One hundred and fifty pairs of matched HCC and adjacent non-cancer liver tissues were histopathologically and clinically diagnosed at The First Affiliated Hospital of Sun Yat-Sen University from January 2004 to June 2006. Plasma samples from a peripheral vein were also collected from the 150 HCC patients. Plasma samples were obtained from healthy volunteers who underwent physical examination at the First Affiliated Hospital of Sun Yat-Sen University. The 150 patients included 95 males and 55 females. The mean age of the patients was 58 years (range, 20–78 years). Clinicopathological classification and staging were carried out according to the 6th edition of the American Joint Committee on Cancer (AJCC) TNM classification system. Another independent 48 patients with histologically proven HCC were included in this study. These 48 pairs of tumor tissues from HCC patients and paired adjacent non-cancer specimens were collected for real-time RT-PCR analysis as previously described (18). The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University. Informed consent was obtained from all patients prior to surgery. All patients were recruited into this study after providing informed consent.

All peripheral blood samples were acquired following a standard collection protocol. Briefly, samples were collected and anticoagulated by ethylene diamine tetraacetic acid (EDTA) and centrifuged for 10 min at 3000 rpm. The serum fractions were aliquoted and stored at −80°C until analysis. The concentrations of serum MIF were measured by quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kits (Quantikine, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocols. The levels of serum VEGF were determined using ELISA kits (Genzyme Corp., USA).

The representative areas of each HCC specimen or paired adjacent non-cancer liver tissue were punched with a tissue cylinder (1 mm in diameter) from formalin-fixed/paraffin-embedded tumor tissues or paired adjacent non-cancer tissue blocks. The selected tissue cores were precisely arrayed into a new recipient microarray block using a tissue arrayer (Beecher Instrument, Silver Spring, MD, USA). Each sample was arrayed in triplicate.

Immunohistochemical analysis was performed to study MIF and VEGF expression in 150 human HCC tissues and paired adjacent non-cancer tissues. Briefly, paraffin-embedded tissue-microarray blocks of HCC tissues and paired adjacent non-cancer tissues were consecutively cut into 4-μm sections. Slides were baked at 60°C for 1–2 h and then deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 20 min at room temperature. Slides were incubated overnight at 4°C with primary antibodies (Abcam, Cambridge, UK; catalog no. ad55445; 1:500 dilution) and VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200 dilution) diluted in phosphate-buffered saline (PBS). After washing, the tissue slides were subsequently treated with the secondary antibody (anti-Rb or mouse IgG/HRP; Zhongshan, 1:2000) for 1 h at room temperature, and then with 3,3′-diaminobenzidine (DAB) solution followed by counterstaining with hematoxylin. Analysis was performed with a Zeiss Axioscope 2 microscope at a ×400 magnification, respectively. The degree of immunohistochemical staining was semi-quantitatively assessed and scored independently by two observers. For levels of MIF and VEGF expression, staining intensity was scored according to the following criteria: no staining, 0; weak staining, 1; moderate staining, 2; and strong staining, 3.

Total RNA was extracted from the tissue samples using Trizol (Invitrogen) according to the manufacturer’s instructions. Real-time PCR amplifications were performed in ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using EvaGreen™ qPCR Master Mix (Biotium, Hayward, CA, USA). The primers for human VEGF were 5′-TCGAGACCCTGGTGGACATC-3′ (forward) and 5′-TGTTGGACTCCTCAGTGGGC-3′ (reverse). MIF primers were 5′-CAGAACCGCTCCTACAGCAAG-3′ (forward) and 5′-CGGCTCTTAGGCGAAGGTG-3′ (reverse) and β-actin primers were 5′-ACAATGTGGCCGAGGACTTT-3′ (forward) and 5′-GGAGAGGACTGGGCCATTCT-3′ (reverse). The optimal PCR amplification for VEGF and MIF was 95°C for 30 sec followed by 40 cycles (95°C for 5 sec, 60°C for 30 sec). The expression of β-actin was used as the internal control. The relative expression levels of VEGF and MIF mRNA were calculated according to the comparative Ct method, and the expression of target genes was normalized to β-actin expression levels in each sample.

Data are presented as means ± standard deviation (SD). All statistical analyses were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). χ² test or Fisher’s exact test was used for comparisons between immunohistochemical and serum results and clinicopathological parameters. Spearman’s bivariate correlation test was used to evaluate the correlation between VEGF and MIF. Differences in VEGF mRNA and MIF mRNA expression between the groups were analyzed by the Student’s t-test. A P-value <0.05 was considered to indicate a statistically significant result.

Using ELISA, the levels of serum VEGF and MIF were evaluated in 150 patients with HCC and 30 normal volunteers. The serum VEGF and MIF levels were significantly higher in patients with HCC when compared with their levels in the normal controls (Table I). Overexpression of serum VEGF and MIF was significantly associated with tumor size, intrahepatic metastasis, vascular invasion and TNM stage (Table II). Furthermore, the high levels of VEGF in the serum were positively related with serum MIF expression in HCC (r=0.579, P<0.05).

In subsequent studies, we detected the role of VEGF and MIF in the clinical progression of HCC. We examined 150 paraffin-embedded, archived HCC tissues, including 30 cases of stage I, 80 cases of stage II and 40 cases of stage III tumors, using immunohistochemical staining. High levels of VEGF were present in the cytoplasm of the malignant cells in 75% (112/150) of HCC tissues (Fig. 1b and c). In contrast, VEGF was negatively or only weakly detectable in adjacent non-cancer tissues (Fig. 1a). In addition, the index values of VEGF staining were significantly increased with the progression of tumor grades I to III (P=0.028). Moreover, VEGF expression was strongly correlated with tumor size (P=0.027), vascular invasion (P=0.032), and serum AFP levels (P=0.043). However, our analyses did not show significant associations between VEGF expression and other clinical features including age, gender, history of hepatitis, liver cirrhosis and tumor multiplicity (Table III).

MIF was localized in the cytoplasm of positive staining HCC cells (Fig. 1d–f). MIF was detected in 81% (121/150) of HCC cases (P<0.001). Our studies showed that high levels of MIF expression were associated with tumor size (P=0.022), tumor grade (P=0.013), presence of intrahepatic metastasis (P=0.039) and vascular invasion (P=0.027) and TNM stage (P=0.013). There were no further associations with other clinicopathological parameters (Table III). Spearman correlation analysis confirmed that VEGF expression was positively correlated with MIF protein expression (r=0.619, P=0.022) in the HCC tissues.

To confirm the effect of VEGF and MIF on the progression of HCC and their correlation, we examined their mRNA levels in 48 HCCs and paired adjacent non-tumor tissues by real-time RT-PCR. The mRNA level of VEGF was significantly increased in the HCC tissues when compared with the level in the paired adjacent non-tumor tissues (P<0.01) (Fig. 2A). In HCC tissues, VEGF mRNA expression increased according to increasing TNM stage (Fig. 2C). The mRNA level of VEGF was significantly increased in metastatic HCC tissues when compared with the level in the nonmetastatic tissues (Fig. 2B). Consistent with VEGF, the MIF mRNA level was markedly higher in the HCC tissues when compared with the level in the adjacent non-tumor tissues (P<0.001) (Fig. 2A). MIF mRNA expression was significantly elevated in later TNM stages (P<0.001) (Fig. 2C). MIF mRNA was higher in the metastatic HCC tissues when compared with that in the nonmetastatic tissues (Fig. 2B). A positively correlation was noted between VEGF and MIF mRNA expression (r=0.72, P=0.066).