from tissues and cells lines with RNAiso-Plus (TAKARA, DaLian, China). Single-stranded cDNA was then synthesized from 1 μg extracted mRNA by using the qRT-PCR cDNA synthesis kit (TAKARA, DaLian, China) according to the manufacturer’s instructions. Real-time PCR was performed by the Applied Biosystems 7500 Sequence Detection system with the iQTM SYBR green supermix (Bio-Rad Laboratories, Hercules, CA, USA). Primers were prepared as described previously[12]. Thermal cycling conditions included 95℃ for 30s and 47 cycles at 95℃ for 5s, followed by 60℃ for 30s and 72℃ for 34s. The relative expression of WTX and miR-20a-5p were evaluated based on the threshold cycle (Ct), and calculated as 2−ΔΔCT. All of the samples get three independent experiments with four technical replicates each day.
Subcutaneous and orthotopic xenograft tumor mouse model
Subcutaneous tumor models were established in 4-week-old Balb/Cathymic nude mice (nu/nu) (the Animal Center of Southern Medical University, Guangzhou, China). 10 days after injection, the tumor volumes were measured twice a week. The diameter was measured using a slide caliper and the volume was calculated using the following formula: tumor volume (mm3) = (L×W2)/2[29]. The mice were sacrificed within one month and the tumors were harvested for farther analysis.
Orthotopic GC mouse models were established in 4-week-old Balb/Cathymic nude mice. The mice were anesthetized with ketamine (70μg/kg), opened a small incision in the abdomen and revealed the stomach. 100µl cell suspension (1×106 cells) were injected into the muscular of the stomach. Then reset the stomach, added penicillin and closed the abdominal. The model mice were sacrificed two months after the surgery. The stomachs were collected, and tumors were measured and fixed for further histopathological study.
MRNA array
Total RNA was extracted from the AGS.veh and AGS.W cell lines using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions, followed by DNaseI digest, purification with RNeasy Kit (Qiagen, Hilden, Germany), quantities, qualities. Then, RNA was used for cDNA synthesis and produce biotin-tagged cRNA with GeneChip IVT Labeling kit (Affymetrix), fragmented cRNA with controls were hybridized to each GeneChip array according to the manufacturer's instructions. Hybridization, data capture, and analysis were performed by CapitalBio Corporation (Beijing, China). Affymetrix Human Genome U133 Plus 2.0 Array (Santa Clara, CA) which contains more than 54,000 probe sets covering more than 47,000 transcripts and variants, which represent more than 38,500 genes, was used for microarray analysis. The differential express genes were selected by using threshold values of ≥2 and ≤−2-fold change and a FDR significance level of <5%. The AGS.veh and AGS.W cell differential express genes profiles data were deposited on Gene Expression Omnibus under the accession code GSE114353.
MiRNA array
Total RNA of GC and matched gastric mucosa tissue labeled by Hy3 with miRCURY LNA miRNA power labeling kit (Exqion, USA). Human lung fibroblast cell line (HLF) was used as controls. MiRNA microarrays (CCDTM-miRNA850-V4p1.4) were provided by Infectious Disease and Immunogenetics section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, USA55. The miRNA arrays hybridization and data analysis were followed the manufacturer's instructions and the previous protocol[30]. The microRNA expression profiles data were deposited on Gene Expression Omnibus under the accession code GSE94882.
Dual-luciferase reporter system analysis.
WTX gene 3′UTR fragment, which covered miR-20a-5p binding site, was amplified from genomic DNA by PCR. The primers sequences were listed as described previously[12]. The specifically mutated WTX gene 3′UTR fragment was obtained by using Quick Change site directed mutagenesis kit (TOYOBO). They were subcloned into the pGL3-control vector (Promega) to construct miR-20a-p-5p-GL3-WTX-3′UTR-WT or miR-20a-p-5p-GL3-WTX-3′UTR-Mut vectors, respectively. Co-transfect the vectors and miR-20a-5p into MGC823 or SGX7901 cells, harvest and analyzed the luciferase according to the manufacturer’s instructions. Three repeat experiments were conducted independently.
KEGG pathway enrichment analysis
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were performed using the web-based tool DAVID 6.8 (https://david.ncifcrf.gov). The results were visualized in a Bar plot by using ggplot2 in R language.
Statistical analyses
Association between the clinicopathological parameters and WTX expression was analyzed by using either the c2 or Fisher’s exact test. The Wilcoxon signed rank test was used to compare WTX expression in gastric cancer and matched normal tissues. Survival curves were analyzed using the Kaplan-Meier method and they were assessed by log rank testing. P-values<0.05 were considered to be statistically significant.