Clinical samples and immunohistochemistry
Formalin-fixed and paraffin-embedded liver cancer tissues were obtained from patients undergoing surgery in Renmin Hospital, Hubei University of Medicine, ShiYan, China. Six HBsAg positive HCC tissues and six HBsAg negative HCC tissues were selected for analysis. The selected pathological tissues were negative for hepatitis A, C, D, and E viruses and negative for human immunodeficiency virus (HIV). As previously described, immunohistochemical staining and Western blot analysis were performed . The antibody used for staining was anti-GP73 (#ab109628, Abcam). The study was conducted according to the principles of the Helsinki Declaration and approved by the Institutional Review Committee of Hubei Medical University according to its guidelines for protecting human subjects. Each participant received written informed consent.
Xenograft nude mice
Male BALB/cA-nu mice (19.3-23.6g) aged 5 weeks were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). The tumorigenicity assay in nude mice was performed as described previously . Animal conservation and sacrifice are carried out according to the methods approved by the Animal Care and Use Committee of Animal Experimental Center of Hubei Medical University.
Cell culture and transfection
Primary human hepatocytes (PHHs) were purchased from the Research Institute for Liver Diseases (Shanghai, China) and cultured as described previously . HepG2 cells, Huh-7 cells, HepG2.2.15 cells, and HEK293T cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). L02 cells and HepG2-NTCP cells were kindly provided by Dr. Jianguo Wu of Wuhan University, China. HepG2 cells, HepG2-NTCP cells, HepG2.2.15 cells, Huh-7 cells, L02 cells, and HEK293T cells were cultured as previously reported . Lipofectamine® 3000 (Thermo Fisher Scientific, USA) was employed to transfect cells with plasmid or small interfering RNAs according to the manufacturer's instructions.
Specific primers were used to amplify the coding region of GP73 by PCR (forward: 5’-GCG GAA TTC ATG ATG GGC TTG GGA AAC-3'; reverse: 5’-CCG CTC GAG TCA GAG TGT ATG ATT CCG-3'). The GP73 PCR product was inserted into the pCMV-Tag2B vector.
For stably overexpressing cell line construction, the lentivirus plasmid pWPXLD (Addgene plasmid # 12258) was modified by inserting a T2A peptide between the gene of interest and the GFP gene. Then, the GP73 gene was amplified by PCR and inserted into the modified lentiviral plasmid pWPXLD.
All constructs were confirmed by DNA sequencing.
ELISA, Western Blotting and Immunofluorescence
For ELISAs, cell culture supernatants were collected to detect the levels of HBeAg and HBsAg with an ELISA kit (Kehua Bio-Engineering, Shanghai, China). Serum GP73 was detected by an ELISA kit were purchased from Hotgen Biotech, China.
Western blot analysis was performed as described previously . Antibodies were used to detect HA tags (#SAB2702196, Sigma), FLAG tags (#F7425, Sigma), GP73 (#ab109628, Abcam), NF-κB (p50) (#ab7549, Abcam), IFN-β (#ab180616, Abcam), IFN-λ1 (# MA5-30682, Invitrogen), IL-6 (#ab9324, Abcam), TNF-α (#ab6671, Abcam) and β-actin (#A1978, Sigma). ImageJ (http://rsb.info.nih.gov/ij/) software was employed for band intensity quantification of the Western blot results.
Quantitative RT-PCR analysis
Total RNA of cells was extracted using TRIzol reagent (Invitrogen). RT-PCR primers as follows: GP73 forward: 5'-CAC AAG GGA AGG GAA ACG TG-3', GP73 reverse: 5'-CGA AGC CTC TTC CAC CTA CA-3'; Klf4 forward: 5'- ATT ACC CAT CCT TCC TGC CC -3', Klf4 reverse: 5’-CAC GAT CGT CTT CCC CTC TT-3'; Sox2 forward: 5’-AGC TCG CAG ACC TAC ATG AA-3', Sox2 reverse: 5’-TGG AGT GGG AGG AAG AGG TA-3'; Nanog forward: 5’-ACC CAG CTG TGT GTA CTC AA-3', Nanog reverse: 5’-CTG CGT CAC ACC ATT GCT AT-3'; c-Myc forward: 5’-ATT CTC TGC TCT CCT CGA CG-3', c-Myc reverse: 5’-AGC CTG CCT CTT TTC CAC A-3'; Oct4 forward: 5’-AG AAC ATG TGT AAG CTG CGG-3', Oct4 reverse: 5’-GGT TCG CTT TCT CTT TCG GG-3'; IFN-β forward: 5'-CTG CAA CCT TTC GAA GCC TT-3', IFN-β reverse: 5'-AAG CCT CCC ATT CAA TTG CC-3'; IFN-λ1 forward: 5'-GCT GGT GAC TTT GGT GCT AG-3', IFN-λ1 reverse: 5'-AAG ACA GGA GAG CTG CAA CT-3'; IL-6 forward: 5'-AGT CCT GAT CCA GTT CCT GC-3', IL-6 reverse: 5'-CTA CAT TTG CCG AAG AGC CC-3'; TNF-α forward: 5'-GTC AAC CTC CTC TCT GCC AT-3', TNF-α reverse: 5'-CCA AAG TAG ACC TGC CCA GA-3'; NF-κB forward: 5'-AAT GGT GGA GTC TGG GAA GG-3', NF-κB reverse: 5'-TCT GAC GTT TCC TCT GCA CT-3; GAPDH forward: 5'- GGA AGG TGA AGG TCG GAG TCA ACG G-3', GAPDH reverse: 5'- CTC GCT CCT GGA AGA TGG TGA TGG G-3'. Expression level data were normalized to the GAPDH expression level in each sample.
HBV DNA was detected by TaqMan real-time PCR using the following primers: 5'-AGA AAC AAC ACA TAG CGC CTC AT-3', 5'- TGC CCC ATG CTG TAG ATC TTG-3' and probe 5'-TGT GGG TCA CCA TAT TCT TGG G-3'.
Virus production and transduction of cell lines
For HBV infection of HepG2-NTCP, the culture supernatant of the HepG2.2.15 cell line was concentrated 100-fold by ultracentrifugation. The concentrated HBV stock titer (genome equivalents/ml, GEq/ml) was assessed using QRT-PCR. The multiplicity of infection (MOI) was defined as the genome equivalents per cell (GEq/cell). HepG2-NTCP cells were infected, as described previously .
For stable cell line generation, lentiviral particles were produced and transfected as previously reported .
A CytoFlex (Beckman Coulter, USA) or MoFlo flow cytometer (Beckman Coulter, USA) was employed to analyze GFP signals or cell immunophenotyping. Single-cell suspensions were tested by cell immunophenotyping analysis after cells were stained with fluorescently labeled antibodies: CD90-APC (clone 5E10, eBioscience, 1:100), CD133-APC (clone AC133, Miltenyi Biotec, 1:100), CD117-APC (clone 104D2, eBioscience, 1:100). Flow cytometry data were analyzed using CytExpert software (Beckman Coulter, USA).
Sphere formation assay
Sphere formation assays were performed as previously reported . HepG2-GFP cells and HepG2-GP73 cells were plated in ultra-low attachment 6-well plates (Corning Inc., Corning, NY, USA) at a density of 2000 cells/well. For Huh7-GFP and Huh7-GP73, a single cell was successively sorted into ultra-low attachment 96-well plates (Corning Inc., Corning, NY, USA) by a MoFlo flow cytometer (Beckman Coulter, USA).
All experiments were performed in triplicate. All data were recorded as the means ± standard deviation (SD) unless otherwise stated. Prism 5 software (GraphPad Software) was used for a statistical test. P < 0.05 was considered statistically significant.