| Objective:To detect the expressions of NEDD9 in hepatocellular carcinoma and adjacent noncancerous tissue(ANT),analyze the relations and clinical significance of overexpression of NEDD9 and clinical pathological features of hepatocellullar carcinoma.To detect the level of NEDD9 in hepatocellullar carcinoma cell lines and study the effect of silencing on the expression of NEDD9 on the proliferation,migration,invasion and apotosis of hepatocellular carcinoma cell lines by RNA interference technique.METHODS:A cohort of 112 patients with HCC who underwent hepatectomy in Tumor Hospital of Guangxi Medical University from 2010 to 2012 was enrolled in this study. All tissue samples were histologically verified HCC. Immunohistochemical and qRT-PCR was used to examine the expression of NEDD9 in hepatocellular carcinoma and adjacent noncancerous tissue(ANT).Western blot and qRT-PCR was examined the expression of NEDD9 in hepatocellullar carcinoma cell lines and HL-7702. small interfering RNA duplexes (siRNA) were designed to silence the endogenous NEDD9. MTT Assay was used to assess antiproliferaton after silencing NEDD9. The apoptosis rate was measured by Flow cytometer. Transwell was used to examine the effect of silencing NEDD9 on the migration and invasion.RESULTS:(1) Except for SK-hep-1, other cell lines including Hep-G2, MHCC-97-H, SSMC-7721 and Huh7 had higher expression levels of NEDD9 than HL-7702 did. Similarly, mRNA and protein levels of NEDD9 were elevated in HCC tissue. levels of NEDD9 protein were up-regulated in four randomly chosen samples. Real-time PCR analyses showed that level of NEDD9 mRNA in HCC tissue was significantly higher than that of ANT in the entire cohort (P=0.002<0.05) and that NEDD9 mRNA increased over twofold in 42.9% (48/112) samples of HCC patients. Taken together, the expression levels of NEDD9 were up-regulated in HCC cell lines and tissue.The expression of NEDD9 in HCC tissue was significantly higer than that in adjacent noncancerous tissue (P=0.002<0.05). (2) The expression of NEDD9 was associated with serum alpha fetal protein (AFP, P=0.029), BCLC stage (P=0.036), size of tumors (P=0.041) and histological grade (P=0.002), but not with age, gender, hepatitis B, Child-Pugh stage, number of tumors, tumor capsule and tumor embolus.The expression level of NEDD9 was correlated with DFS and OS,Patients with high expression of NEDD9 (6-12) had shorter DFS and OS than patients with low expression of NEDD9 (0-5) (DFS:P=0.04; OS:P<0.001). In addition, COX univariate analysis indicated that tumor embolus (P=0.033), tumor size (P=0.001), BCLC stage (P=0.003), serum AFP (P=0.013), histological grade (P<0.001) and NEDD9 expression (P<0.001) were closely related to poor prognosis.(3) The expression level of NEDD9 was significantly downregulated byNEDD9-siRNA. Migration assay demonstrated that numbers of migrated cells were decreased in both MHCC97-H and HepG2 after cells were treated with NEDD9-siRNA. Similarly, invasion assay showed that numbers of MHCC-97-H and HepG2 cells in NEDD9-siRNA group were much less than numbers in corresponding control groups (p<0.001). The MTT assay demonstrated the proliferation of MHCC-97-H and HepG2 cells was significantly suppressed by NEDD9-siRNA (p<0.001). Flow cytometer demonstrated the apoptosis rate of MHCC-97-H and HepG2 cells was raised by NEDD9-siRNA.CONCLUSION:NEDD9 is commonly upregulated in HCC tissu and is associated with prognosis of HCC patients. The expression of NEDD9 was significantly downregulated by NEDD9-siRNA. Proliferation, Migration, invasion of MHCC-97-H and HepG2 cells was significantly suppressed by NEDD9-siRNA. The apoptosis rate was raised by NEDD9-siRNA.In summary, NEDD9 may be a prognostic marker and potential therapeutic target in HCC. |
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