B7-H3 promotes aggression and invasion of hepatocellular carcinoma by targeting epithelial-to-mesenchymal transition via JAK2/STAT3/Slug signaling pathway

According to the following the inclusion and exclusion criteria, 116 HCC tumor tissues and corresponding adjacent noncancerous liver tissues used in immunohistochemical analysis were randomly obtained from patients undergoing liver curative resection between 2004 and 2008 hospitalized in department of hepatobiliary surgery, Bethune International Peace Hospital and the Fourth Hospital of Hebei Medical University. (a) distinctive pathologic diagnosis of HCC, (b) without anticancer treatment before liver resection, (c) underwent primary and curative resection for HCC between 2004 and 2008, and (d) complete clinicopathologic and follow-up data. 20 normal liver tissues were also taken from the biopsy tissues of healthy living donors for transplantation. All specimens were collected in the operating theater immediately (≤15 min) after resection of the tumors and then were snap frozen in liquid nitrogen or fixed in 10% buffered formalin solution and embedded in paraffin for histological analysis. The histologic grade of tumor differentiation was determined by the Edmondson-Steiner grading system. Liver function was assessed by the Child-Pugh score system. Tumors were classified according to the WHO classification and the International Union against Cancer tumor-node-metastasis (TNM) classification. If patients had multiple lesions in the liver, we selected the main nodule for our study. All samples were obtained with informed consent and their use was approved by the ethics committee of the institution. The median follow-up period was 33.5 months (range, 9–62 months; standard deviation [SD], 11.6 months). At the last follow-up (Dec 31st, 2012), 79 (68.1%) patients finally died, including 32 due to liver failure or bleeding from the gastrointestinal tract and the remaining 47 cases due to tumor recurrence.

Human HCC cell lines (HepG2 and SMCC-7721) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured according to the instructions from American Type Culture Collection (ATCC). The cells were maintained in high-glucose DMEM (Gibco) supplemented with 10% fetal calf serum (Gibco). The cells were incubated at 37°C in a humidified chamber containing 5% CO₂.

Goat anti-B7-H3 was purchased from R&D company for immunohistochemistry. B7-H3 monoclonal antibody (Biolegend MIH35) was used in vitro experiment. Rabbit anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Slug, and anti-Snail antibodies were purchased from Abcam company. Rabbit anti-phospho-Stat3 (Tyr705), Stat3, Phospho-Jak2 (Tyr1008), and Jak2 were purchased from Cell Signalling Technology. Anti-GAPDH antibody was purchased from Santa Cruz Biotechnology.

Immunohistochemistry was performed using the Dako EnVision™ method according to the manufacturer’s instructions. In brief, 4-μm thick consecutive sections were cut by microtome, dewaxed in xylene and rehydrated through graded ethanol solutions. Antigens were retrieved by heating the tissue sections at 100°C for 30 min in EDTA solution. Sections were cooled down and immersed in 0.3% H₂O₂ solution for 20 min to block endogenous peroxidase activity, and then rinsed in PBS for 5 min, blocked with 5% BSA at room temperature for 20 min, and incubated with primary antibodies against B7-H3 (final concentration in use, 5 μg/ml) at 4°C overnight. Negative controls were performed by replacing the specific primary antibody with PBS. After three PBS washes, sections were incubated with secondary antibodies for 30 min at room temperature. Diaminobenzene was used as the chromogen and hematoxylin as the nuclear counterstain. Sections were dehydrated, cleared and mounted.

Evaluation of B7-H3 staining in cancer cells was evaluated by authorized pathologists who had no knowledge of the patients’ clinical status and outcome. B7-H3 expression scores were given separately for the stained area and for the intensity of staining. Quantification was made as follows; ≤33% of the cancer cells: 1, >33 to ≤66% of the cancer cells: 2, >66% of the cancer cells: 3; intensity of staining: absent/weak: 1, moderate: 2, strong: 3. The intensity of B7-H3 staining was considered weak when either cytoplasmic expression or rare membranous condensation was present, moderate when incomplete and discontinuous moderate membranous expression was present, and strong when complete membranous expression of the molecule was present. Each section had a final grade that derived from the multiplication of the area and intensity scores. Sections with a final score of ≤3 were classified as tumors with low B7-H3 expression, whereas sections with a final score of >3 were classified as tumors with high B7-H3 expression.

To further analyze the role of B7-H3 in HCC malignancy, HepG-2 and SMMC7721 cells were tranfected with B7-H3 shRNA plasmid using Lipofectamine²⁰⁰⁰ (Invitrogen, CA). Each shRNA vector is cloned in pGFP-V-RS (Origene Technologies, Inc.) plasmid under U6 promoter for mammalian cell expression. The set sequence of the B7-H3 siRNA contains 5 vials of gene-specific siRNA expression vectors in pGFP-V-RS plasmid. We selected the most efficient one to carry out the following experiment. This sequence of the B7-H3 siRNA is 5′-TGAAACACTCTGACAGCAAAGAAGATGAT-3′. The plasmid contains a non-effective siRNA cassette against green fluorescent protein as a scrambled negative control (Origene Technologies, Inc.) In brief, about 3 × 10⁵ cells were seeded per well in a 6 well plate. After 24 h, the cells were transfected with 1.5 μg of cDNA or siRNA plasmid for 6 h, and the media were replaced with fresh growth medium. At 48 h after transfection, cells were harvested for analysis. Transfection efficiency was evaluated by RT-PCR and western blotting assay, respectively.

The MTT assay was used to study the effect of B7-H3 siRNA interference on HCC cell proliferation. Cells were seeded at a density of 5 × 10³ cells per well in 200 μl of DMEM medium into 96-well plates, and cultured overnight. At different time points, the medium was replaced with 100 μl fresh medium containing 0.5 mg/ml MTT (Sigma, USA). Four hours after the addition of MTT, the supernatants were removed and discarded. 150 μl of dimethylsulfoxide (DMSO, Sigma) was added to each well to dissolve the crystals. Cell viability was determined by scanning with a microplate reader at a wavelength of 490 nm. Each experiment was performed in triplicate and repeated at least three times.

The induction of apoptosis by B7-H3 siRNA transfection was evaluated with a Cell Death Detection ELISA^Plus kit (Roche, Germany) according to the manufacturer’s instruction. Cells were cultured in 96-well plates, starved by serum deprivation for 24 h. Then, cells were transfected with B7-H3 siRNA or scrambled non-target siRNA for 24, 48 and 72 h as indicated. Measurements were made using an ELISA reader at 405 nm. Each group was repeated in six wells and the results were averaged.

B7-H3 or control siRNA transfected HCC cells were preincubated with serum-free medium for 24 h, and cell layers were scraped with a pipette tip when cells reached 80% confluence. Cells were then washed twice with PBS and incubated in serum-free medium at 37C in a 5% CO₂ incubator for 24 h. Photographs were taken at different times from 0 to 48 h. Wound width was measured at 200 × magnification using a BX50 microscope (Olympus®). Ten measurements were made at random intervals along the wound length. Data were averaged and expressed as the mean wound distances. This experiment was done in triplicate.

Invasion assays were done using transwell matrigel invasion chambers with a pore size of 8 μm coated with 1 μg/cm² to 2 μg/cm² martigel (BD, USA). Cells were detached and resuspended in serum-free DMEM. A total of 2 × 10⁴ cells in 100 μl of serum-free media were seeded onto the upper portion of a 24-well matrigel chamber. The lower compartment contained DMEM with 10% FBS. After incubation at 37°C for 24 h in a 5% CO₂ atmosphere, the non-invading cells and gel were removed from the upper chamber with cotton tipped swabs. The cells were rinsed with PBS and cells on the filters were fixed with methanol for 30 min and stained with crystal violet solution. The number of invading cells on the filters was counted in 5 random fields per filter at 200× magnification in triplicate wells of each group.

HCC cells were washed with PBS twice and lysed with 1 mL RIPA lysis buffer containing protease and phosphatase inhibitor for 30 min on ice. After removing the insoluble material by 12,000 × g centrifugation for 30 min at 4°C, the supernatants were collected. Cell lysate protein content was determined using a Bicinchoninic acid (BCA) protein assay kit. Equivalent amounts of whole cell extracts were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h and then incubated with respective primary antibody overnight at 4°C followed by the incubation with the appropriate HRP-conjugated secondary antibody for 2 h at room temperature. Blots were visualized with an ECL detection kit (Pierce, USA) and GAPDH was used as a loading control.

For MMP-2 and MMP-9 activity detection, cell lysate from treated cells were prepared and subjected to electrophoresis with 8-12% SDS polyacrylamide gels containing 0.1% gelatin. After electrophoresis, the gels were washed with 2.5% Triton X-100 for 30 min to remove SDS and incubated in a reaction buffer at 37°C for 16 h. Finally, the gels were stained with 0.5% Coomassie brilliant blue R-250 solution containing 10% acetic acid and 20% methanol for 30 min and then destained with 7.5% acetic acid solution containing 10% methanol. Areas of gelatinase activity appear as clear bands against the blue-stained gelatin background.

The Mann–Whitney U test, χ2 test or Spearman rho test were performed for comparative statistical evaluations among groups and for correlation analysis with histological and clinical parameters (age, gender, tumor stage, tumor grade, and postoperative survival). The Kaplan-Meier method was used to analyze survival data and the log-rank test was taken to control differences in patients’ survival. Other data are shown as the mean and range or mean ± SD of 3 independent experiments. Statistical comparisons were performed using Student’s t test. All p values were determined by 2-sided tests with significance considered at p < 0.05 using SPSS 20.0 for Windows.