Study object
A total of 63 patients with liver cancer hospitalized in The 962nd Hospital of the PLA from January 2014 to January 2016 were included in this study. All included patients met the following criteria: 1. without history of combined chronic diseases; 2. without history of chemotherapy or physical therapy before surgery in The 962nd Hospital of the PLA; 3. without family history of related tumors. Based on the 7th edition of the Union for International Cancer Control (UICC) tumor lymph node metastasis (TNM) staging standard (2010), patients were classified. All surgically resected liver cancer tissues and adjacent normal tissues (more than 5 cm away from cancerous tissues) were rapidly stored in liquid nitrogen at -80℃. Detailed information for patients is listed in Table 1.
Table 1
The clinical feature of liver cancer patients
Clinicopathological features | Cases (n) |
Sex | Male | 35 |
Female | 28 |
Age (years) | ≤ 60 | 41 |
> 60 | 22 |
Tumor size | ≤ 5 cm | 36 |
> 5 cm | 27 |
Venous infiltration | Yes | 36 |
No | 17 |
Metastasis | Yes | 40 |
No | 21 |
Microarray-based analysis
Microarray-based analyses were performed using Affymetrix. RNA was extracted from tissues with TRIzol reagent (Thermo Fisher Scientific, USA) and hybridized with GeneChip Human Gene 2.0 ST Array (Thermo Fisher Scientific) at 48℃ and 60 rpm, which was then washed and stained followed by scanning with a GeneChipTM Scanner 3000 7G system (Thermo Fisher Scientific). Data analysis was subsequently carried out using Expression Console Software with background correction and normalization of raw data by robust multichip analysis (RMA, City of Industry, CA, USA). mRNAs with differential expression were identified according to t-test. The heatmap of differential mRNAs was plotted with p < 0.01 and |Fold change| > 2 as threshold.
Experimental antibodies
The following antibodies were used in this study: primary antibodies against HBx (ab39716, dilution at 1:500 for Western blot analysis; 1:100 for RNA Binding Protein Immunoprecipitation (RIP) assay; 1:200 for Immunohistochemistry (IHC); Abcam, Cambridge, UK), XB130 (#12684, dilution at 1:1000 for Western blot analysis; 1:50 for RIP; 1:100 for IHC; Cell Signaling Technology, Boston, MA, USA), AKT (#9272, 1:1000, Cell Signaling Technology), phosphorylated-AKT (Ser473) (#4060, 1:2000, Cell Signaling Technology), and β-actin (sc-47778; 1:500, Santa Cruz, CA, USA) as well as a secondary antibody (ab205718, 1:10000, Abcam).
IHC
The obtained liver tissue samples were fixed in 4% paraformaldehyde for 1 h and embedded in dehydrated paraffin. Tissues were then sectioned and immersed with blocked permeable solution for 30 min, which were the incubated with 3% hydrogen peroxide for 10 min and blocked by 5% goat serum. A total of 50 µL primary antibody was added into the sections for an incubation for 1 h at room temperature, followed by an incubation of secondary antibody for 30 min at 37℃. Next, sections were added with 50 µL peroxidase, and the color was developed with Diaminobenzidine (DAB) for 5 min. Washed by phosphate buffers saline (PBS), tissue sections were counterstained with hematoxylin. After 30 s of differentiation with ethanol hydrochloride, the cells were dehydrated, mounted and observed under an inverted microscope (IX53, Olympus, Tokyo, Japan).
Cell culture and transfection
Human liver cancer cell line HepG2 and normal hepatocyte MIHA were incubated in Roswell Park Memorial Institute-1640 medium at 37℃ with 5% CO2. The cell suspension of HepG2 cells was cultured in a 37℃ incubator with 5% CO2 for 24 h, after which cells were seeded onto culture dishes for overnight culture. The plasmids containing small interfering RNA targeting HBx (si-HBx) and XB130 (si-XB130) fragments and empty plasmids were transfected into HepG2 cells using Lipofectamine 2000 (Thermo Fisher Scientific) when cell confluence reached 60%. At 4 h post-transfection, the medium was renewed, and cells were further incubated for 48 h for subsequent experiments. The pcDNA3.1 plasmids (GenePharma, Shanghai, China) were used, while the sequence fragments were synthesized by Sangon Biotechnology (Shanghai, China).
Western blot analysis
Cells were dissolved on ice in Radioimmunoprecipitation assay buffer (50 mmol/L Tris-Cl [pH = 7.5], 120 mmol/L NaCl, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 2 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, and 1% NP-40) containing proteinase inhibitor cocktail (Roche, Basel, Switzerland). The protein content of the cleavage products was measured by bicinchoninic acid. A total of 30 µg of protein was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (Millipore, Bedford, MA, USA) membrane, which was blocked with 5% bovine serum albumin (BSA) after marker separation. Subsequently, the membrane was incubated with the above-mentioned primary antibodies at 4℃ for 16 h, and then with secondary antibody at room temperature for 1 h. Immunoreactive protein bands were determined using enhanced chemiluminescence system (Thermo Fisher Scientific, USA), which were quantified by QuantityOne v4.6.2 imaging software (Bio-Rad, USA).
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA from tissues and cells was extracted with TRIzol reagent (Thermo Fisher Scientific, USA), followed with measurement of quality and concentration of RNA by a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific). RNA was reversely transcribed into complementary DNA (cDNA) using the miRcute miRNA cDNA first-strand synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China). Quantitative PCR was performed using the SYBR-Green I Master Mix kit (Thermo Fisher Scientific), whereas PCR was performed in the GeneAmp PCR system (PE2400, USA). The results of electrophoresis were scanned by an ultraviolet peroxide with Gel Doc 100 gel detector (Bio-Rad), with gray values of each band calculated by Image J software. Primers for HBx and XB130 (Table 2) were synthesized by GenePharma (Shanghai, China).
Table 2
Primer sequences for RT-qPCR
Gene | Sequences |
XB130 | F: 5'-CGGACTCAGACUCUTGCCUTU-3' |
R: 5'-CUGUAGCTUACCGTTGUUCG-3' |
HBx | F: 5'-ACCGACCTTGAGGCCTACTT-3' |
R: 5'-GCTTGGCAGAGGTGAAAG-3' |
GAPDH | F: 5'-GACCTGACCTGCCGTCTA-3' |
R: 5'-AGGAGTGGGTGTCGCTGT-3' |
Notes: RT-qPCR, Reverse transcription quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase |
RIP
For Co-IP analysis, HepG2 cells were lysed by incubation with protein G-agarose beads (Roche Diagnostics, Switzerland) at 4℃ for 1 h, followed with cell centrifugation. The collected supernatant was incubated overnight at 4℃ with 4 µg anti-HBx antibody or immunoglobulin G (IgG; Thermo Fisher Scientific). The immune complexes were precipitated by incubation with 50 µL protein G-agarose at 4℃ for 3 h. Agarose beads were precipitated by centrifugation and washed three times with lysis buffer. Next, the beads were suspended in 2 × Laemmli sample buffer (Sigma-Aldrich) for 5 min. The protein G-agarose beads were removed from the complex by a 10,000 g centrifugation for 5 min, and the supernatant was loaded onto 10% SDS-PAGE for Western blot analysis.
Cell counting kit (CCK)-8 assay
Cells were seeded on 96-well plates containing 100 µL Dulbecco’s modified eagle medium (DMEM) at a density of 3 × 103 cells/well. After 24, 48 and 72 h of incubation at 37℃ and 5% CO2, the original medium was discarded and a DMEM containing 10 µL CCK8 solution (Takara, Japan) was added. After another 3 h of incubation, the optical density (OD) value of cells at 450 nm was measured using a microplate reader RT-6100 (Rayto, Shenzhen, China), and the growth curve was plotted.
Colony formation assay
A total of 5,000 cells were mixed with 0.5% soft agar (Solarbio, Beijing, China), which were added to a 6-well plate covered with 0.8% agar. The top of the agar was added with 2 mL complete medium, followed by cell culture in standard culture environment for 2 weeks. Afterwards, colonies were photographed and counted under an inverted microscope (IX53, Olympus).
Wound healing experiment
Cells at 90% confluence were seeded into a 6-well plate and cultured overnight at 37℃ with 5% CO2. Wounds with interval of 5 mm were made using a pipette along the layer of adherent cells. The cells within the scratch were removed by PBS, and fresh medium was added to further the culture. Cell migration to wound was evaluated by photographing at 0 and 24 h.
Transwell assay
The invasive ability of HepG2 cells was evaluated by Transwell assay. Cells (5 × 104 cells) were seeded in 0.5 mL serum-free medium and then added to the apical Transwell chamber, while 0.75 mL medium containing 5% fetal bovine serum added to the basolateral chamber. The Transwell chamber was incubated for 18 h and fixed with 4% paraformaldehyde for 1 h, followed by the removal of cell debris and addition of hematoxylin (BD Biosciences, San Jose, CA, USA) for a 1-h staining at room temperature. Cells were photographed under a microscope (IX53, Olympus) with five visual fields randomly selected.
Flow cytometry for detection of cellular apoptosis
Cell apoptosis was assessed using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kits (Solarbio). Briefly, cells were detached with 0.25% trypsin, resuspended in PBS with 1 × 106 cells/mL, and fixed with 0.7 mL absolute ethanol for 24 h. After a centrifugation at 180 g for 30 s, the supernatant was discarded. Cells were resuspended in 1 mL PBS followed by another centrifugation. The precipitated cells were suspended with 100 µL 1 mg/mL RNase A, and the cell suspension was incubated with 400 µL 50 µg/mL Annexin V-FITC for 15 min in the dark. Following that, 400 µL 50 µg/mL PI was added into cells and incubated for 10 min in void of light. The cellular apoptosis was determined by a flow cytometer (CytoFLEX, BECKMAN COULTER).
Flow cytometry for detection of cellular cycle
Cells in logarithmic growth phase were collected and seeded in a 6-well plate at 1 × 105 cells/well, which were cultured overnight at 37℃ with 5% CO2. After trypsinization, cells were collected and centrifuged in a flow tube at 1,000 r/min for 5 min. Following addition of 70% precooled ethanol, the precipitates were fixed at 4℃ for more than 18 h, and then added with RNAase (50 mg/L) at 10 µL/well and PI (50 mg/L) at 300 µL/well under shaking. After 30 min of reaction at room temperature in void of light, DNA detection was performed by a flow cytometer (CytoFLEX, BECKMAN COULTER), and the proportion of cell cycle in each phase was analyzed by Modifit software (Verity Software House, USA).
Statistical analysis
The SPSS22.0 (IBM SPSS Statistics, Chicago, IL, USA) was employed for statistical analysis. Measurement data derived from repeated independent experiments were expressed as mean ± standard deviation. Survival analysis was performed as by means of Kaplan-Meier analysis, while unpaired t-test was used for comparison between two groups. One-way or two-way analysis of variance (ANOVA) and Tukey's post-test were used for data comparison among multiple groups, and log rank test was used for statistical post-analysis, with p < 0.05 as the indicator of statistical significance.