Patients and Human specimens
Paraffin-embedded and fresh human HCC samples were collected from 115 patients undergoing HCC resection at the Jiangxi Province Tumor Hospital of Nanchang University from June 2013 to July 2020. Fresh specimens obtained after resection were frozen in liquid nitrogen and stored at -80°C for further investigation. Informed consent of the patients was obtained and the investigation was permitted by research ethics committee of the Jiangxi Province Tumor Hospital of Nanchang University. All patients were followed up for 5 years.
Cell culture and treatment
HCC cell lines including Huh7, MHCC97H, HepG2, HCCLM3, Hep3B and human normal hepatocyte cell lines HL-7702 were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences and the Shanghai Institute of Cell Biology in China. The identity of the cell lines was confirmed by short tandem repeat analysis. All cell lines were cultured in Dulbecco’s modified Eagle’s Medium (Gibco) containing 10% fetal calf serum (FBS, HyClone, USA) at 37 °C in a humidified incubator containing 5% CO2.
quantitative real-time PCR (qRT-PCR)
Total RNA was extracted by the standard Trizol-based protocol (Invitrogen, USA). Complementary DNA (cDNA) was synthesized using the PrimeScript RT Reagent Kit (Invitrogen, USA) and qRT-PCR was performed using SYBR Premix Ex Taq (TaKaRa Bio, Shiga, Japan), according to the manufacturer's instructions. Information about the gene-specific primers were in Supplementary Table 1.
Western blot was performed as previous study . Extraction of total cellular proteins was extracted by RIPA buffer (Beyotime, Shanghai, China) containing protease and inhibitor mixes (Thermo Fisher Scientific, New York, USA) on ice. BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA) was performed to evaluate protein concentration. Equal amounts of proteins were separated by sodium dodecylsulfonate (SDS) polyacrylamide gel electrophoresis and transferred onto apolyvinylidene flusoride (PVDF) membrane by electroblotting (Millipore, Bedford, MA, USA). Primary antibodies were added and incubated throughout a night at 4°C. Primary antibodies including anti-OTUD3 monoclonal antibody (1:500; HPA028543, Sigma), anti-ACTN4 monoclonal antibody (1:1000, 15145, CST), anti-p65 monoclonal antibody (1:1000, 8242, CST), anti-p-p65 monoclonal antibody (Ser536)(1:1000, 3033, CST), anti-IκBα monoclonal antibody (1:1000, 4812, CST), anti-p-IκBα monoclonal antibody (Ser32) (1:1000, 2859, CST) and anti-Tubulin monoclonal antibody (code ab7291, 1:2000 dilution, Abcam). After being incubated with the second antibody (CST, MA, USA) for 1h at room temperature, the intensity of protein bands was analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA).
Immunohistochemistry (IHC) staining
Paraffin-embedded sections (4 mm thick) of human HCC tissues and normal adjacent tissues were deparaffinized. Sections were subjected to antigen retrieval in microwave-heated citrate buffer (pH6.0) for 30 mins. After incubation for 30 mins in goat serum (Solarbio, Beijing, China), tissued sections were incubated by primary antibodies overnight at 4℃. Next, HRP-conjugated secondary antibody (Boster) was used to incubate sections for 2h at room temperature. DAB Detection Kit (Maxim) was adopted for immunostaining for 2 mins. The proportion of positive areas were scored semi-quantitatively by 3 pathologists who were blind to the clinical parameters. In brief, 100 cells were counted randomly at 200X microscopic fields and were classified into five groups according to the percentage of positive staining cells in HCC tissues as follows: 0 = negative; 1 – 3 = 1 – 25%; 4 – 6 = 26 – 50%; 7 – 9 = 51 – 75%; 10 – 12 = ≥76%. The score ranging from 0 to 6 was considered as a low-expression group, whereas the score ranging from 7 to 12 was considered as a high-expression group.
Stable cell lines and plasmids
HCC cell lines with stable OTUD3 overexpression (OE) or knockdown were established by transfection of lentivirus containing OTUD3 overexpression plasmid (GV640 vector, Genechem, Shanghai, China) or short hairpin RNA (shRNA) (pGLVH1 vector, GenePharma, Shanghai, China). Cells infected by lentivirus were selected using puromycin (Invitrogen, Carlsbad, CA, USA) for one month. Transient OTUD3 overexpression or knockdown was performed by transfections of OTUD3 OE or knockdown plasmids using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's recommended protocol .
Paraffin-embedded sections of xenografted tumour tissues were deparaffinized, followed by antigen retrieval in microwave-heated EDTA buffer (pH8.0) for 30 mins. After incubation for 15 mins in 0.1% TritonX-100 (Solarbio, Beijing, China) and 1% goat serum (Solarbio, Beijing, China) for 30 mins, tissued sections were incubated in dark by primary antibodies overnight at 4℃, followed by secondary incubation for 1h with Alexa Fluor 594 goat anti-rabbit IgG (1:500, Life Technologies). Nuclear were stained with Hoechst 33342 (Life Technologies) for 1 min. Tissues were visualized using a confocal laser scanning microscope (SP-II; Leica Microsystems, Wetzlar, Germany).
Cells were seeded in 96-well plates at an initial concentration of 5x104 cells per well. After culturing for 24 hours, 5-ethynyl-20-deoxyuridine (EdU; Ribobio) was used to culture cells for 2 hours, followed by three washes with PBS. After cells being incubated with 1xApollo reaction cocktail for 30 minutes, Hoechst 33342 (5 mg/mL) was used to stain the DNA contents of the cells in each well for 25 minutes and was visualized through a confocal laser scanning microscope (SP-II; Leica Microsystems, Wetzlar, Germany).
Cell Counting Kit-8 assay
CCK8 assay was used to evaluate cell viability after 24, 48, 72, 96, 120h. Transfected cells were seeded into 96-well plates at an initial concentration of 3x103 cells per well. 10μl of CCK-8 solutions (Dojindo Laboratories, Kumamoto, Japan) was added to each well according to the manufacturer’s instructions. After incubation in cell incubator for 1h, the absorbance at wavelength of 450 nm was recorded.
The wound-healing assay
Transfected cells were incubated into 6-well plates until growing to 80% to 90% confluence. Then 200μl pipette tip was used to scratch across the cells surface followed by three washed with PBS. Subsequently, the cells were incubated at 37 °C and the wound range was imaged by phase-contrast photography at 0h, 24h and 48h. Three randomly selected wound areas were analyzed.
In vitro migration and invasion assays
Cell migration assay and invasion assays were performed using a transwell system (Corning, NY, USA) with or without Matrigel matrix (BD bioscience) coated above the membrane. Stably transfected cells were suspended in pure DMEM at a concentration of 1x105 /ml. 500μl cell suspension was added in the upper chamber. Fresh medium containing 10% FBS was added in the lower chamber as a chemoattractant. After incubation for 48h, the non-migrated cells on the upper surface of the membrane were removed, and the cells on the lower surface were fixed with methanol and stained by 0.1% crystal violet. The cells in five random microscopic fields were counted and imaged using a light microscope with a DP70 CCD system (Olympus Corp).
Cell lysis was incubated with 50µl protein A+G Agarose (Thermo Scientific) and 1 µg of the indicated antibody overnight at 4°C. The protein A/G-agarose were collected by centrifugation. Loading buffer was added to the tube and heated for 15 mins at 100°C. Then the immunoprecipitated proteins were examined by SDS-PAGE and immunoblotting analysis. The intensity of protein bands was analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA).
Xenografts mouse model
HCCLM3 cells (2 x106 cells per mouse) expressing a luciferase reporter stably transfected with LV-shNC or LV-shOTUD3 lentivirus were subcutaneously injected into the flanks of 8-week-old female BALB/c-nude mice (n=6 per group). The in vivo imaging system (IVIS, PerkinElmer, USA) was employed to monitor and image the growth of tumours regularly. Tumours were measured by caliper every five days to examine tumour volume using the formula: V = [length/2] × [width2]. Finally, all of the tumour xenografts were harvested and weighted at the 30th day. All animals were randomly divided into different groups by a technician under blinding condition. Animal experiments were approved by the Ethics Committee for Animal Experiments of the Second Affiliated Hospital of Nanchang University.
HCCLM3 cells (2 x106 cells per mouse) expressing a luciferase reporter stably transfected with LV-shNC or LV-shOTUD3 lentivirus were injected into the tail vein of 8-week-old female BALB/c-nude mice (n=6 per group). IVIS was employed to monitor and photograph the tumour progression in mice. Organs of mice were harvested after 5 weeks and metastatic nodes in lung sections were evaluated after HE staining.
All animals were randomly divided into different groups by a technician under blinding condition. Animal experiments were approved by the Ethics Committee for Animal Experiments of the Second Affiliated Hospital of Nanchang University.
All results are shown as mean ± SD from at least three independent experiments. Log-rank test was employed to analyze survival of patients. Student’s t-test was used in statistical analyses between two groups. One-way ANOVA was employed for multiple comparisons. GraphPad Prism (version 5) was used in all statistical analyses and P< 0.05 was considered significant.