The Effects Of Grp78 Knockdown On Invasion And Metastasis Of Hepatocellular Carcinoma

[Background and Objective]Human hepatocellular carcinoma(HCC)is the fifth most common cancer worldwide and causes more than 500,000 deaths annually.Currently the ideas,strategies,modes,techniques and frameworks of HCC therapy have undergone many great changes.The therapeutic modes of multitude special course of studies and multielemen have significantly changed the therapeutic efficacy.However,the outcome of HCC is still very poor for the invasion and metastasis of HCC in early stage.So far invasion and metastasis have been regarded as the most important reasons for HCC recurrence,but the mechanism of invasion and metastasis of HCC remains unclear.Thus it is urgent for the researchers to clarify the underlying mechanism and explore novel therapeutic approaches to block invasion and metastasis of HCC.Glucose-regulated protein 78(GRP78)that is localized in the endoplasmic reticulum is important in the folding,maturation,and transport of proteins out of cells.Lots of studies have indicated the altered GRP78 expression and functions in tumorgenesis and progression.Recently advances have revealed that GRP78 is expressed on the surface of metastatic tumors and elevated expression of GRP78 is associated with increased lymphatic node metastasis.These data suggest GRP78 may play roles in the regulation of tumor invasion and metastasis.In this article,we examined the expression pattern of GRP78,FAK in 44 cases of hepatocellular cancer tissue specimens by immunohistochemistry and down-regulated the expression of GRP78 in human HCC cell line BEL7402 by small interfering RNA(siRNA)to explore the roles of GRP78 in the invasion and metastsis of HCC.[Materials and Methods]Part One.The Correlationship of Grp78 and FAK in HCC tissue samples.We examined the expression pattem of Grp78,FAK in 44 cases of hapatocellular cancer tissue specimens by immunohistochemistrty.The correlation of Grp78 and FAK were analysed.We examined the expression pattern of FAK-p-tyr397 in 44 cases of hapatocellular cancer tissue specimens by immunohistochemistrty.The correlation of FAK phosphorylation at tyr397 with the differention was investigated.Part Tow.The Effects of Specific Down-regulation of Grp78 by siRNA on Invasion and Metastasis of Hepatocellular Carcinoma.To investigate the roles of Grp78 in invasion and metastasis of HCC, we down-regulated the expression of Grp78 by siRNA in human HCC cell line BEL7402,the invasion and metastasis behaviors of HCC in Grp78 knock-down cells was analyzed by Transwell assay and wound healing assay.FAK phosphorylation was analyzed by immunoprecipitation.The activity of RhoA was determined by GST pull-down assay.The expression status of E-cadherin,N-cadherin and Vimentin was examined by Western Blot.Part Three.The Molecular Mechanism of Grp78-siRNA Induced Inhibition of Invasion and Metastasis in Human Hepatocellular Carcinoma Cells.To investigate the roles of Grp78 in the invasion and metastasis in HCC,Grp78 was knockdown by siRNA in human HCC cell line BEL7402, the status of cell adhesion,cell spreading,and focal adhesion turnover the expression.To explore the effects of Grp78 down-regulation on the expressions and activities of matrix metalloproteinase-2,9 and c-jun,we analysed the expressions and activities of them in Grp78-siRNA transfected cells by Western Blot and gel zymography. [Results]Part One.The Correlationship of Grp78 and FAK in HCC tissue samples.We find that the expression of Grp78 and FAK were significantly correlated with the differentiation of HCC(r=0.456,P=0.001;r=0.312, P=0.039)and the expression of Grp78 was also significantly correlated with with FAK(r=0.434,P=0.001),but has no correlation with FAK-p-tyr397. Part Two.The Effects of Specific Down-regulation of Grp78 by siRNA on Invasion and Metastasis of Hepatocellular Carcinoma.We found that the invasion and metastasis capabilities of HCC in Grp78 knock-down cells were significantly decreased compared with parental cells.Phosphorylation of FAK was significantly inhibited and RhoA activity was promoted in Grp78 knock-down cells.The expression levels of N-cadherin,Vimentin were significantly lower,while E-cadherin was higher in Grp78 knock-down cells than in normal cells.Part Three.The Molecular Mechanism of Grp78-siRNA Induced Inhibition of Invasion and Metastasis in Human Hepatocellular Carcinoma 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 Grp78 knock-down cells.We found decreased expressions and activities of MMPs.We also found decreased expression of Phosphorylation of c-junk.[Conelusion]All of our results revealed that:1.The expression of Grp78 and FAK were significantly correlated with the differenciation of HCC;But FAK-p-tyr397 has no correlationship with the differentiation of hepatocellular carcinoma;2.The expression of Grp78 and FAK is significantly correlated in human hepatocellular carcinoma; 3.The invasion and metastasis capabilities of human hepatocellular carcinoma in Grp78 knock-down cells were significantly decreased which is realized by inhibiting EMT and depressing FAK signal path.4.Inhibition of focal adhesion turnover,retarded cell polarity formation and reduced expressions and activities of matrix metalloproteinase-2,9 play key roles in Grp78-siRNA induced inhibition of invasion and metastasis of human hepatocellular carcinoma cells;5.Grp78 silencing may inhibit the invasion and metastasis of HCC and may be a potential target for HCC gene therapy.