Overexpression Of NEDD9 Promotes Cell Invasion And Metastasis In Hepatocellular Carcinoma Expression Pattern Of DIXDC1 In Hepatocellular Carcinoma And Its Correlation With Prognosis

Objective Hepatocellular carcinoma(HCC)is currently one of the most commonly diagnosed cancer in the world.Dispite the advances in diagnosis and tumor ablation procedures have provided an opportunity for a cure or.prolonged life span,the prognosis of HCC patients remains typically poor,largely due to the high rate of tumor recurrence and metastasis.Neural precursor cell expressed,developmentally downregulated 9(NEDD9)is a focal adhesion scaffold protein which has been associated with metastasis of solid tumors.Recently,NEDD9 was reported to be upregulated in HCC,However,the precise function of NEDD9 in HCC is largely unknown.Therefore,in the present study,we aimed to explore the potential role and underlying mechanism of NEDD9 in HCC cell invasion and metastasis both in vitro and in vivo.Methods NEDD9 expression level in human HCC cell lines and HCC tissueswas evaluated by qPCR,Western blotting and immunohistochemical analyses.The clinicopathological significance and prognostic value of NEDD9 were investigated in a cohort containing 140 patients with HCC.The different expression pattern of NEDD9 between non-invasive HCC and invasive HCC was also evaluated.Following construction of NEDD9 over-expression and.knock-down HCC cell lines,we investigated the role of NEDD9 in cell proliferation,invasion and metastasis by MTS assay,transwell migration and matrigel invasion assays in vitro and spleen injection-liver metastasis assay in nude mice.We further explored the underlying mechanisms responsible for the effects of NEDD9 on invasion and metastasis of HCC by qPCR,immunofluorescent analysis,Western blotting and immunohistochemical analysis.Results The relative expression of NEDD9 was significantly higher in most HCC cell lines compared with the normal liver cell lines by qPCR and Western blotting analyses.The upregulated expression of NEDD9 was further evaluated by immunohistochemistry in 140 paired HCC clinical samples and results showed that NEDD9 expression was significantly correlated with serum AFP(p=0.008),tumor thrombi(p=0.005),TNM stage(p=0.014).According to the existence of tumor thrombi in HCC or not,we found the different expression pattern of NEDD9 between non-invasive HCC and invasive HCC tissues.For prognostic analyses,it was observed that patients with high NEDD9 expression had a higher tendency of disease recurrence and 'much shorter survival time by Kaplan-Meier curve assessment.Moreover,a multivariate Cox proportional hazards model revealed that NEDD9 expression was a independent risk factor for disease-free survival in HCC patients.For the subsequent gain-and-loss function studies,the NEDD9 overexpression lentivirus plasmid was successfully constrcted,and the NEDD9 overexpression and knoc-kdown HCC cell lines was established in HCC-LM3 and Huh7 cells.MTS cell proliferation assay demonstrated that NEDD9 knockdown could significantly inhibit cell growth.Transwell migration and matrigel invasion assays showed that NEDD9 depletion resulted in reduced cell migration and invasion,whereas overexpression of NEDD9 exerted the opposite effects.The spleen injection-liver metastasis assay in nude mice showed that ectopic expression of NEDD9 formed more intrahepatic metastases compared with the control group.Mechanically,further studies revealed that NEDD9 inversely regulates E-cadherin in HCC cells and HCC tissues,and FAK was involved in the regulation of E-cadherin induced by NEDD9.Conclusions NEDD9 expression level is upregulated both in HCC cell lines and HCC tissues.High NEDD9 expression is associated with the invasiveness of HCC in clinical samples.NEDD9 could promote the proliferation,invasive and metastastic ability in HCC cell lines.NEDD9 inversely regulates E-cadherin expression,which indicated that NEDD9 may promotes the invasion and metastasis of HCC cells through the downregulation of E-cadherin.Objective The poor prognosis of HCC patients after surgical resection is largely due to the high rate of tumor recurrence and metastasis.As a molecular prognostic indicator for patients with malignant tumor,cancer biomarker exert its important significance for clinical diagnosis and follow-up.DIXDC1(Dishevelled-Axin domain containing 1)is a DIX(Dishevelled-Axin)domain possessing scaffold protein that actsas a positive regulator of the Wnt pathway during neural development.Although DIXDC1 had been investigated in several cancers,to the best of our knowledge,it has not yet been studied in human HCC.Therefore,in this retrospective study,we aimed to investigate the expression pattern of DIXDC1 and assess the clinical significance of DIXDC1 expression in HCC patients.Methods We performed data mining and analyzed DIXDC1 mRNA expression level from the publicly available Oncomine database.The expression level of DIXDC1 was analyzed in 25 paired fresh-frozen HCC tissues and corresponding non-cancerous tissues by qPCR and Western blotting analyses.By using immunohistochemical analysis,we further evaluate the phenotypic expression of DIXDC1 in another independent set of 140 pairs of HCC specimens,and investigated the clinicopathological significance and prognostic value of DIXDC1 in HCC patients.Results Data containing three independent investigations from Oncomine database demonstrated that DIXDC1 mRNA was downregulated in HCC compared with matched non-cancerous tissues.Similar results were also obtained in 25 paired HCC tissues and corresponding non-cancerous tissues by qPCR and Western blot analysis.The reduced expression of NEDD9 was further evaluated by immunohistochemistry in 140 paired HCC clinical samples and results showed that DIXDC1 expression was significantly correlated with s tumor size(p=0.024),tumor differentiation(p<0.001),tumor thrombi(p=0.019),TNM stage(p=0.019),and BCLC stage(p=0.008).For prognostic analyses,it was observed that patients with low NEDD9 expression had a higher tendency of disease recurrence and much shorter survival time by Kaplan-Meier curve assessment.Moreover,a multivariate Cox proportional hazards model revealed that DIXDC1 protein expression was one of the independent prognostic factors for overall survival of HCC patients.Conclusions Our study for the first time revealed that DIXDC1 expression was frequently down-regulated in HCC tissues.Low expression of DIXDC1 was significantly associated with disease progression and poor postoperative outcome of HCC patients.DIXDC1 might serve as a novel prognostic molecular marker for HCC patients after surgical resection.