Abstract
BackgroundThe molecular prognostic markers and carcinogenesis of intrahepatic cholangiocarcinoma (ICC) have not been well documented. The purpose of this study was to investigate the prognostic value of the eyes absent homolog 4 (EYA4) gene in ICC and its biological effects on ICC growth in vitro and in vivo.MethodsOne hundred twelve patients with ICC who underwent hepatectomy were enrolled in the study. EYA4 mRNA and EYA4 protein levels in ICC and adjacent non-tumoral tissues were evaluated using real-time quantitative polymerase chain reaction and immunohistochemical staining, respectively. EYA4 protein levels in ICC cells were determined using western blot analysis. The associations between EYA4 expression and clinicopathologic features of ICC were analyzed. To identify independent prognostic factors, univariate and multivariate analyses were performed. The biological effects of EYA4 on ICC cells were evaluated by establishing stable EYA4-overexpressing transfectants in vitro, and EYA4’s effects on tumor growth were evaluated by intra-tumoral injection of EYA4-expressing plasmids in a NOD/SCID murine model of xenograft tumors.ResultsICC tissues had significantly lower EYA4 mRNA and protein levels compared with adjacent non-tumoral tissues (both P < 0.001). Univariate and multivariate analyses showed that EYA4 protein level, tumor number, adjacent organ invasion, lymph node metastasis, and tumor differentiation were independent prognostic factors for disease-free survival and overall survival (all P < 0.05). In vitro, EYA4 overexpression inhibited tumor cell growth, foci formation, and cell invasiveness. In vivo, intra-tumoral injection of EYA4-expressing plasmids significantly inhibited ICC growth in the murine xenograft model compared with the control group (P < 0.05).ConclusionEYA4 gene functioned as a molecular prognostic marker in ICC, and its overexpression inhibited tumor growth in vitro and in vivo.
Highlights
The molecular prognostic markers and carcinogenesis of intrahepatic cholangiocarcinoma (ICC) have not been well documented
Low eyes absent homolog 4 (EYA4) expression in hepatocellular carcinoma (HCC) tissues was associated with short disease-free survival (DFS) and overall survival (OS), and multivariate analysis showed that EYA4 expression was an independent prognostic factor in HCC patients [15]. These results suggested that the EYA4 gene might play an important role in tumor occurrence and progression, but its prognostic value in ICC and its biological effects on ICC cells remain unknown
EYA4 overexpression suppressed growth of ICC cells in vitro Using two stable EYA4-overexpressing transfectants of ICC cell lines RBE-EYA4 and SSP-EYA4, we studied the biological effects of the EYA4 gene on ICC cells
Summary
The molecular prognostic markers and carcinogenesis of intrahepatic cholangiocarcinoma (ICC) have not been well documented. The purpose of this study was to investigate the prognostic value of the eyes absent homolog 4 (EYA4) gene in ICC and its biological effects on ICC growth in vitro and in vivo. Some clinicopathologic parameters, including serum carbohydrate antigen 19-9 (CA19-9) level, tumor number, adjacent organ invasion, lymph node metastasis, and tumor differentiation, have been shown to be prognostic factors for ICC [5,6,7], the molecular prognostic markers and potential mechanisms of ICC have not been well documented. Some studies showed that EYA family members could combine with sine oculis (SIX1) and dachshund (DACH) to function as transcriptional factors to regulate specific gene expression in mammalian organogenesis [10, 11].
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