Role of hepatitis B surface antigen in the development of hepatocellular carcinoma: regulation of lymphoid enhancer-binding factor 1

Thirty surgical resected HCC tissues from different individuals were provided by Shanghai Cancer Institute. Tissue samples were categorized as tumorous (T) or matched adjacent peritumorous liver tissues (pT) by hematoxylin and eosin (HE) stained sections under the microscope. The size and regions of the resection of the tumorous and peritumorous tissues were decided by the surgeons based on each individual case under the regulation of the ethics committee. All these HCCs were associated with HBV infection as defined by serum HBsAg positive. Normal liver tissues (NL) from liver transplantation donors (n = 9) were obtained from Shanghai Cancer Institute and First Affiliated Hospital, Zhejiang University School of Medicine (kindly provided by Dr. Shusen Zheng). All samples collected followed the regulations of the ethics committees of both hospitals.

Resected liver tissue samples were immediately immersed in 4% formalin and fixed for 18 to 24 h and paraffin-embedded. Immunohistochemical staining was carried out on tissue sections by using anti-LEF-1 polyclonal rabbit antibody (1:50, Abcam, Cambridge, UK) or anti-HBsAg monoclonal antibody (1:50, Changdao Biotech, Shanghai, China) to detect the expression of LEF-1 and HBsAg respectively.

After treated with 10 U DNase I (TaKaRa, Dalian, China) at 37°C for 30 min, 2 μg total RNA was reverse transcribed into cDNA by SuperScript II reverse transcriptase (Invitrogen, Carisbad CA, USA) according to the manufacturer's protocol. Quantitative real-time PCR was carried out using specific primer pairs designed by PrimerBank [11]. For real-time PCR, 2 μl of 10-fold dilutions of the cDNA products were assayed using the Premix Ex Taq Perfect Real Time PCR kit (TaKaRa, Dalian, China). To assess the association of HBsAg and LEF-1 isoforms in HCC tissues, two pairs of primers were designed to detect different LEF-1 isoforms. Primers LP1 and LP2 were designed to target the β-catenin binding domain, which could differentiate the 38 kDa truncated LEF-1 isoform from the 55 kDa full-length LEF-1 [12]. Another pair of primers LP3 and LP4 was targeted to the 3' UTR region of LEF-1 mRNA, and thus could detect both the full length and the isoforms. The house keeping gene GAPDH was used as an internal control. All experiments were performed twice independently. Primers used in this study are listed in Table 1.

The Wilcoxon signed rank tests were performed to evaluate the difference of expression levels of LEF-1, cyclin D1 and c-myc between HCC tissues and normal tissues. Tests were considered of statistical significance when their p values were less than 0.05.

Immunohistochemical staining of the HCC tissues showed that HBsAg was detected in 13 of 30 HCC tissues, either in tumor cells or peritumor cells. HBsAg was detected only in 5 out of the 13 tumor tissues, while in the paired peritumor tissues, HBsAg was observed in all 13 samples (Table 2). LEF-1 was detected in both tumor cells and peritumor cells of all 30 HCC tissues, with no significant difference between tumor cells and peritumor cells. When LEF-1 expression level was analyzed in the HBsAg positive tissues, it was simultaneously associated with the expression levels of HBsAg (Figure 1 and Table 2). The exspression of LEF-1 was found more pronounced in peritumor tissues, compared to that in the tumor tissues among HBsAg positive HCC samples, whereas, no significant differences of LEF-1 expression were observed between tumor cells and peritumor cells in the other 17 HBsAg negative tissues. Cellular distribution pattern of LEF-1 protein was compared between peritumor cells and tumor cells of HBsAg positive tissues. LEF-1 protein was located either exclusively in the nucleus or both in the nucleus and cytoplasm of tumor cells, whereas in peritumor cells LEF-1 was located predominantly in the cytoplasm (Figure 2 and Table 2). When the expression of LEF-1 protein was compared with that of HBV negative normal liver tissues, marked up-regulation of LEF-1 was observed both in tumor tissues and the peri-tumor tisseus among all of 30 HCC tissues. The cellular location of LEF-1 in normal liver cells was in the cytoplasm, more closely representing that in peritumor cells (Figure 2).

The expression pattern of LEF-1 isoforms was studied in HCC tissues by quantitative real-time PCR. Results showed that compared to that of normal liver tissues by real-time PCR, both 38 kDa truncated isoform and 55 kDa full-length LEF-1 were markedly increased in tumor cells and peritumor cells (Figure 3). However, when compared to that in the peritumor cells, the 38 kDa truncated isoform of LEF-1 was more markedly induced in tumor cells, (Figure 3A), while the 55 kDa full-length LEF-1 did not show significant changes (Figure 3B). To further investigate the association of the expression pattern of LEF-1 isoforms and HBsAg expression, LEF-1 isoforms were analyzed in 13 HBsAg positive HCC tissues. The 38 kDa truncated isoform of LEF-1 was significantly up-regulated in tumor cells compared to that in the peritumor cells, while the 55 kDa full-length LEF-1 did not exhibit changes between tumor and peritumor cells (Table 2). However in the other 17 HBsAg negative HCC tissues, no significant changes were observed in either isoforms.

To further study the deregulation of Wnt pathway induced by aberrant up-regulation of LEF-1, expression levels of c-myc and cyclin D1 in HCC tissues and normal liver tissues were compared by real-time PCR. Results showed that compared to that of normal livers, the expression of cyclin D1 and c-myc was increased significantly in both tumor cells and peritumor cells of HCC tissues (Figure 4). In addition, the expression level of cyclin D1 was much higher in peritumor cells compared to that of tumor cells, and c-myc expression showed a similar pattern (Figure 4).