Zinc finger protein ZBTB20 expression is increased in hepatocellular carcinoma and associated with poor prognosis

Abstract

Background: Our previous studies showed that ZBTB20, a new BTB/POZ-domain gene, could negatively regulate α feto-protein and other liver-specific genes, concerning such as bio-transformation, glucose metabolism and the regulation of the somatotropic hormonal axis. The aim of this study is to determine the potential clinical implications of ZBTB20 in hepatocellular carcinoma (HCC).

Methods: Quantitative real-time RT-PCR and Western blot analyses were used to detect expression levels of ZBTB20 in 50 paired HCC tumorous and nontumorous tissues and in 20 normal liver tissues. Moreover, expression of ZBTB20 was assessed by immunohistochemistry of paired tumor and peritumoral liver tissue from 102 patients who had undergone hepatectomy for histologically proven HCC. And its relationship with clinicopathological parameters and prognosis was investigated.

Results: Both messenger RNA and protein expression levels of ZBTB20 were elevated significantly in HCC tissues compared with the paired non-tumor tissues and normal liver tissues. Overexpressed ZBTB20 protein in HCC was significantly associated with vein invasion (P=0.016). Importantly, the recurrence or metastasis rates of HCCs with higher ZBTB20 expression were markedly greater than those of HCCs with lower expression (P=0.003, P=0.00015, respectively). Univariate and multivariate analyses revealed that ZBTB20 overexpression was an independent prognostic factor for HCC. The disease-free survival period and over-all survival period in patients with overexpressed ZBTB20 in HCC was significantly reduced.

Conclusions: The expression of ZBTB20 is increased in HCC and associated with poor prognosis in patients with HCC, implicating ZBTB20 as a candidate prognostic marker in HCC.

Figure 1

Typical scored immunohistochemical staining of liver tissue specimens. Formalin-fixed and paraffin-embedded tissues were incubated with primary anti-ZBTB20 polyclonal antibodies (diluted 1:350) at 4°C overnight. The visualization signal was developed with diaminobenzidine (DAB) and the slides were counterstained in hematoxylin. Total score was calculated from sub-scores of staining distribution (0-4) and intensity (0-3).

Figure 2

ZBTB20 expression in HCC tissues and HCC cell lines. A. Western blot analysis of ZBTB20 in HCC cell and normal liver cell lines. B. Evaluation of ZBTB20 mRNA in HCC. C. Western blot analysis of ZBTB20 in HCC and normal liver tissues. T Tumor tissue; NT non-tumor tissue; NL normal liver. D. Expression index of ZBTB20 protein. ZBTB20 protein expression level in HCC was significantly higher than those in NT (P < 0.001). The data are representative of three independent experiments.**: T compared with NT or NL, p <0.05.

Figure 3

Immunohistochemistry detection of ZBTB20 expression in normal liver tissue, cirrhotic liver tissue, HCC (T) and in noncancerous hepatic tissues (N). A. Normal liver tissue. B. Cirrhotic liver tissue. The expression of ZBTB20 was weakly positive in hepatocytes. C. HCC cells stained strongly positive with a large granular pattern. D The boundary area between the tumor and non-tumor lesion. Hepatocytes adjacent to HCC lesion were stained with a small granular pattern. Original magnification, ×200. T, tumor; N, Paired-nontumor tissue.

Figure 4

Expression of ZBTB20 in HCC and correlation with AFP expression. A. The consecutive slices of two samples immunostained by anti-ZBTB20 or anti-AFP antibody. a&b. Positive staining of AFP in HCC, while negative staining of ZBTB20; c&d. Positive staining of ZBTB20 in HCC, while negative staining of AFP. Magnification: ×100 & ×200. B. Scatter plot of the correlation of IRS of ZBTB20 with AFP. By Spearman's correlation analysis, IRS of ZBTB20 was negatively correlated to AFP (r = - 0.33, P = 0.001).

Figure 5

Cumulative overall and disease-free survival curves of patients with high or low intratumoral or peritumoral features. (A and B) Low intratumoral ZBTB20 was associated with prolonged overall and disease-free survival (P = 0.001 and P<0.001, respectively). (C and D) Low peritumoral ZBTB20 was associated with disease-free survival (P = 0.002) but not with overall survival(P = 0.225).