Effects Of GRP78 Secreted By HCC Cells On The Invasion And Metastasis Of HCC

ObjectiveTo investigate the effect of GRP78 secreted by hepatocellular carcinoma cells on the invasion and metastasis of HCC and explore the underlying mechanism.MethodsHCC cell lines SMMC7721 and PLC5 were treated with recombinant human GRP78.The invasiveness of hepatocellular carcinoma was detected by xCELLigence RTCA DP.Cell migration were examined by scratch wound assay.The formation of invadopodia was examined by immunofluorescence and observed with confocal microscope.The expression and phosphorylation level of Cortactinus FAK and Src were detected by Western blotting and the expression of EGFR on the surface of hepatocellular carcinoma cells was detected by flow cytometry.The levels of EGFR on cell surface were evaluated by Flow cytometry analysis.ResultsxCELLigence RTCA DP analysis revealed that treatment of SMMC7721 and PLC5 with recombinant human GRP78 promotes the invasion of HCC in a dose-dependent manner and the optimal concentration was 400 μg/m L.Scratch wound assay showed that at a concentration of 400 μg/m L facilitated the migration of SMMC7721 and PLC5.Immunofluorescence assay and immunoblotting assay showed that rh GRP78 promoted the formation of invadopodia and the phosphorylation of Cortactin(Y421),FAK(Y397)and Src(Y416).Furthermore,xCELLigence RTCA DP analysisalso revealed that the pro-invasive effect of recombinant human GRP78 was inhibited by blockade of EGFR using EGFR monoclonal antibody.ConclusionsThe exocrine GRP78 of hepatocellular carcinoma cells can promote the invasion and metastasis of hepatocellular carcinoma andpromote the formation of invadopodia and the phosphorylation of FAK,Src and Cortactin.EGFR signaling plays important role in this process.