Expression, Subcellular Localization And Clinical Implication Of Wnt Antagonist Dickkopf-3in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies threatening human health worldwidely. Our previous study of Dickkopf-3(DKK3) gene methylation and clinical significance in hepatocellular carcinoma suggest that methylation of the DKK3gene can be used as a protential marker for diagnosis or prognosis. Meanwhile we also noted that, contrary to the significant specificity of DKK3methylation in cancer tissues, the differences of DKK3’s mRNA expression between cancer and corresponding adjacent tissues seemed not obvious, moreover no correlation between gene methylation and mRNA’s expression was found. To clarify the regulation of DKK3gene expression and its role in tumorigenesis, our study analyze the correlation between DKK3gene methylation and its protein expression, and further investigated clinical implication of DKK3expression in HCC by examining the expression and subcellular localization of DKK3in HCC.AIM:To examine the expression and subcellular localization of Wnt antagonist Dickkopf-3in hepatocellular carcinoma, analyze the correlation between DKK3gene methylation and its protein expression, and investigate clinical implication of DKK3expression in HCC.METHODS:First, DKK3protein expression in liver cancer cell line SMMC-7721, HepG2, normal liver cell line HL-7702, peripheral blood mononuclear cell (PBMC) and43pairs of HCC and corresponding adjacent tissues were examined by immunofluorescence or immunohistochemistry. Then, DKK3methylation status were assayed by methylation-specific PCR in31cases of HCC and paired adjacent tissues, and the correlation between DKK3methylation and its protein expression were statistically analyzed; Finally, The correlation between DKK3protein expression and clinical pathological data was analyzed.RESULTS:The high expression level of DKK3protein occurred in67.4%(29/43) of HCC tissues; such frequency was significantly higher than that of41.8%(18/43) in the paired adjacent tissues (P=0.017). For the single case of paired tissue, DKK3expression has similar levels in HCC tissues and adjacent tissues is24cases(55.81%), there is15cases(34.88%) that DKK3expression is higher in HCC tissues than adjacent tissues, but there is only4cases(9.30%) that DKK3expression is lower in HCC tissues than adjacent tissues. This comparison may indicate that, DKK3protein tended to express in cell nucleus in HCC tissues and SMMC-7721, HepG2cell lines. DKK3gene methylation were detected in90.3%(28/31) of HCC tissues, which was significantly higher than that in64.5%(20/31) of the corresponding adjacent tissues (P=0.015). There was no correlation between DKK3methylation status and its protein expression(P>0.05). There was correlation between DKK3expression in HCC tissues and relapse(P=0.002), A/G and ALP(P=0.025;0.038), but no correlation between DKK3expression in HCC tissues and other clinical and pathological features of the patients(gender, age, tumor number, liver cirrhosis, the Child-pugh classification, tumor gross type, tumor growth pattern, tumor size and TNM stage, et al)(P>0.05). Kaplan-Meier survival curve showed that, the overall survival of patients with high expression of DKK3is48.3%, the free-disease survival of patients with high expression of DKK3is20.7%; the overall survival of patients with low expression of DKK3is71.4%, the free-disease survival of patients with low expression of DKK3is64.3%. In contrast, patients with low expression of DKK3had higher overall or free-disease survival than those with high expression (overall survival P=0.049; free-disease survival P=0.001).CONCLUSIONS:The expression of DKK3protein in liver tissues was higher than the paired adjacent tissues, and its subcellular location tended to express in cell nucleus. In addition, there was no correlation between DKK3methylation status and its protein expression. These results suggest that DKK3may not play a tumor-suppressor role as one of typical Wnt antagonist in hepatocellular carcinoma, and the methylation status is not sufficient to lead to complete gene inactivation. Finally, DKK3protein expression in HCC tissues may be used as a prognostic maker for hepatocellular carcinoma.