Role Of Ezrin Gene In Invasion And Metastasis Of Hepatocellular Carcinoma And Biological Impact Of Evodiamine In Hepatocellular Carcinoma Cells HepG2

Part Ⅰ The Expression and clinical significance of Ezrin、 CD44in Hepatocellular CarcinomaObjective:To investigate the correlation of the expression and clinicopathologic significance of Ezrin in hepatocellular carcinoma.Methods:SP immunohistochemical staining was performed in70cases of hepatocellular carcinoma to detect the expression of Ezrin、 CD44. The expression of Ezrin、CD44in70cases of hepatocellular carcinoma were analyzed statistically.Results:1. The Ezrin、CD44were located in cytoplasm;2. There was no significant difference of the expression level of the Ezrin in hepatocellular carcinoma between tumor size and age (P>0.05), But significant difference was found in lymph node involvement(P<0.05);3. Significant difference of the expression level of the CD44v6in hepatocellular carcinoma was found in lymph node involvement (P<0.05). but there was no significant difference in TNM stages(P>0.05);4There was positive correlation between expression level of the Ezrin and CD44(P<0.05).Conclusion:1、Over-expression of Ezrin and CD44has a correlation with their lymphnode involvement and prognosis;2、 Co-detected method of the expression levels of the two proteins can be important significant in detecting the action of hepatocellular carcinoma analyze the potential capacity of transmission and results they give remedy to help clinical doctor to cure the patients. Part II The Effeets and Mechanism of Specific Down-Regulation of Ezrin siRNA on Invasion and Metastasis of Hepatocellular Carcinoma.Objective:To explore the roles of Ezrin in the invasion and metastsis of HCC by down-regulated the expression of Ezrin in human HCC cell line HepG2via small interferong RNA (siRNA).Methods:To investigate the roles of Ezrin in invasion and metastasis of HCC, We down-regulated the expression of Ezrin by siRNA in human HCC cell line HepG2, the invasion and metastasis behaviors of HCC in Ezrin knock-down cells was analyzed by Transwell assay and wound healing assay. Src Phosphorylation was analyzed by immunoprecipitation. The expression status of E-cadherin, N-cadherin and Vimentin was examined by Western Blot. To investigate the roles of Ezrin in the invasion and metastasis in HCC, Ezrin was knock down by siRNA in human HCC cell line HepG2, the status of cell adhesion, cell spreading, and focal adhesion turnover the expression. To explore the effects of Ezrin down-regulation on the expressions and activities of matrix metalloproteinase-2,9and c-jun, we analysed the expressions and activities of them in Ezrin siRNA transfected cells by Western Blot and gelzymography.Results:We found that the invasion and metastasis capabilities of HCC in Ezrin knock-down cells were significantly decreased compared with parental cells. Phosphorylation of Src was significantly inhibited The exPression levels of N-cadherin,Vimentin were significantly lower, while E-cadherin was higher in Ezrin knock-down cells than in normal cells.The results showed promoted cell adhesion of cancer cells with extracellular by cell adhesion assay and increased numbers sizes of focal adhesion by immunofluorescent microscopy. We also observed retarded spreading in Ezrin knock-down cells. We found decreased expressions and activities of MMPs. We also found decreased expression of Phosphorylation of c-junk.Conclusion:1. The invasion and metastasis capabilities of human hepatocellular carcinoma in Ezrin knock-down cells were significantly decreased which is realized by in inhibiting EMT and depressing Src signal path.2.Inhibition of focal adhesion turnover,retarded cell polarity formation and reduced expressions and activities of matrix metalloproteinase-2,9play key roles in Ezrin siRNA induced inhibition of invasion and metastasis of human hepatocellular carcinoma cells; Ezrin silencing may inhibit the invasion and metastasis of HCC and may be a potential target for HCC gene therapy. Part Ⅲ The Biological Impact of Evodiamine in Hepatocellular Carcinoma cells HepG2Objective:To investigate the effect of Evodiamine on ezrin expression and its relation to HepG2cells proliferation, invasiveness and migration.Methods:1、MTT assay was used to observe the cell growth inhibition;2、Colony assay was to observe the cell colony forming units(CFUs);3Matrigel invasion assays and wound-healing experiment were used to study the movement and invasion in Hep2cells;4Expressions of ezrin and phosphate-ezrin protein were detected by Western blotting and ezrin mRNA was detected by reverse transcriptase-polymerase chain reaction(RT-PCR) in HepG2cells treated by Evodiamine.Results:1、Evodiamine inhibited HepG2cell growth with time-and dose-dependent manners. IC50values was37.283μM after48h of Evodiamine exposure;2、Evodiamine inhibited HepG2CFUs and showed dose-dependent manner;3The migration ability of HepG2cells were inhibited, which was detected by scratch method.4Reverse transcriptase polymerase chain reaction(RT-PCR) and Western blotting results revealed that Evodiamine notably down-regulated Ezrin expression at both rnRNA and protein levels, and inhabited its phosphorylation as well as.Conclusion:Evodiamine may suppress hepatocellular carcinoma cell growth by inhibiting its proliferation in a time-and dose-dependent manner,and down-regulate the expression level of ezrin mRNA and ezrin protein as well as its phosphorylation, which is related with the effects of Evodiamine on hepatocellular carcinoma cells. These results suggest that Evodiamine is likely to be valuable for the treatment of human hepatocellular carcinoma.