Cell culture
Gastric cancer cell lines (AGS and MGC803) were cultured in DMEM (Thermo Fisher Scientific, Cat: 11,965,092) containing 10% fetal bovine serum (Biological Industries, BISH0744) and 1% penicillin/streptomycin (Invitrogen). HEK-293 cells were cultured in DMEM supplemented with 10% FBS (Biological Industries, BISH0744) and 1% penicillin/streptomycin. AGS and BCG803 gastric cancer cells were authenticated by short tandem repeat (STR) profiling. The STR profile of our AGS cells was found to be 100% consistent with the STR data of AGS cells from the China Infrastructure of Cell Line Resources. MGC803 cell STR profiling data are not accessible in public databases, including ATCC. Cells were regularly tested for mycoplasma contamination using a LookOut Mycoplasma PCR Detection Kit (MP0035, Sigma) and were used only when negative.
RNA isolation and quantitative real-time PCR (qRT–PCR)
Total RNA was extracted with an RNeasy Plus Mini Kit (Tiangen, DP451) following the manufacturer’s specifications. Reverse transcription was performed using HiScript II Q RT SuperMix (Vazyme, R223-01). qRT–PCR was carried out using SYBR qPCR Master Mix (Vazyme, Q511-02) and a 7500 Fast Real-Time PCR System (Applied Biosystems, Singapore). 36B4 was used as an internal control. The sequences of the primers used for qPCR were as follows: 36B4 F: GGC GAC CTG GAA GTC CAA CT; R: CCA TCA GCA CCA CAG CCT TC. CTGF F: CTC GCG GCT TAC CGA CTG; R: GGC TCT GCT TCT CTA GCC TG. CYR61 F: AGC AGC CTG AAA AAG GGC AA; R: AGC CTG TAG AAG GGA AAC GC. DUB1 F: GAG GCC GGG GCT CTG A; R: ACT GGG ATG TGC AGA CTT GG. TAZ F: AGA GTC GGG TCG GGA TTT GT; R: AGG CCG GAT TCA TCT TCT GGG.
Plasmids and siRNA
The DUB1 and TAZ plasmids were acquired from HANBIO Company (https://www.hanbio.net). The DUB1 and TAZ deletion constructs were subcloned from the full-length plasmid DNA. The HA-K48 and HA-Ub plasmids were used in a previous study. Lipofectamine 2000 (1,662,298, Invitrogen) was used for plasmid transfection. Small interfering RNAs were used for knockdown of specific genes. The DUB1 siRNA sequences were as follows: (1) CCG GCA AGC UGC GAA UAU UTT and AAU AUU CGC AGC UUG CCG GTT; and (2) GCA CAC CAC UGA AGA GAU UTT and AAU CUC UUC AGU GGU GUG CTT. The negative control siRNA sequences were as follows: UUC UCC GAA CGU GUC ACG UTT and ACG UGA CAC GUU CGG AGA ATT. RNAiMAX reagent (13,778,150, Invitrogen) was used for siRNA transfection. For lentiviral DUB1 silencing, shDUB1 was inserted into the vector pLKO.1, which was cotransfected with the pMD2.G envelope plasmid and psPAX2 packaging plasmid into HEK293 cells. The DUB1 shRNA-expressing lentivirus was harvested after 48 h. Gastric cancer cells were incubated with 2 ml of antibiotic-free medium containing 200 µl of lentiviral suspension.
Western blot analysis
Standard western blotting techniques were utilized to analyze relative protein expression in cells. The following antibodies were used for western blot analysis: anti-Flag-M2 (A8592, Sigma, 1:1000), anti-HA (2,013,819,001, Roche, 1:1000), anti-Myc (9E10, Santa Cruz, 1:1000), anti-GAPDH (0411, Santa Cruz, 1:1000), anti-TAZ (Cell Signaling Technology, CST83699, 1:1000), and anti-DUB1 (Sigma, HPA12082, 1:1000). Protein signals were detected with an ECL kit (Millipore Co., Billerica, Massachusetts, USA).
Luciferase reporter assays
For TEAD luciferase activity assays, MGC803 and AGS cells expressing siDUB1 or siControl were transfected with the TEAD luciferase reporter vector for 24 h. Cells were then harvested for assays. Luciferase reporter assays were performed using a dual luciferase assay kit (Promega). The pRL-null vector expressing Renilla luciferase (Promega) was used as the internal control for normalization of the transfection efficiency.
Wound healing and Transwell assays
For the wound healing assay, MGC803 and AGS cells expressing siDUB1 or siControl were seeded in a 6-well plate until confluent, and a wound was then made by scratching with a sterile tip. Images of the cells were acquired at the indicated time points after wounding. The distance between the two edges of the scratch wound was measured using ImageJ software. The Transwell system (8 μm pore size, Corning) was employed for cell migration and invasion assays. For invasion assays, the membranes in the upper chambers were coated with Matrigel (BD Biocoat, USA). After 24 h, the gastric cancer cells that had migrated to the bottom surface of the insert membrane were fixed, stained with crystal violet and counted under a 20 × objective. The experiments were performed in triplicate.
Cycloheximide assay
MGC803 cells were transfected with siDUB1 or siControl for 24 h. After that, cycloheximide was added to the culture medium at a final concentration of 100 μmol/L. Cell lysates were collected at 0, 3, 6 and 9 h after cycloheximide treatment. HEK293 cells were transfected with 2 µg of the Flag-DUB1 or Flag vector. After 24 h, the cells were treated with cycloheximide at a final concentration of 100 μmol/L. Cell lysates were collected at 0, 3, 6 and 9 h after cycloheximide treatment.
Immunofluorescence (IF) staining
MGC803 cells on coverslips were fixed with 4% paraformaldehyde and incubated with primary antibodies against DUB1 (Sigma, A300-940A) and TAZ (CST, 71,192) at 4 °C overnight. After that, the cells were washed with PBS. Then, the cells were incubated with a fluorophore-conjugated secondary antibody (Invitrogen, Carlsbad, CA). Finally, the cells were counterstained with DAPI (Life Technology). Images of stained cells were acquired with a confocal laser scanning microscope (Leica TCS SP8 STED). The -integrated fluorescence density was measured by ImageJ software.
Clone formation assays
MGC803 and AGS cells were seeded in six-well plates overnight and treated with 50 nM DUB1 siRNA or 50 nM siControl. After twenty-four hours, the gastric cancer cells were washed with PBS, trypsinized and plated at a low density (5000 cells/well in a six-well plate). The cells were cultured for 10 days, and the medium was refreshed every two or three days. Colonies were stained with crystal violet. The colonies in a given area were counted for each condition.
Co-IP assay
The coimmunoprecipitation assay was performed as previously described. Total lysates of MGC803 cells were collected and precleared with rabbit IgG for 2 h and were subsequently immunoprecipitated with an anti-DUB1 antibody (Sigma, A300-940A) overnight, with rabbit IgG (Santa Cruz) as the negative control. Bound proteins were analyzed by western blotting with an anti-TAZ antibody (CST, 71,192). For the overexpression experiment, HEK293 cells were transfected with 5 µg of Flag-DUB1 (full-length or domain deletion mutants) and Myc-TAZ (full-length or domain deletion mutants) in 10 cm dishes. Cell lysates were precleared with IgG and subsequently incubated with an anti-Flag-M2 antibody (A8592, Sigma, 1:1000), with mouse IgG as the negative control. Bound proteins were analyzed by western blotting.
Pull-down assay
TAZ protein was purchased from NOVUS Biological Company (Cat: NBP2-22,949). GST protein and GST-fusion DUB1 truncate domains expression plasmids were expressed in E coli and purified by GST agarose beads. The mixture of TAZ protein and GST-fusion DUB1 domains were incubated at 4 degree for 30 min. The resin was washes with PBS containing 30 mM imidazole. Then the mixture was washed with PBS containing 0.01% Triton 100. The bound proteins were eluted and subject to western blot analysis.
In vitro ubiquitination assays
For in vitro ubiquitination assays, cells were separately transfected with vectors, including the Myc-TAZ, Flag-DUB1 and HA-Ub expression vectors, for 24 h. Cells were then treated with MG132 (10 μM) for 6 h, and the level of Myc-TAZ ubiquitination was determined by IP with an anti-HA antibody (2,013,819,001, Roche, 1:1000) followed by western blot analysis with an anti-Myc antibody (9E10, Santa Cruz, 1:1000).
In vivo tumorigenesis assay
For the in vivo tumorigenesis assay, MGC803 cells were infected with shControl or shDUB1 lentivirus. After 48 h of infection, the cells were treated with 1 µg/ml puromycin for 3 days. MGC803 cells (2 × 106) were injected into the right dorsal flanks of 4-week-old female BALB/c nude mice. Tumor formation in the nude mice was monitored over a 4-week period. The tumor volume was calculated with the following equation: tumor volume = 0.5 × length × width2. The mice were sacrificed five weeks after injection. After the mice were sacrificed, the tumors were weighed and photographed. The experiments were performed under the protocols approved by the ethics committee of Xinxiang Medical University.
Cell proliferation assay
MGC803 and AGS cells were transfected with siDUB1 or siControl in 24-well plates. Twenty-four hours after transfection, the cells were counted, and 4000 cells were seeded into 96-well plates. Relative cell viability was measured at the indicated time points. Cell numbers were determined using CCK8 cell proliferation reagent and measuring the absorbance at 450 nm. Cell proliferation was further analyzed by EdU incorporation and flow cytometry assays. The numbers of gastric cancer cells were determined by using a 5-ethynyl-20-deoxyuridine (EdU) assay kit (RiboBio, Guangzhou, China). For quantitative analysis of images, each data point was considered to represent the fluorescence-positive area calculated from a minimum of five randomly selected fields from three individual experiments. EdU incorporation and FACS assays were performed according to the manufacturer’s instructions. The experiments were performed in triplicate. For cell cycle analysis, MGC823 cells were transfected with 50 nM siDUB1 or siControl. After 24 h, the cells were fixed with 70% ethanol and stained with propidium iodide. Twenty-four hours post transfection, the cells were stained with propidium iodide and annexin V. A BD LSR FACS instrument was used to measure the fluorescence intensity.
Tissue microarray (TMA) and immunohistochemical (IHC) analyses
One hundred paraffin-embedded gastric cancer samples were acquired from Shanghai Outdo Biotech Company (http://www.superchip.com.cn). All gastric tumor samples were examined by pathology specialists. The pathological grade and lymph node metastasis status of each sample were obtained from Shanghai Outdo Biotech Company. The usage of the samples was approved by Shanghai Outdo Biotech Company with written informed consent from all patients. Specific antibodies against TAZ (CST, 71,192) and DUB1 (Sigma, HPA12082) were used to detect the staining density in human samples. Scores were calculated based on the staining intensity and percentage of positive tumor cells in the whole tissues, which were evaluated according to the Fromowitz standard. The staining intensity was graded as follows: no staining, 0; weak positive staining, 1; moderate positive staining, 2 and strong positive staining, 3. The percentage of positive cells was divided into four categories: 0–25% staining, 1; 26–50% staining, 2; 51–75% staining, 3; and 76–100% staining, 4. Staining scores of 1–2 were regarded as indicating low expression, while staining scores of 3–4 were regarded as indicating high expression. All staining was assessed at 200X magnification, and at least three fields from each core were counted.
RNA sequencing and data analysis
Global gene expression analysis (siControl and siDUB1) was performed with the RNA sequencing platform from BGI (Beijing Genomics Institute). The RNA sequencing data have been deposited in the Gene Expression Omnibus (GEO) database (accession number: GSE143947). Differentially expressed genes (P < 0.01 and fold change > 2) were subjected to Ingenuity Pathway Analysis (IPA). For gene set enrichment analysis of RNA-seq data, the conserved Hippo signature gene sets were used and downloaded from Molecular Signatures Database v7.4. GSEA was implemented using GSEA 4.1.0 software with default parameters. A volcano plot was generated using the ‘ggplot2’ package in R (threshold P < 0.05 and fold change > 1.5).
In vitro deubiquitination assay
The proteins were over-expressed in HEK293 cells and immunoprecipitated with antibodies. Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were mixing with 20X E1 Enzyme, 10X Mg2 + -ATP Solution, 10X Ubiquitin Solution, 1ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 ul reaction buffer. The reaction was carried out at 37 °C for 1 h and products were analyzed by western-blot assays with anti-TAZ antibody.
The precise sites of protein for deubiquitination
HEK293 cells were transfected with indicated WT vectors or mutation vectors for ubiquitination assays. The poly-ubiquitinated TAZ was detected through the analysis of western blotting. After 24 h, cells were then treated with MG132 (10 μM) for 6 h, and the level of Myc-TAZ ubiquitination was determined by IP with an anti-HA antibody (2,013,819,001, Roche, 1:1000) followed by western blot analysis with an anti-Myc antibody (9E10, Santa Cruz, 1:1000).
Analysis of TCGA data and progression-free survival data
Gene expression data for 385 gastric cancer patients in TCGA were downloaded from the website (http://gepia.cancer-pku.cn/index.html). The DUB1 mRNA levels in normal gastric tissue and gastric cancer tissues of different stages were generated with GEPIA online software. The progression-free survival (PFS) data of patients stratified by TAZ and DUB1 expression were generated from the KMPLOT online analysis database (https://kmplot.com). The Affy IDs of TAZ and DUB1 were 202133_at and 227093_at, respectively. The PFS survival data of gastric cancer patients stratified by DUB1 and TAZ expression were generated from the KMPLOT database.
Statistical analysis
No specific statistical tests were used to predetermine the sample size. Statistical analysis was performed using GraphPad Prism 7 software or SPSS version 23.0. Data are expressed as the mean ± s.e.m. values. Differences between two independent groups were evaluated with Student’s t test. The Kaplan − Meier method with the log-rank test was applied for survival analysis. Differences were considered to be statistically significant when P < 0.05 (*P < 0.01; **P < 0.001).