Reagents
Please see Extended Methods.
Clinical Specimens
10 pairs of HCC samples were obtained from the first affiliated hospital of Dalian Medical University (Dalian, China). All samples were collected with the informed consent of the patients and the experiments were approved by Research ethics committee at the first affiliated hospital of Dalian Medical University. Human HCC Tissue Microarrays were obtained from Shanghai Outdo Biotech Company or Shanxi ChaoYing Biotechnology Company.
Cell Culture Experiments
Human HCC cell lines MHCC-97H, HUH7, BEL-7402, Hep3B, HepG2 and SMMC-7721 were purchased from the cell bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (CTCC, Shanghai, China). THLE-2 and SNU-449 were obtained from ATCC. MHCC-97H, SNU-449 and MHCC-97L cell lines were maintained in RPMI 1640 medium (GIBCO, USA). 293T, HUH7, BEL-7402, Hep3B, HepG2 and SMMC-7721cells were cultured in DMEM (GIBCO, USA). Cell lines were maintained in culture supplemented with 10% FBS (GIBCO, USA) and 1% penicillin/streptomycin (Thermo) at 37℃ with 5% CO2 in a humidified incubator (Thermo).
Plasmids
shRNA
Lentiviral shRNAs were cloned in pLKO.1 within the AgeI/EcoRI sites at the 3’end of the human U6 promoter. The targeted sequences were shown in the table above.
All the expression vectors used in this study (including SFB-PPARγ, Myc-USP22, pLoc-USP22 and Lenti-PPARγ) were constructed using Gateway Technology (Invitrogen). Briefly, cDNAs with attB homologous sequence were generated by PCR and then subcloned into pDONR221 vector as the entry clones. Subsequently, the entry clones were recombined into gateway destination vectors with various tags (SFB and Myc) or lentiviral vectors (pLoc and Lenti). For Myc-USP22C185S vector, mutation in the USP22 cDNA was generated by overlap extension PCR and then cloned into Myc vector as above.
Generation of cell lines expressing shRNAs, Ploc-USP22 or Lenti-PPARγ.
HEK293T cells were used to package virus. 2-3*107 cells were plated in 10 cm culture dish and the plasmid was co-transfected with psPAX2 and pVSVG by Lipofectamine 2000 according to the manufacturer’s protocol. Conditioned medium containing recombinant lentiviruses was collected 48 hours after transfection and filtered through non-pyrogenic filters with a pore size of 0.45 μm (Merck Millipore, Billerica, MA, USA). Samples of these supernatants were applied immediately to target cells together with Polybrene (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 8 mg/ml, and supernatants were incubated with cells for 12 hr. After infection, cells were placed in fresh growth medium and cultured as usual. Selection with 2μg/ml puromycin (InvivoGen) or 8μg/ml Blasticidin (InvivoGen) was initiated 48 h after infection.
Cell proliferation and cloning formation assay
For the clone assay, cells were seeded at a density of 500 cells/well in 12-well plates in complete RIPM 1640 medium. And the medium was changed every three days. After 14 days, cells were fixed and stained with crystal violet.
Click-It EdU Alexa Fluor Imaging Kit was used for the EdU Cell Proliferation Assay. All reactions were performed using this kit in accordance with the manufacturer’s instructions.
Immunohistochemistry
The samples were then fixed with 4% PFA, and embedded with paraffin. Standard IHC staining procedures were performed according to the instructions of IHC Kit. USP22 (1:100), ACC (1:750), ACLY (1:750) and PPARγ (1:50) were used as the primary antibodies. EDTA and Citrate solution were used for antigen retrieval depend on antibody instruction. H-score was used to assess the staining intensity.
Xenograft Tumor Model
Pathogen-free male athymic nude mice (4-5 weeks old, 18‑22 g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All the mice were housed in specific pathogen‑free (SPF) environments at the Institute of Genome Engineered Animal Models for Human Disease of Dalian Medical University. During the tumor formation assay, BEL-7402 (5 x 106) MHCC97L (5 x 106) MHCC97H cells (1 x 106) were injected into the flank of the mice. The tumor volumes were measured using a caliper every 3 days. The mice were sacrificed after four weeks, and tumor volumes were then measured.
Immunoprecipitation and S-Protein pull down assay
Immunoprecipitation and SFB pull-down experiment was performed as described previously. Cells were lysed in E1A lysis buffer (250 mM NaCl, 50 mM HEPES [pH 7.5], 0.1% NP-40, 5 mM EDTA, protease inhibitor cocktail [Sigma]). The antibodies to USP22 and PPARγ were used for immunoprecipitation. The SFB pull-down experiment was done as described previously. Briefly, 293T cells were transfected with SFB-tagged protein and lysed in NETN buffer (200 mM Tris-HCl [pH 8.0], 100 mM NaCl, 0.05% NP-40, 1 mM EDTA, protease inhibitor cocktail [Sigma]) for 20 min at 4°C. Crude lysates were subjected to centrifugation at 14,000 rpm for 15 min at 4°C. Supernatants were incubated with S-Protein Agarose for 4 h (Millipore, USA). The agaroses were washed three times with NETN buffer. Proteins were eluted by boiling in 1× SDS running buffer and subjected to SDS-PAGE for immunoblotting.
In vivo deubiquitination assay
HA-UB and SFB-PPARγ were co-transfected with MYC-USP22 into HEK-293T cells, after 36h cells were treated with 10 μM MG132 for 6 h. SFB-PPARγ was purified with S-Protein Agarose and western blotted with antibodies against HA, MYC and FLAG.
Immunofluorescence
Cells were cultured and fixed on the 8-chamber slide. Cells were permeabilized by 0.1% Triton X-100 and blocked by 10% goat serum before incubation with primary antibodies. Secondary antibodies (goat anti-mouse Alexa 488 and goat anti-rabbit Alexa 555) were obtained from Invitrogen. Cell nucleus were counterstained with DAPI. Images were acquired under a point-scanning confocal microscope on an upright stand.
RNA isolation and quantitative real-time PCR
RNA was isolated from tumor cells using Trizol reagent. Reverse transcription PCR was performed using the Revert Aid First Strand cDNA synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) according to the protocol. Quantitative real-time PCR was performed using StepOnePlus and the DNA double‑strand‑specific reagent SYBR‑Green I for detection (Roche Applied Science, Penzberg, Germany). Fold changes were calculated using the Cq method. The results were normalized to β-Actin levels. The primer sequences were as follows:
USP22
F 5’-AGCAGCGGATTCACCATCTC-3’
R 5’- TGATGTATGCGATCACCAGTGT-3’
PPARγ
F 5’- GATGCCAGCGACTTTGACTC-3’
R 5’- ACCCACGTCATCTTCAGGGA-3’
ACC
F 5’- ATGTCTGGCTTGCACCTAGTA-3’
R 5’- CCCCAAAGCGAGTAACAAATTCT-3’
ACLY
F 5’- TCGGCCAAGGCAATTTCAGAG-3’
R 5’- CGAGCATACTTGAACCGATTCT-3’
FASN
F 5’- AAGGACCTGTCTAGGTTTGATGC-3’
R 5’-TGGCTTCATAGGTGACTTCCA-3’
SCD
F 5’- TCTAGCTCCTATACCACCACCA-3’
R 5’-TCGTCTCCAACTTATCTCCTCC-3’
RNA Sequencing
RNA was extracted from MHCC-97H cells using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing library preparation used the NEBNext Ultra RNA Library Prep Kit for Illumina followed by manufacturer’s instructions (NEB, Ipswich, MA, USA). Sequencing was done on the Illumina HiSeq instrument using a 2x150 Paired End (PE) configuration with 30–40 million reads per sample by GENEWIZ, LLC. (South Plainfield, NJ, USA). Sequencing libraries were constructed from total RNA using SMART-RNAseq Library Prep Kit (Hangzhou KaiTai, AT4201). In briefly, the mRNA were isolated from total RNA with Sera-Mag Magnetic Olido(dT) particles, and then chemically fragmented. The fragmented RNA was reverse-transcribed into cDNA using random primer containing a tagging sequence at their 3’ends. And the cDNA libraries were subsequently amplified using the KAPA high-fidelity DNA polymer. Quality of the libraries was validated by the 2100 Bioanalyzer (Agilent Technologies). Subsequently, High-throughput sequencing was performed using a NovaSeq 6000 (Illumina). After sequences were mapped using hisat2 (version 4.8.2) against the Mus_musculus.GRCm38.dna.primary_assembly.fa, the reads for each library were converted to FPKM (fragments per kilobase of exon per million fragments mapped) by running Cuffdiff 2.1.137 to determine gene expression. Biological pathway analysis was performed using clusterProfiler. RNA sequencing and library construction were performed by technical staff at Hangzhou KaiTai Bio-lab.
Tracing with 13C6-Glucose
Medium was changed to RPMI 1640 containing 13C6-Glucose (2g/L) when the cell density was about 80%, and 24 hours later cell culture plates were washed with PBS and snap-frozen in liquid nitrogen and stored at-80˚C.
Sample preparation for metabolomics analysis
The lyophilized powder was redissolved in 30μl organic solvent (Chloroform/Methanol=2/1) by 30 second vortex, then added 60μl organic solvent (Acetonitrile/Isopropanpl/H2O=65/30/5). After centrifugation, the supernatant can be directed for UPLC (Waters, USA) -Q Exactive HF MS (Thermo Fisher Scientific, USA) analysis.
UPLC-MS based information acquisition
Waters BEH C8 column (100 mm × 2.1 mm, 1.7 μm) was used for separation. The Mobile phases were Acetonitrile/H2O (60:40, v/v) for phase A and Isopropanpl/Acetonitrile (90:10, v/v) for phase B, both containing 10 mM ammonium acetate. The flow rate was 0.26 mL/min. The column temperature was at 55°C. The gradient started with 32% B and maintained for 1.5min, then increased to 85% B within 14 min, arrived to 97% at 15.6 min. After maintaining for 2.4 min, it returned to the initial 32% B. The MS capillary temperature was 320℃ with the auxiliary air heating temperature set at 350℃. The sheath gas and auxiliary gas flow rate were set as 45 and 10 units. Full scan resolution was set as 120K and the negative mode was used, m/z scan range was 70-1100 Dalton and the spray voltage was 3 kV.
Statistical Analysis
Pearson Correlation Coefficient was used to evaluate the relationship between USP22 and PPARγ, ACC, ACLY and FASN protein and mRNA expression levels in human HCC tissues and TCGA database. ANOVA-post-hoc pairwise comparison analysis was used to compare the means of more than two groups. Student's t-test (unpaired, two-tailed) and Wilcoxon-test (paired two-samples) was used to compare the mean value of two groups. The differences in survival were calculated using the Kaplan-Meier test. Survival analysis in mouse model was performed by log-rank test. Data representative of two or more independent experiments. Bars and error represent mean ± standard deviations (SD) of replicate measurements. Statistical significance was defined as *p < 0.05, **p < 0.01. All of the relative protein expression was normalized by ImageJ (version no.: 1.8.0_112; https://imagej.nih.gov/ij/). Statistical analysis was performed using the SPSS 21.0 software package (SPSS, Inc., Chicago, IL, USA).