The Expression And Function Of NEDD9 In Hepatocellular Carcinoma

Background: The incidence of hepatocellular carcinoma(HCC) is increasing worldwide, each year about 1 million new diseases was examined. It is also the most common and one of the highest malignant degree of malignant tumor in China. In clinical work, we can always find that many hepatocellular carcinoma patients with intrahepatic metastasis or distant organ metastasis during or shortly after treatment. Although some intervention measures are adopted facing the clinical recurrence of hepatocellular carcinoma, such as TACE, TAE, and biological treatment, but the overall effect of these manners is still very poor. The key molecular mechanism and molecular network of recurrence and metastasis is not entirely clear of HCC. So exploring the molecular mechanism of recurrence and metastasis of HCC, taking effective prevention and treatment is the major problem to be solved in the medical community.NEDD9 is known as Neural precursor cell Expressed Developmentally Down regulated 9,which is also called HEF1(Human enhancer of filamentation 1)and Cas-L,whose m RNA length is 2505 nt. The gene is conservative in all vertebrates, in human chromosome 6 p25-24. NEDD9 molecules is a member of Cas(Crk- associated substrate)family, ang plays a very important role in the metastasis, growth and carcinogenic transformation of tumor cells. Recently some scholars founded that NEDD9 was associated with tumor metastasis in melanoma as a metastasis gene by comparative oncogenomics method. There was NEDD9 amplification and overexpression in human metastatic melanoma. NEDD9 expression reduced by RNAi can reduce the transfer ability of melanoma. NEDD9 and Ras specific signaling pathways work together can enhance the transfer ability of original melanin cells and metastatic melanoma cels. And other scholars study found that NEDD9 is a downstream molecule which can promote migration of malignant glioma cells. Reducing the expression of NEDD9 by si RNA can significantly inhibit cell migration of malignant glioma. However, an article in Nature reports the NEDD9 expression is reduced in a breast cancer cell line with pulmonary metastasis. So NEDD9 may plays a different role in different tumor cells. Recently, the role of NEDD9 in tumor invasion and metastasis caused extensive concern, but the role of NEDD9 and its related molecules in HCC and its regulation network have not been reported.Objectives:To examine the NEDD9 expression in HCC tissues and ANHTs, to analyze the relationship between NEDD9 expression and clinicopathological factors and prognosis; Useing gene transfection and RNA interference technology, knockdown and overexpress the NEDD9 expression, to observe the change of tumor cell proliferation, migration and invasion ability in citro and in vivo; to evaluat NEDD9 as a significance of liver cancer gene therapy targets; To explore the possible mechanism of NEDD9 mediate HCC cell proliferation, migration and invasion.Methods:Through the RT- q PCR and Western blot method to detect NEDD9 m RNA and protein expression level of HCC tissues and ANHTs; Using immunohistochemical staining method to analyze of the relationship between the expression level of NEDD9 and some of the clinical pathological factors; Evaluation by using the survival analysis to explore the effect of NEDD9 on the prognosis of HCC recurrence and survival; To change the NEDD9 expression level of different cancer cells by the method of genetic recombination, and determined the influence of NEDD9 on cell proliferation, migration and invasionability of HCC in vitro by MTT, plate clone formation experiment, scratch repair and Transwell Chambers in membrane experimentesl, respectively.To explore the effect of NEDD9 on cell proliferation, invasion and metastasis of HCC in vivo. To explore the possible molecular mechanisms about how NEDD9 promotes proliferation, migration and invasion of HCC.Results:Part 1: In the experiment, we found that The expression levels of NEDD9 m RNA and protein in HCC tissues were signi?cantly higher than those in matched adjacent non-tumor hepatic tissues. High NEDD9 expression was correlated with larger tumor size, advanced tumor grade, metastasis, intrahepatic venous invasion, and high UICC TNM stages in HCC patients. Patients with high NEDD9 expression levels exhibited poorer recurrence-free and overall survival than those with low NEDD9 expression, and NEDD9 expression status was an independent prognostic factor for survival. Importantly, this correlation remained significant in patients with early-stage HCC or with normal serum AFP levels.Part 2: We found that the expression level of NEDD9 m RNA and protein in the hepatocellular carcinoma cell line was significantly higher than that of normal liver cells. Compared to normal liver cells, HCC cells showed a stronger ability of proliferation, migration and invasion. The difference of proliferation, migration and invasion ability of cells is coincidence with the NEDD9 m RNA level and protein expression level. Using the method of lenti-virus transfection, MHCC-97 H and Huh7 cells were transfected stablely, NEDD9 expression were overpressed and knocked down respectively. Raised NEDD9 gene expression of two types of HCC cells enhanced the proliferation, migration and invasion ability obviously, and lowered NEDD9 gene expression after transfection of two kinds of HCC cells decreased the proliferation, migration and invasion ability in vitro. Subcutaneous transplantation tumor experiments in vivo results show that the MHCC-97 H has certain ability to form tumor, the ability of lower NEDD9 expression MHCC-97 H to form tumor was significantly weaker than the control group, the tumor size wassignificantly smaller. Nude mice pulmonary metastasis of liver cancer found that, normal NEDD9 MHCC-97 H HCC cells have a certain risk of pulmonary metastasis, lower expression NEDD9 of MHCC-97 H HCC cells did not result in a nude mice pulmonary metastasis of liver cancer.Part 3: In the third part of the experiment, we found that NEDD9 may regulate the expression of Fascin protein and its structure reorganization to change the skeleton structure of HCC cells, so as to promote the liver cancer cell migration and invasion. We also found that the increased NEDD9 expression in HCC cells resulted in the increasing of Snail and Slug expression levels, but E-cadherin expression level was then be decreased; When NEDD9 expression was decreased, the expression level of transcription factor Snail and Slug level will also be decreased, and E-cadherin expression level will rised. We found that, increase the expression of NEDD9 in HCC cells, the expression levels of MMP-2 and MMP-9 will rise, however when downregulated NEDD9 expression level, the expression level of MMP-2 and MMP-9 was also be lowered.Conclusions:NEDD9 expression level in HCC tissue increased significantly comparing to ANHT, the expression level of NEDD9 correlated with tumor size, tumor pathologic differentiation degree, metastasis, portal vein invasion and TNM staging. NEDD9 expression level of HCC can predict the recurrence and survival of hepatocellular carcinoma. NEDD9 promoted HCC cell proliferation, migration and invasion and other biological characteristics in vitro and in vivo. NEDD9 may promotes HCC cell proliferation, invasion, migration and other biological characteristics by regulating the expression levels of Fascin protein, rebuilding the skeleton structure, inducing the EMT process, and regulating the expression of MMP-2 and MMP-9.