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...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 EVA4-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 diseasefree 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.展开更多
Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)spe...Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.展开更多
基金funded by the National Natural Science Foundation of China (Nos.30672051 and 81472261)
文摘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 EVA4-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 diseasefree 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.
基金supported by National Natural Science Foundation of China(Grant Number 81472261)Natural Science Foundation of Guangdong Province(Grant Numbers 2014A030310033 and 2015A030313032)+1 种基金Science and Technology Planning Project of Guangdong Province(Grant Number 2013B021800122)Science and Technology Planning Projects of Guangzhou City(Grant Number 201604020044).
文摘Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.
