EYA4 gene functions as a prognostic marker and inhibits the growth of intrahepatic cholangiocarcinoma

Background: 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. Methods: One 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. Results: ICC 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). Conclusion: EYA4 gene functioned as a molecular prognostic marker in ICC, and its overexpression inhibited tumor growth in vitro and in vivo.