1,The Role Of NEDD9 In Invasion And Metastasis Of Hepatocellular Carcionma 2,The Expression Of Soluble Human Single Antibody Against Hepatocellualr Carcinoma And Activity Analysis

Hepatocellular carcinoma is one of the most frequent fatal malignancies with high frequency of metastatic recurrence in our country. Most of the mortality associated with HCC arises from uncontrolled metastases. The prevention of the metastatic recurrence of HCC is rare. It is argent for researches and clinicians to explore the molecular mechanism underlying HCC metastasis to conquer the metastasis and recurrence of HCC.ObjectiveTo examine the HCC tumour tissues and HCC cell lines for the expression of NEDD9 and using the siRNA technical to knockdown the expression of NEDD9 in HCC cell line. We will assess the impact of RNAi-mediated NEDD9 knockdown on migration, invasion and proliferation of HCC cells. At last we will explore the relationship between NEDD9 and other molecules related to invasion and metastasis.Methods1. Primary tumour tissues from 43 nonmetastatic HCC and 25 metastatic ones were obtained from patients who underwent surgery in our hospital. Immunohistochemistry was performed to detect the expression of NEDD9.2. Using semi-quantitative RT-PCR to examine the HCC cell lines and corresponding untransformed liver cell line QZG for mRNA expression of NEDD9.3. Two different NEDD9-specific shRNA duplexes were designedhomologous to the NEDD9 mRNA consensus sequence. The annealed oligos were cloned into the plasmid pSilencer3.1-H1 neo. The constructed plasmids were confirmed by DNA sequencing. According to the manufacturer's instruction, the shRNA plasmids were transfected into MHCC97 cells and using RT-PCR and western-blot analysis to examine the inhibition of NEDD9 expression.4. Wound-healing assays and in vitro invasion assays were performed to explore the effect of NEDD9 knockdown on HCC cell migration and invasion.5. MTT assays were used to investigate NEDD9 knockdown on cell proliferation.6. The effect of NEDD9 knockdown on cell cycle of MHCC97 cells was examined by FACS analysis.Results1. NEDD9 was highly expressed in metastatic HCC. A positive rate of NEDD9 expression in nonmetastatic HCC was lower than that in metastatic HCC. The average staining score in metastatic HCC was significantly higher than that in nonmetastatic HCC.2. Over-expression of NEDD9 in human hepatocellular cancer cell lines. We examined the three HCC cell lines and corresponding untransformed liver cell line QZG for mRNA expression of NEDD9 using semi-quantitative RT-PCR. All HCC cell lines showed marked up-regulation of NEDD9 expression. By contrast untransformed liver cell line QZG showed lower level of NEDD9 expression3. NEDD9 specific shRNA vectors were constructed successfully. NEDD9 expression was down-regulated at mRNA and protein level by transient transfection. 4. NEDD9 knockdown inhibits human hepatocellular carcinoma migration and invasion.5. The growth curve of each cell group showed that cell proliferation was slower in the cells transfected with shN1 and shN2 vectors as compared with the cells transfected with control vector shGFP. RNAi-mediated knockdown of NEDD9 expression in MHCC97 decreased cells in S phase compared to cells transfected with shGFP vector.ConclusionOur results indicate that siRNA mediated down-regulation of NEDD9 can inhibit migration, invasion and proliferation of HCC cells which NEDD9 are over-expressed. NEDD9 may play an important role in HCC cells migration, invasion and proliferation Tumor specific scFv has a promising future in the field of tumor diagnosis and therapy. A single-chain antibody (scFv) which is specific for hepatocellular cancer was selected and obtained from the human scFv phage display library in our laboratory previously. The scFv was expressed in prokaryotic expression vector pET-28a, but the product was mainly inclusion body. We can only obtain the active product through complex re-nature procedure.ObjectiveTo construct the prokaryotic expression vector which product is soluble. After expression and purification, we will analysis the biology activity of the scFv.MethodsThe cDNA sequence covering the full open reading frame of scfv-D25 cDNA was digested with EcoRI-Hind III and inserted into the EcoRI-Hind III site of pET-32a to obtain the expression vector pET-32a/scFv-D25. The targeted protein expression was induced by the addition of IPTG in E.coli BL21. The expression of scFv-D25 was identified by SDS-PAGE electrophoresis analysis. After the purification of soluble scfv-D25 by Ni-NTA, we analyzed its activity.ResultsThe scFv fragments were cloned into the expression vector and identified by agarose gel analysis. The soluble scFv-D25 was expressed in E.coli BL21 and purified by Ni-NTA. We obtained the high purified product. The scFv-D25 was identified by indirect ELISA and immuno-fluorescent analysis.ConclusionExpression system of pET32a/scFv-D25 has been successfully constructed. The product is mainly soluble. Soluble scFv-D25 was expressed and purified. The purification can bind to HCC cells specifically. This work laid a solid foundation for its further application in the diagnosis and therapy of HCC.