Tissue samples
In RT-PCR method, 25 pairs of tumor and adjacent non-tumor tissues in PC patients were acquired from Fudan University Shanghai Cancer Center. In IHC analysis, human PC tissue microarrays (n = 67) and corresponding control healthy (n = 25) were prepared by Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). According to IHC results, USP51 expression in PC tissue were divided into USP51 high (n = 37) and USP51 low (n = 30) group, then the overall survival rate for PC patients were assessed.
Immunohistochemistry (IHC) assay
Expression of USP51 in human PC tissue microarrays, as well as expression of Ki67 in tumor of a Xenograft model was assessed, using IHC method. Paraffinembedded samples (cut into 4 mm thick) from human pancreatic or mice tumor tissue were routinely dewaxing, rehydration, and then blocked using 3% H2O2. IHC was conducted using DAB substrate (Long Island Biotech) and hematoxylin (714094, BASO) following manufacturer's instructions. The primary antibody for the IHC was rabbit Ki67 polyclonal antibody (ab16667, abcam) and rabbit USP51 polyclonal antibody (NBP1-86167, NOVUS).
Cell culture and treatment
Five human PC cell lines (ASPC1, BXPC3, MiaPaca2, PANC1 and SW1990) and one human normal pancreatic cell line (HPC-Y5) were acquired from ATCC (Manassas, VA, USA). MiaPaca2, SW1990 and ASPC1 cells were allowed a monolayer culture at 37 oC under 5 % CO2 in a medium containing 90 % (v/v) of DMEM (Hyclone, Logan, UT), 100 U/ml of penicillin (Solarbio, Beijing, China) and 10 % (v/v) of heat-inactivated fetal bovine serum (FBS) (Gibco Laboratories).
To confirm that USP51 acted through β-catenin to exert carcinogenesis in PC in vitro, ASPC1 cells transfected with USP51 overexpression vector (oeUSP51) were subjected to 10 μmol/l of XAV939 (S1180, selleck).
Cell Counting Kit -8 (CCK-8) assay
After treatment, proliferation of MiaPaca2, SW1990 and ASPC1 was assessed using Cell Proliferation and Cytotoxicity Assay Kit (CP002, SAB, Beijing, China), according to a Manufacturer's instruction.
Cell cycle detection
After treatment, MiaPaca2, SW1990 and ASPC1 were dyed at 25 oC in darkness using 50 μg/ml of propidium iodide solution (C001-200, 7sea Biotech, Shanghai, China) for 10 min. Red fluorescence (488 nm) was recorded on the FL1 channel of flow cytometry (Accuri C6, BD Biosciences, San Jose, CA, USA). Cell cycle of MiaPaca2, SW1990 and ASPC1 cells were assessed using FLOWJO software (Version 6 TreeStar).
Lentivirus
The primers of human USP51 gene (NM_201286.3) were: 5’-CGGAATTCATGGCCCAGGTTCGAGAAAC-3’ (forward) and 5’-CGGGATCCAAGAAGTTTCTCGAACCTGGGC-3’ (reverse), which were cloned into pLVX-Puro vector (Clontech). pLVX-Puro-USP51 was packaged into lentiviruses in 293T cells according to a provided instructions, and the lentiviruses expressing USP51 were transfected into ASPC1 cells using Invitrogen Lipofectamine® 2000 Reagent following the a manufacturer's proposal. Meanwhile, lentiviruses without USP51 coding sequence were considered as a control Vector.
Three designed shRNA targeting human USP51 mRNA (NM_AB449916.1) were used for siRNA-mediate USP51 depletion (siRNA-USP51), which were cloned into PLKO.1 vector (purchased from Suzhou Synbio Technologies (Suzhou, China)). pLKO.1-shUSP51 was packaged into lentiviruses in 293T cells, and then transfected into ASPC1 cells as mentioned above. Meanwhile, lentiviruses expressing non-specific siRNA were used as a negative control (siNC).
Real-time (RT)-PCR
Total RNA from pancreatic tissue, PC cell lines (ASPC1, BXPC3, MiaPaca2, PANC1 and SW1990), or human normal pancreatic HPC-Y5 cells was extracted by Trizol Reagent (1596-026, Invitrogen) and reverse transcribed using cDNA synthesis kit (Fermentas). The primer targeted USP51 (NCBI NM_201286.3) were: 5’-CCTCAGACACGGAGAAGC-3’ (Forward, F) and 5’-GGACCCTGACCAAACTCG-3’ (Reverse, R); Pos: 478-575; Size: 98 bps; The primer targeted β-catenin (NM_001904.3) were: 5’-CGACACCAAGAAGCAGAGATG-3’ (F) and 5’- GGGACAAAGGGCAAGATTTCG-3’ (R); Pos: 1688-1831; Size: 144 bps; The primer targeted cyclin D1 (NM_053056.2) were: 5’-GCGAGGAACAGAAGTGCG-3’ (F) and 5’-TGGAGTTGTCGGTGTAGATGC-3’ (R); Pos: 203-394; Size: 192 bps; Primers targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NCBI NM_001256799.2) were: 5’-AATCCCATCACCATCTTC-3’ (F) and 5’-AGGCTGTTGTCATACTTC-3’ (R), Pos: 436-653; Size: 218 bps. mRNA levels of USP51, β-catenin and cyclin D1 were quantified through SYBR Green PCR Kit (Thermo Fisher) on an ABI7300 system (Applied Biosystem), and normalized by GAPDH using the 2-△△Ct method [7].
Western blot analysis
25 μg of total protein in lysis of pancreatic tissue (20 mg) or PC cells sample, determined by BCA protein assay kit (PICPI23223, Thermo, MA, USA), was separated on 10 % SDS-PAGE. Electrophoretically pure including USP51, β-catenin, cyclin D1 and GAPDH were sent onto PVDF membranes (Millipore, USA) and incubated with anti-USP51 antibody (PA5-35274, Invitrogen), anti-β-catenin antibody (#8480, CST), antibody against cyclin D1 (#2922, CST) and antibody against GAPDH (#5174, CST) at 4 oC overnight followed by a second antibody (A0208, Beyotime, Shanghai, China) for 1 hour at 25 oC. Expression of USP51, β-catenin and Cyclin D1 was normalized by GAPDH and quantified using ECL system (GE Healthcare/Amersham Biosciences).
Co-Immunoprecipitation (Co-IP) and ubiquitination assay
The association between USP51 and β-catenin in MiaPaca2 cells was assessed using Co-IP. Immune complexes of total protein (100 μg) in MiaPaca2 lysis supernatant was obtained using Protein A/G PLUS-Agarose (sc-2003, Santa Cruz). Antibodies used in IP were: IgG (sc-2027, Santa Cruz Biotechnology), anti-USP51 antibody (Orb181545, biorbyt) and antibody against β-catenin (ab227499, Abcam). Antibodies used in Western-blot were anti-USP51 antibody (PA5-35274, Invitrogen) and anti-β-catenin antibody (#8480, CST). Same amount of total protein was reserved for input control.
To study the effect of USP51 on the levels of β-catenin ubiquitination (Ub-β-catenin), Ub-β-catenin, precipitated in the immune-complexes, was determined using Anti-ubiquitin antibody (ab7780), according the western blot.
Xenograft model
Nude mice with 4-6-week-old were provided by Shanghai Laboratory Animal Company. MiaPaca2 cells transfected with siNC/siUSP51 (7 × 106 cells, 100 μl) were subcutaneously injected into nude mice (n = 6 in each group), and then fed separately. Tumor formation in siNC and siUSP51 groups was monitored from 12 th day to 33 th day. On 33 th day, all mice were killed. Tumor weight (g) was recorded. Histological changes in pancreatic tumor tissue were recorded using Hematoxylin-eosin (H&E) staining. IHC was conducted to evaluate the expression of Ki67, using antibody against Rb Ki67 (ab16667, abcam).
2.8 Statistics and data analysis
Data was showed as mean ± standard error of the mean (SEM), calculated from at least 3 parallels. Comparison between two groups was evaluated using Student's test with P < 0.05 being statistical significance. Overall survival of PC patients was analyzed by Kaplan-Meier method using medcalc sofeware.