OTUB2 Regulates YAP1/TAZ to Promotes the Progression of Esophageal Squamous Cell Carcinoma

Patients and Tissue Specimens

Between January 2008 and December 2018, a total of 183 paired tissue samples including ESCC tissues and corresponding adjacent normal tissues were obtained from newly diagnosed patients who received no chemotherapy or radiotherapy, at the Department of Pathology, of the First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, PRChina. The study protocol was performed under the approval of the Ethics Committee of The First Affiliated Hospital of Xinjiang Medical University. Inclusion criteria were confirmed ESCC diagnosis and no previous chemotherapy or radiotherapy, and availability of complete clinicopathological. Exclusion criteria included other cases presenting tumor metastasis and cases in which the results of immunohistochemical staining were inconclusive due to detachment were excluded. Patient prognostic related factors were analyzed using telephone follow-up to record patient survival, and OS (overall survival, OS) was defined as the time from first diagnosis of ESCC to death or final follow-up until December 31,2019.

Bioinformatics Analysis

TIMER database (http://timer.cistrome.org) and the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/) were used to search the expression of OTUB2 in esophageal cancer; the GeneMANIA database (http://genemania.org/) was used to search the interaction between OTUB2 and YAP1/TAZ (WWTR1:TAZ also known as WWTR1).

Immunohistochemistry

Anti-OTUB2 (bs6236R) purchased from Bioss Reagents Co., Ltd.; anti-YAP1 (ab52771), anti-TAZ (ab242313) were purchased from the abcam Company (UK). and Anti-CTGF (23936–1-AP) were purchased from the ProteinTech, Antibody dilution concentrations were 1:50. PBS, citric acid antigen repair solution, goat serum, hydrogen peroxide, generic type II resistance were all purchased from Bioss Reagent Co., Ltd. The specific experimental protocols were as follows: after processing, which included dewaxing, antigen retrieval, and H2O2 treatment, the paraffin-embedded tissue slides were incubated with OTUB2, YAP1 TAZ and CTGF antibody overnight at 4 °C. Then, the slides were washed with phosphate buffered saline (PBS) and incubated with a biocatalytic secondary antibody. Finally, the tissue sections were treated with DAB and were then counterstained with hematoxylin. The immunoreactions were evaluated independently by two pathologists. The score was calculated based on the staining intensity and the percentage of positive cells, the staining intensity was divided into non-staining (colorless), weak-staining (light yellow), moderate-staining (light brown), and strong-staining (dark brown), and the scores were 0, 1, 2, and 3, respectively. The scoring of positive cells was as follows: 0 for 0, 1 for 0–30%, 2 for 30–60%, and 3 for 60–90%. According to the staining index SI, the staining results were scored as negative 0–3 points and positive 4–9 points, of which 4–5 points indicated low expression, 6–7 points indicated moderate expression, and 8–9 points indicated high expression. SI = percentage of positive cells × dye intensity.

Cell Culture and Transfection

Human ESCC cell lines KYSE30, KYSE150, and KYSE450, and the human esophageal epithelial cell line SHEE, were purchased from the Shanghai Cell Bank (Shanghai, China). All cell lines were grown in DMEM (Gibco), supplemented with 10% fetal bovine serum (BI), and incubated at 37 °C in a humidified incubator containing 5% CO2. Specific small interfering RNA (siRNA) for OTUB2, and control siRNA were synthesized by Shanghai Genechem Co., Ltd. We first used western blotting to detect OTUB2 expression in three ESCC lines, the cell lines with the highest expression of OTUB2 were selected for lentivirus transfection to knockdown OTUB2, and the sequences with the highest knockdown efficiency were selected for subsequent molecular experiments and cell function studies.

Target sequence of shRNA:

Name Sequences shNC 5′-TTCTCCGAACGTGTCACGT-3’ shOTUB2#1 5′-TCCCACTACAACATCCTTT-3’ shOTUB2#2 5′-AGATGGATACCGCCCTGAA-3’ shOTUB2#3 5′-TGGTGGAACTGGTAGAGAA-3’ Western Blotting

Briefly, the ESCC cell line was digested with trypsin and washed with PBS three times. The cell lysate was added to ice at 4 °C for 20 min, then centrifugation and collected the supernatant. BCA protein quantification was used. Next, the protein samples were separated by 10% SDS PAGE (40 μg/lane), and electrophoretically transferred to polyvinylidene difluoride membranes (PVDF). Subsequently, 5% skim milk was used to seal the membrane for 2 h, and the corresponding primary antibody was incubated overnight at 4 °C. TBST was used to wash membranes three times, and the corresponding secondary antibody was added and incubated at room temperature for 1 h. Protein expression was detected by ECL chemiluminescence.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Total RNA of ESCC cells was extracted using TRIzol reagent according to the manufacturer’s instructions. Subsequently, 1 μg RNA was used to synthesize cDNA in accordance with the manufacturer’s instructions. The reaction was terminated at 85 °C for 5 s and then at 37 °C for 30 min. RT qPCR reaction was carried out according to the “two-step” procedure. (TRIzol Reagent, PrimeScriptTM one stEP RT-PCR kit, purchased by Invitrogen; QuantiNova SYBR Green PCR Kit from QIAGEN, Germany). The primer sequences used are listed as follows:

Primer Sequences GAPDH Forward: 5′-GAAGGTGAAGGTCGGAGTC − 3′ Reverse: 5′-GAAGATGGTGATGGGATTTC −3′ OTUB2 Forward: 5′-AACATCCTTTATGCAGCCGAT-3′ Reverse: 5′-GCTGCACATTTTCTTGCTACAGT-3′ YAP1 Forward: 5′-CTCACAGCAGAACCGTTTCCC-3′ Reverse: 5′-AGCCAAAACAGACTCCATG-3′ TAZ Forward: 5′-TCCCAGCCAAATCTCGTGATG-3′ Reverse: 5′-AGCGCATTGGGCATACTCAT-3′ CTGF Forward: 5′-TGTGCACCGCCAAAGATGGT-3′ Reverse: 5′-TCTTCCAGTCGGTAAGCCGC-3′ Immunofluorescence

ESCC and SHEE cells were seeded in a confocal laser dish for 24 h and then fixed at 4 °C for 30 minutes with 4% tissue cell fixative. After washes with 0.5% Triton X-100/PBST, cells were blocked with 5% BSA for 30 min at room temperature and incubated with OTUB2 antibody (1:300) at 4 °C overnight. Slides were then incubated with fluorescent secondary antibody (1:500) for 2 h, and then incubated with DAPI at room temperature for 10 minutes. Finally, images were taken under an inverted fluorescence microscope.

CCK-8 Assay

Cell growth was monitored using a CCK-8 kit (Boiss Reagents Co., Ltd). When the cells grew to logarithmic phase, the cells were digested with trypsin to prepare cell suspensions and counted. They were divided into the KYSE150 group, KYSE150 sh-non group (shNC), and KYSE150 shOTUB2 group (shOTUB2#1). Briefly, cells were plated at 5000 cells/well in 96-well plates and cultured for 72 h. After every 24 h, the 10 μL CCK-8 reagent was added and the optical density value at 450 nm for each well was obtained using a microplate reader. The CCK-8 assay was performed for three times in triplicate.

Plate Clone Formation Assay

ESCC cells were seeded in 6-well plates at the density of 1000 cells/well. After continuous culture for 7 days, the cell clones were fixed with 4% tissue cell fixative and incubated with crystal violet. Clones containing > 50 cells were counted under an inverse microscope. The plate clone formation assay was performed three times in triplicate.

Flow Cytometry

ESCC cells (5 × 105/well) were seeded into 6-well plates and cultured for 48 h at 37 °C. After the ESCC cells were collected, cells were stained with an Annexin V-FITC apoptosis detection kit (BD Biosciences, USA) and flow cytometry was used for apoptosis detection.

Wound Healing Assay

After transfection for 48 h, the ESCC cells were seeded into a six-well plate at a density of 1.0 × 106 cells/mL until the confluence reached 90%. A 20-μL pipette tip was used to draw a straight-line wound. The cell debris then washed with PBS and they were cultured continuously in medium. The wound areas and migrated cells in each group were assessed after 24 h and 48 h.

Transwell Assay

Transwell assays were performed following the manufacturer’s instructions to analyze the invasive ability of ESCC cells. A Transwell plate with 8-mm pore size chambers precoated with Matrigel (Corning Incorporated, Corning, NY, USA) were used. In brief, the number of cells per well in the Transwell chamber was 1 × 105 and 500 μL complete culture medium containing 10% FBS was added to each lower chamber of the Transwell dish. Cell suspension was added to the upper chamber and placed in the cell incubator for 24 h. Next, the medium was removed and the cells were fixed with methanol and stained with 500 μL of 0.1% crystal violet solution and allowed to stain cells at room temperature for 10 min. The cells which failed to migrate or invade the Matrigel were removed with a swab, then the Migrated and invasive cells were pictured and their numbers calculated from five randomly selected microscopic fields.

Statistical Analysis

All experiments were performed in triplicate. Statistical analysis was conducted using SPSS version 22.0 software and GraphPad prism version 8. For comparisons between two groups, a Student’s t test or chi-squared test was used. P < 0.05 were considered to be statistically significant.

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