Cell lines and culture

The human HCC cell lines Huh7 and MHCC97H, and the human liver cell line L-02 were obtained from the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sijiqing Biological Engineering Materials Co., Hangzhou, China) at 37°C in 95% air and 5% CO₂.

Gene infection

A multiplicity of infection (MOI) of 40 was determined experimentally for MHCC97H cells. Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and incubated to reach approximately 80% confluence. After removing the medium, adenovirus expressing NDRG2 (Ad-NDRG2) or the negative control gene Lac Z (Ad-LacZ) was added in serum-free DMEM, incubated for 2 h, replaced with fresh DMEM supplemented with 10% FBS and incubated for 48 h.

Gene transfection

Huh7 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. Cells were transfected with NDRG2 siRNA or negative control siRNA (TaKaRa) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. NDRG2 siRNA sequences were: 5'-GCUCUCUGGAAAUUCUGAGUUGAUA-3' (sense) and 5'-UUAAGAGCAUAUCUCGCCAGGAUGU-3' (antisense). Negative control siRNA sequences were: 5'-UUCUCCGAACGUGUCACGUTT-3' (sense) and 5'-ACGUGACACGUUCGGAGAATT-3' (antisense). Cells were exposed to siRNA in DMEM for 6 h, after which the medium was replaced with DMEM containing 10% FBS and the cells were incubated for 48 h.

RNA isolation and Quantitative RT-PCR

Total RNA was isolated from cells using Trizol Reagent (Invitrogen) and quantified. cDNA was synthesized from 5 μg of RNA using AMV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer's instructions. The cDNA was used as a template for real-time quantitative PCR using the Prism 7500 real-time PCR instrument (Applied Biosystems Inc., Foster City, CA, USA) and the Universal Mastermix (ABI). Primers were designed using Primer Express Software (ABI). Primer sequences were: NDRG2, 5'-GAGATATGCTCTTAACCACCCG-3' (sense) and 5'-GCTGCCCAATCCATCCAA -3' (antisense), product size: 90 bp; CD24, 5'-ACCTGTTTCCATTCAACAAGAGCAC-3' (sense) and 5'-TCTGAGATCGCACCACTGCAC-3' (antisense), product size: 164 bp; β-actin, 5'-AGCGAGCATCCCCCAAAGTT-3' (sense) and 5'-GGGCACGAAGGCTCATCATT-3' (antisense), product size: 285 bp. The PCR reaction consisted of 12.5 μl of SYBR Green PCR Master Mix, 300 nM each of forward and reverse primers, and 1.5 μl of template cDNA in a total volume of 25 μl. The thermal cycling conditions were as follows: initial denaturation step at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 34 seconds. Data were normalized to β-actin which was used as a loading control.

Western blot analysis

Cells and liver tissues (see below) were lysed in 200 μL of buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM MgCl₂, 0.5% NP-40, 0.1 mM phenylmethyl sulfonylfluoride (PMSF) and protease inhibitor cocktail. A total of 20 μg of lysate (as measured by BCA protein assay; Pierce, Rockford, IL, USA) was loaded per lane onto 12% SDS polyacrylamide gels for separation by electrophoresis and transfer onto Hybond nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). Following transfer, membranes were incubated with 5% fat-free milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h at 37°C. Primary antibody was then added and incubated overnight at 4°C. Primary antibodies were anti-NDRG2 (Abnova, Taiwan, China), anti-CD24 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Boster, Wuhan, China). After washing three times with PBS, membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) for 1 h. The blots were developed with chemiluminescence substrate solution (Pierce) and exposed to X-ray film for visualization.

Adhesion assay

Next, 24-well plates were coated with collagen I (5 μg/cm²). Cells exposed to adenovirus or siRNA for 48 h were seeded at a density of 1 × 10⁵/well and then incubated for 80 min. Five duplicate wells were set up for each group. At the end of the experiment, cells were washed twice with PBS to remove non-adherent cells. The remaining cells were counted under a microscope in five randomly-chosen fields per cm² of substrate surface area. Experiments were repeated three times and data were summarized as mean ± SD.

Migration assay

Cells (1 × 10⁵) exposed to adenovirus or siRNA for 48 h were plated in 6-well plates and grown to confluence. The monolayer was wounded by scratching with a sterile pipette tip lengthwise along the chamber. After wounding, cells were washed twice with PBS and cultured at 37°C for 24 h. Images were captured immediately after cell wounding (0 h) and 24 h after cell wounding. Wound width (μm) was measured using OpenLab software.

Invasion assay

In vitro invasion assays were performed using 24-well transwell units with Matrigel-coated polycarbonate filters (Corning Costar, Cambridge, MA). Cells exposed to adenovirus or siRNA for 48 h were seeded in the upper chamber of the transwell at 1 × 10⁵ cells in 500 μl of serum-free medium, while the bottom chamber was filled with 200 μl of medium containing 10% FBS; After 24 h incubation, transwells were fixed with methanol for 15 min and stained with gentian violet for 10 min. Cells in the upper chamber were removed using a cotton swab and cells that invaded through the Matrigel to the other side of the filter were manually counted. Experiments were performed in triplicate. Data represent the average number of cells from three filters.

HCC clinical specimen preparation

From 2007 to 2010, 50 patients (34 men and 16 women from 21 to 72 years of age) with primary HCC were enrolled in this study at the Xijing Hospital of the fourth military medical university. Tumors were resected and primary HCC was confirmed by a pathologist. The study was approved by the research ethics committee of Xijing Hospital. Proteins were extracted with standard techniques as soon as the liver samples were excised. All patients were prospectively monitored using the α-fetoprotein (AFP) assay. Tumor differentiation was classified according to the Edmondson grading system, with slight modification [21, 22], into two groups: well-differentiated HCC (grades I and II) and poorly-differentiated HCC (grades III and IV). NDRG2 and CD24 expression was scored as positive if > 10% of the cells showed moderate to strong staining. Expression was scored as weak if either cytoplasmic or membranous staining was noted in < 10% of the cells. Expression was scored as negative if neither cytoplasmic nor membranous staining was observed [7].

Immunohistochemical analysis

Four micrometer-thick tissue sections were subjected to immunofluorescent staining analysis. Free-floating liver sections were blocked with 5% normal goat serum in PBS containing 0.3% Triton X-100 for 1 h at room temperature. The sections were then incubated with the following primary antibodies overnight at 4°C: mouse anti-NDRG2 (1:200) or rabbit anti-CD24 (1:200). For double immunofluorescent staining, two antibodies were added at the same time. After incubation with species-specific secondary antibodies conjugated to Cy2 or Cy3 (1:400, Jackson ImmunoResearch, West Grove, PA) for 3 h at room temperature, the fluorescent signals were visualized using a confocal laser microscope (Olympus, Center Valley, PA). NDRG2+ and/or CD24+ immunoreactive areas were obtained bilaterally from every fifth section in a random square unit (125 × 125 μm) and the percentage of immunoreactive area to the total area was calculated. NDRG2/CD24 double-labeled cells were counted manually. Cy2 and Cy3 relative fluorescent intensity was measured using the NIH image-J software.

Statistical analyses

Statistical analyses were performed using SPSS 11.0 software. Data were summarized as mean ± SD. The χ² test, one-way ANOVA and post hoc Bonferroni test were used for comparison between groups. P < 0.05 was considered statistically significant.

NDRG2 and CD24 expression in HCC and normal liver cells

The expression of NDRG2 and CD24 was examined in three liver cell lines. MHCC97H and Huh7 are liver cancer cells. A preliminary experiment indicated that MHCC97H was highly invasive while Huh7 was weakly invasive (data not shown). L-02 is a normal, non-invasive human liver cell line. Results showed that the mRNA and protein expression of NDRG2 in MHCC97H cells was lower than in Huh7 cells. L-02 cells showed the highest level of NDRG2 among the three cell lines. CD24 expression was higher in MHCC97H cells compared to Huh7 cells while L-02 expressed the lowest level of CD24 (Figure 1).

NDRG2 regulates CD24 expression in HCC cells

To understand the regulation of NDRG2 and CD24, MHCC97H cells, which express a low level of NDRG2, were transiently infected with adenoviruses expressing NDRG2. Increased NDRG2 mRNA and protein expression was detected in these cells while expression of CD24 mRNA and protein was suppressed (Figure 2A-C). By contrast, transfection of NDRG2 siRNA into NDRG2-positive Huh7 cells increased CD24 expression (Figure 2D-F).

NDRG2 modulates the adhesion, migration and invasion of HCC cells

The behavior of Ad-NDRG2-infected MHCC97H cells was assessed. Restoration of NDRG2 expression significantly inhibited cell adhesion, migration and invasion (Figure 3); By contrast, siRNA-treated Huh7 cells showed increased adhesion, migration and invasion compared to control cells (Figure 4).

NDRG2 and CD24 show a different expression pattern in HCC clinical specimens

Since CD24 appeared to be regulated by NDRG2 in HCC cell lines, the expression of NDRG2 and CD24 was studied in HCC clinical specimens by indirect immunofluorescence. Double NDRG2/CD24 immunostaining indicated that CD24 was highly expressed in tumors compared to normal adjacent tissues. Decreased NDRG2 expression was detected in tumors while increased expression was detected in normal adjacent tissues. Co-expression of NDRG2 and CD24 was observed in the cytoplasm (Figure 5A). NDRG2 fluorescence intensity was significantly lower in tumors than in normal adjacent tissues (Figure 5B). By contrast, CD24 fluorescence intensity in tumors was higher than in normal adjacent tissues (Figure 5C). To confirm these results, proteins extracted from liver tissues were detected by western blotting analysis. Data showed that NDRG2 expression was decreased in tumor tissues compared to normal adjacent tissues; however, CD24 was enhanced in tumor tissues (Figure 5D).

Low NDRG2 expression correlates with high CD24 expression in HCC and with histopathological features