Expression of B7-H6 expression in human hepatocellular carcinoma and its clinical significance

The HCC tissue array HLiv-HCC180Sur-05 was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, P. R. China). A total of 90 HCC patients (from 25 to 73 years of age, with a median age of 56) who underwent surgery between February 2007 and March 2009 were enrolled in the present study. No patient received preoperative chemotherapy or radiotherapy, and all tumor tissues were confirmed as HCC using hematoxylin and eosin (H&E) staining after surgical resection. Incomplete tissue samples and several missing tissue samples were excluded during the heat-induced antigen retrieval. A final total of 87 cases were included in the statistical analysis. Table 1 presents the detailed clinical parameters of the patients.

The rabbit anti-human B7-H6 polyclonal antibody was purchased from Abcam (Cambridge, MA, USA). The HRP-conjugated goat anti-mouse/rabbit secondary antibody (K500711, ready to use) was obtained from Dako (Glostrup, Denmark). A rabbit anti-human GAPDH antibody (Sigma, St. Louis, MO, USA) was used for Western blot analysis. The RNeasy Mini Kit was supplied by Qiagen (Valencia, CA, USA), and SYBR Green Master Mix kits were provided by TaKaRa (Dalian, China). DMEM and fetal bovine serum (FBS) were purchased from Gibco (Cambrex, MD, USA).

Immunohistochemical staining was performed as described in our previous reports [12‐15]. Briefly, the HCC tissue array block was cut into 4-µm-thick sections, and the sections were dewaxed in xylene and rehydrated in a graded series of alcohols. Antigens were retrieved by heating the tissue sections at 100 °C for 30 min in an EDTA (1 mM, pH 9.0) solution. Cooled tissue sections were immersed in 0.3% hydrogen peroxide solution for 15 min to block endogenous peroxidase activity, rinsed with phosphate-buffered saline (PBS) for 5 min and blocked with a 3% BSA solution at room temperature for 30 min. Next, the sections were incubated with a rabbit polyclonal antibody against human B7-H6 (1:150) at 4 °C overnight and were then incubated with an HRP-conjugated goat anti-rabbit secondary antibody. Diaminobenzene was used as the chromogen, and hematoxylin was used as the nuclear counterstain. Finally, the sections were dehydrated, cleared and mounted.

H-score was used to assess the immunostaining intensity of B7-H6 [14, 16], which was calculated as follows: \(\begin{aligned} H \, score\, = \, & \left( {\% {\text{ tumor cells unstained}}\, \times \,0} \right)\, + \,\left( {\% {\text{ tumor cells stained weak}}\, \times \, 1} \right) \\ & + \,\left( {\% {\text{ tumor cells stained moderate}}\, \times \, 2} \right)\, + \,\left( {\% {\text{ tumor cells stained strong}}\, \times \, 3} \right) \\ \end{aligned}\), ranging from 0 (100% negative tumor cells) to 300 (100% strongly stained tumor cells). Two independent senior pathologists examined all HCC tissue sections blindly. The averaged score was calculated and further used for statistical analysis.

RNA interference technology was used to establish stable cell lines. Small hairpin RNA (shRNA) against human B7-H6 gene (NM_001202439.2; GenBank) was obtained from Shanghai Generay Biotech Co., Ltd. (Shanghai, China). The shRNA target sequence against B7-H6 was as follows: 5′-CATCAAGAATATGGATGGCACATTT-3′. And the non-targeted control sequence was 5′-TTCTCCCCGAACAACAACGTGTCACCACCACGT-3′. shRNA was cloned into a lentiviral vector encoding green fluorescent protein (GFP).

Human HCC cell lines HepG2 and SMMC-7721 were obtained from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, which were cultured in standard DMEM supplemented with 10% fetal bovine serum under standard culture conditions (5% CO₂, 37 °C). To establish stable B7-H6 knockdown HCC cell lines, lentivirus was used. The recombinant B7-H6-targeting lentivirus (LV-B7-H6-shRNA virus) or control mock lentivirus (LV-NC virus) were transfected into HepG2 and SMMC-7721 cells. Using an Aria II flow sorter (BD Bioscience, NJ, USA), the GFP-positive cells were subsequently sorted from the two stable cell lines, which were then respectively named as LV-B7-H6-shRNA or LV-NC.

RT-PCR was performed to confirm the knockdown of B7-H6 expression at the mRNA level in HepG2 and SMMC-7721 cells. Total RNA was extracted using TRIzol (Invitrogen) reagent, and purified RNA was then reversely transcribed to cDNA using an RT reaction kit (Promega). RT-PCR was conducted using the ABI 7600 system (Applied Biosystems, USA), and SYBR Green was used as a DNA-specific fluorescent dye. Human GAPDH was selected as a housekeeping gene. Primers were synthesized as follows: GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′ and GAPDH reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′; and human B7-H6 forward: 5′-CTCCTGATTCTGCTGTGGGC-3′ and human B7-H6 reverse: 5′-GTCGGAATGCCTCTTGGTGA-3′. In addition, RT-PCR products were confirmed by electrophoresis on a 1.8% agarose gel containing 0.1% ethidium bromide. Images of the fluorescent bands were captured using a Bio-Rad gel documentation system.

The cell proliferation was performed using Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) according to the manufacturer’s instructions. 3 × 10⁴ cells were seeded into 96-well plates and maintained in 100 μL of standard DMEM supplemented with 10% FBS. Then culture medium was replaced with CCK-8 reagent (10 μL CCK-8) at indicated time points. After incubating for 1 h, the absorbance of each well was examined at a wavelength of 450 nm. The relative absorbance of experimental values to the initial values indicates cell growth or death, respectively. Cell growth was monitored every 24 h over a period of 3 days. Each experiment was repeated for at least three times.

Cell migration ability was assessed using a wound scrape assay to examine the role of B7-H6 in regulating the migration ability of HCC cell lines, as described in our previous studies [12, 17, 18]. Briefly, cells from the LV-B7-H6-shRNA and LV-NC groups were maintained in 6-well plates. A small wound area was created using a 200-µL pipette tip when cells reached a confluence of 90%. Cells were then washed twice with PBS and were further incubated in serum-free DMEM at 37 °C in a 5% CO₂ incubator for 0 h, 12 h, 24 h and 36 h. Images were acquired at the indicated time points, and the wound width was measured using a BX50 microscope (Olympus^®) at 100× magnification with a calibrated eyepiece grid (1 mm/20 μm graduations). A total of 10 measurements were made at random intervals along the wound length, and data from three independent experiments were averaged and expressed as a percentage of the original width. Each experiment was conducted in triplicate.

The Transwell culture system was used to examine the invasive ability of the HCC cell lines after knockdown of B7-H6 expression, as described in our previous studies [12, 17, 18]. Briefly, the upper chamber of Transwell^® inserts with a pore size of 8 μm and a diameter of 6.5 mm was coated with 20 μL of Matrigel (diluted 1:3 in serum-free DMEM) and was incubated at 37 °C for 4 h. Cells from the LV-B7-H6-shRNA and LV-NC groups were harvested by trypsinization after 12 h of serum starvation and were then resuspended in a serum-free DMEM at a density of 1 × 10⁶ cells/mL. Subsequently, 200 μL of the cell suspension was added to the upper chamber of the coated inserts, while 600 μL of DMEM supplemented with 10% FBS was placed in the bottom chamber as a chemoattractant. After incubation at 37 °C for 12 h, 24 h and 36 h respectively in a 5% CO₂ atmosphere, the noninvading cells and Matrigel were removed from the upper chamber with a cotton-tipped swab. The inserts were rinsed with PBS, and the cells on the filters were fixed with methanol for 30 min and stained with crystal violet solution (Sigma). The number of invading cells on the filters was counted in five randomly selected fields per filter at 100× magnification. Each experiment was conducted in triplicate.

Apoptosis-mediated cell death was examined using an APC-Annexin V apoptosis detection kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, cells from the LV-B7-H6-shRNA and LV-NC groups were harvested, washed with PBS and resuspended in binding buffer at a density of 1 × 10⁶ cells/mL. Subsequently, 100 µL of the cell suspension was incubated with 5 μL of APC-annexin V solution and 5 μL of propidium iodide (PI) solution. After a 15-min incubation in the dark, the stained cells were analyzed by flow cytometry (BD FACSCanto II, BD Biosciences, NJ, USA), and the data were analyzed using FlowJo 10.0.6 software.

Briefly, 1 × 10⁶ cells from the LV-B7-H6-shRNA and LV-NC groups were washed with PBS and were then fixed with 70% ice-cold ethanol at 4 °C overnight. After cells were washed twice with PBS, they were then stained with a solution containing 200 μg/mL PI, 0.1% sodium azide, 0.1% Triton X-100 and 10 μg/mL RNAses for 2–4 h in the dark at room temperature. The stained cells were analyzed for subG1, S and G2 phase peaks by flow cytometry (BD FACSCanto II, BD Biosciences, NJ, USA) using ModFit software.

Cells were homogenized briefly in 10 volumes of lysis buffer containing (in mM) 20 Tris–HCl (pH 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na₄P₂O₇, 1 Na₃VO₄, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma Aldrich, St. Louis, MO, USA), 1% (v/v) Triton X-100 and 10% (v/v) glycerol. The homogenates were centrifuged twice at 20,000×g at 4 °C for 15 min, and the supernatants were retained as total protein. Protein concentrations were determined by the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, MA, USA). Western blot analysis was performed under standard conditions with specific anti-B7-H6 (1:2000; Abcam, MA, USA), anti-C-myc (1:1500, Abcam, MA, USA), anti-C-fos (1:2000, Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:2000, Cell Signaling Technology, MA, USA), and anti-GAPDH (1:4000, Sigma, St. Louis, MO, USA) antibodies and HRP-labeled goat anti-mouse/rabbit secondary antibody (1:6000, Sigma Aldrich, St. Louis, MO, USA). The immunoreaction was visualized using an enhanced chemiluminescence detection kit (Thermo Fisher, MA, USA) and exposure to X-ray film, and band densities were quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad).

Statistical analysis was conducted by the GraphPad Prism 5.0 software package (GraphPad Software, Inc., San Diego, USA) using a paired Student’s t-test, Wilcoxon signed-rank test or Chi square test where appropriate for final analysis of the data. A P value < 0.05 was considered statistically significant.

Immunohistochemical staining was used to examine the B7-H6 expression in both HCC tissues and normal liver tissues. Figure 1 shows that the positive staining for B7-H6 was predominantly localized on the membrane and in the cytoplasm of HCC cells. Figure 1a shows high expression of B7-H6 in HCC tissue. Figure 1b indicates moderate expression of B7-H6 in HCC tissue. Figure 1c represents low expression of B7-H6 in HCC tissue. Figure 1d reveals that weak to moderate staining of B7-H6 could also be found in normal liver tissues. Table 1 summarizes the correlation between the patients’ clinical parameters and B7-H6 expression in the human HCC tissues. Our data demonstrated that B7-H6 expression in the human HCC tissues was significantly associated with the age (P = 0.015) and tumor size (P = 0.034) of the patients. We did not find any correlation between B7-H6 expression and the other clinical parameters of the patients. Therefore, our data suggested that the overexpression of B7-H6 was involved in the progression of human HCC. Moreover, we also verified the prognostic value of B7-H6 expression at the mRNA level according to TCGA data from http://​gepia.​cancer-pku.​cn/​; Fig. 2 shows that lower expression of B7-H6 at the mRNA level was significantly associated with better survival in the HCC patients (P = 0.017).

In the present study, we used the human HCC cell lines HepG2 and SMMC-7721 to assess the role of B7-H6 in the regulation of cellular functions. The knockdown of B7-H6 expression was achieved in both cell lines using shRNA via lentiviral infection, and we then sorted the cells based on GFP expression using a flow sorter. The infection efficiency was also confirmed by detecting GFP expression using fluorescence microscopy (Fig. 3a, b). In addition, we confirmed the knockdown efficiency by real-time RT-PCR analysis, and the present data showed that B7-H6 expression at the mRNA level was significantly decreased in the LV-B7-H6-shRNA group compared with that in the LV-NC group in both the HepG2 and SMMC-7721 cell lines (P < 0.01 and P < 0.05 for HepG2 and SMMC-7721, respectively; Fig. 3c, d). In addition, Western blot analysis confirmed that the expression of B7-H6 at the protein level in the LV-B7-H6-shRNA group was decreased compared with that in the LV-NC group in both HCC cell lines (Fig. 3e, f, both P < 0.01).

We assessed the cell proliferation rate of the human HCC cell lines HepG2 and SMMC-7721 after knockdown of B7-H6 expression in both the LV-B7-H6-shRNA and LV-NC groups using a CCK-8 assay. Figure 4a shows that in HepG2 cells, the proliferation rate of the LV-B7-H6-shRNA group was significantly lower than that of the LV-NC group after 48 and 72 h (P < 0.05 and P < 0.01, respectively), and in SMMC-7721 cells, the proliferation rate of the LV-B7-H6-shRNA group was significantly lower than that of the LV-NC group after 48 and 72 h (both P < 0.05). Moreover, Fig. 4b shows that in the wound healing assay, in HepG2 cells, the cell-free area of the LV-B7-H6-shRNA group was significantly wider than that of the LV-NC group at 24 h and 36 h (P < 0.05 and P < 0.01 respectively) after the scratch was made on the cell monolayer, and in SMMC-7721 cells, the cell-free area of the LV-B7-H6-shRNA group was significantly wider than that of the LV-NC group at 36 h (P < 0.01) after the scratch was made on the cell monolayer. The Transwell invasion assay indicated that in both HepG2 and SMMC-7721 cells, the number of crystal violet-stained cells was significantly decreased in the LV-B7-H6-shRNA group compared with that in the LV-NC group (at 24 h, P < 0.01 and P < 0.001 respectively, and at 36 h, both P < 0.001) (Fig. 4c). We further tested the contribution of B7-H6 expression to HCC cell apoptosis and cell cycle regulation. Figure 4d shows that in both HepG2 and SMMC-7721 cells, the LV-B7-H6-shRNA group displayed an increased percentage of cells in the G1 phase and a decreased percentage in the G2/M phases compared with the LV-NC group. Therefore, our present data supported the notion that the knockdown of B7-H6 expression could induce cell cycle arrest in HCC cells.

Because the knockdown of B7-H6 expression significantly suppresses proliferation and induces cell cycle arrest in human HCC cell lines, we next carried out Western blotting to examine the protein expression levels of C-myc, C-fos and cyclin D1, which are involved in the regulation of cell proliferation and the cell cycle. As shown in Fig. 5a, b, in both HepG2 and SMMC-7721 cells, B7-H6 knockdown could significantly decrease the expression of C-myc (both P < 0.01), C-fos (both P < 0.01) and cyclin D1 (P < 0.05 and P < 0.01, respectively), which further confirmed that B7-H6 was involved in the regulation of cell proliferation and the cell cycle.