Cell culture
HBV-unrelated HCC cell lines (HepG2, SMMC7721 and Huh7) and HBV-related HCC cell lines (HepG2.2.15 and Hep3B) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Solarbio, Shanghai, China) supplemented with penicillin (100 U/mL), streptomycin (0.1 mg/mL), and 10 % fetal bovine serum (Gibco, Grand Island, NY, USA) in a 5 % CO2 humidified incubator at 37 °C. G418 (6.5 mg/mL; Solarbio, Shanghai, China) was added to the culture medium to maintain HepG2.2.15 cells. LPS from Escherichia coli 0111:B4 was purchased from Sigma Aldrich (St. Louis, MO, USA). SB203580, SP600125, and PD184352 were purchased from Selleckchem (Houston, TX, USA) and pyrrolidine dithiocarbamate (PDTC) from Tocris (Bristol, UK).
RNA interference
Small interfering RNAs (siRNAs) targeting TLR4 were designed and synthesized by Ribobio (Shanghai, China). Non-targeting siRNAs were used as negative controls (Ribobio, Shanghai, China). Individual siRNAs (50 nM) were transfected into cells with Lipofectamine™ 2000 Transfection Reagent (Invitrogen, MA, USA) according to the manufacturer’s instructions. After incubation for 24 and 36 h, transfected cells were collected for analysis of mRNA and protein expression, respectively.
Western blot analysis
Whole cell lysates were prepared using lysis buffer [50 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, 0.5 % NP-40, 0.1 % SDS, and 5 mmol/L EDTA (pH 8.0)] containing aprotinin (2 mg/L), phosphatase inhibitor Leupeptin (5 mg/L) and phenylmethylsulfonyl fluoride (PMSF; 1 mmol/L; Solarbio). Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, Rockford, USA). Equal amounts of proteins were subjected to 10 or 12 % SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % fat-free milk and incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 2 h. Signals were visualized by chemiluminescence (Thermo Fisher Scientific, Rockford, USA) and quantitated using Image J software. Primary antibodies used in this study were rabbit anti-TLR4 polyclonal antibody (1:1,500; Santa Cruz Biotechnology, CA, USA), rabbit anti-SAPK/JNK polyclonal antibody (1:1,000), rabbit anti-p44/42 MAPK (Erk1/2) polyclonal antibody (1:1,000), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) polyclonal antibody(1:1, 000), rabbit anti-p38 MAP Kinase polyclonal antibody (1:1,000), rabbit anti-Phospho-p38 MAP Kinase (Thr180/Tyr182) polyclonal antibody (1:1,000), rabbit anti-NF-kB p65 polyclonal antibody (1:1,000), and rabbit anti-phospho-NF-kB p65 (Ser536) polyclonal antibody (1:1,000) (Cell Signaling Technology, USA), and rabbit anti-JNK1 + JNK2 (phospho T183 + Y185) polyclonal antibody(1:1,000) (abcam, USA), and mouse anti-β-actin polyclonal antibody (1:1,000) (Proteintech, Wuhan, China). Anti-rabbit (1:10,000) and anti-mouse (1:10,000) IgGs were purchased from Proteintech (Wuhan, China).
Quantitative Real-Time Polymerase Chain Reaction (qPCR)
Total RNA was extracted using TRIzol (Invitrogen) and cDNA synthesis was performed using the PrimeScript RT reagent Kit (Takara, Japan). qPCR was performed using the SYBR Premix Ex Taq ™II (Takara) on an Applied Biosystems 7300 Real-Time PCR System (ABI, USA). The TLR4 primer sequence (PrimerBank ID: 373432602c1) came from PrimerBank (http://pga.mgh. harvard. edu/primerbank/): sense: 5′-AGA CCT GTC CCT GAA CCC TAT-3′; and anti-sense: 5′-CGA TGG ACT TCT AAA CCA GCC A-3′. The primers for amplification of GAPDH were as follows: sense: 5′-GTT GGA GGT CGG AGT CAA CGG A-3′; and anti-sense: 5′-GAG GGA TCT CGC TCC TGG AGG A-3′. All PCR amplifications were performed with an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 62 °C for 30 s.
Cell counting kit-8 (CCK8) assay
Cells were seeded in 96-well plates at a density of 4000–5000 cells per well. Cells were allowed to adhere for 12 h and starved with serum-free medium for additional 12 h followed by treatment with 30 μmol/L of SB203580, SP600125, PD184352, or PDTC. The cells were exposed to 10 μg/mL LPS for different times. CCK8 (Zomanbio, China) was added to each well and incubated for 1 h at 37 °C. The optical density (OD) was measured at a wavelength of 450 nm using a microplate reader.
Flow cytometry analysis
Cells were seeded at a density of 5 × 105 cells per well in 6-well plates. After treatment, cells were fixed in ice-cold 70 % ethanol. Cell cycle distribution was analyzed using the Cell Cycle Analysis Kit (MultiSciences, China) and apoptosis was detected using the Annexin V-PE/7-AAD Apoptosis Detection Kit I (BD BioSciences, USA) according to the manufacturer’s instruction. Stained cells were examined by FACSCalibur flow cytometry.
Tumorigenicity in nude mice
For tumorigenicity assays, 4- to 6-week-old male BALB/C nu/nu nude mice (16–18 g) were purchased from the Experimental Animal Center of Shanghai (Shanghai, China). Mice were randomly divided into 3 groups (n = 5 for each group) to receive a s. c. injection of 5 × 106 HepG2.2.15 cells transduced with replication-defective lentivirus expressing TLR4-shRNA (Le-TLR4) or negative control shRNA (Le-NC) or normal saline (NS). Le-NC (2 × 107TU, 40 μL/mouse), Le-TLR4 (2 × 107TU, 40 μL/mouse) or NS (40 μL/mouse) was injected intratumorally at several points every two days, with an accumulated dose of 1 × 108 TU. Tumor volumes were measured every 5 days with a caliper and calculated according to the formula: 0.5 × length × width2. At 18 days after the cell inoculation, mice were sacrificed. The same treatments were done to other 3 groups of nude mice received HepG2 cells. All experimental manipulations were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Nanchang University, Nanchang, China.
Laser scan confocal microscopy
Cells were seeded on coverslips and allowed to adhere for 12 h. After treatment, cells were fixed with 100 % methanol for 30 min and blocked with 5 % bovine serum albumin (BSA) for 30 min. Coverslips were incubated with mouse anti-HBx monoclonal antibody (1:100, abcam) or rabbit anti-TLR4 polyclonal antibody (1:100, Santa Cruz Biotechnology) in 1 % BSA at 4 °C overnight, followed by with fluorescence labeled secondary antibody (1:100, Earthox, USA) in 1 % BSA for 30 min at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Images were captured using Nikon A1 Confocal Laser Microscope System and adjusted using NIS-Elements Viewer 4.0 (Nikon, Japan).
Co-immunoprecipitation assay
HepG2.2.15 cells were grown on 10-cm cell dishes. After culture for 36 h, cells were harvested and lysed in the immunoprecipitation lysis buffer [20 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1 % NP-40, 0.1 % SDS, 0.01 g/mL sodium deoxycholate, and 2 mmol/L EDTA(pH 8.0)] containing Aprotinin (2 mg/L), Leupeptin (5 mg/L) and PMSF (1 mmol/L). Total cell lysates were pre-cleaned with protein-A + G Sepharose Beads (7seabiotech, China) for 3 h at 4 °C and centrifuged. Aliquots of the supernatant were used as input. The remaining supernatants were incubated with mouse anti-HBx or anti-TLR4 antibody or isotype control IgGs and then with protein A + G Sepharose Beads (50 % slurry) at 4 °C overnight. After centrifuging, the pellets were resuspended with the SDS sample buffer and boiled to remove Sepharose beads. Lysate inputs and immunoprecipitates were then subjected to SDS-PAGE and analyzed by Western blotting. Total cell lysates were used as input control.
Statistical analysis
Data were presented as mean and standard deviation (SD) for normally distribution. Groups were compared by one-way Analysis of variance (ANOVA) and multiple comparisons by LSD-t test using SPSS 21.0 (IBM SPSS for Windows, Version 21.0; IBM Corporation, Armonk, NY, USA). P < 0.05 was considered significant.