Effects Of Lectin BS-I And Grp78 O-GalNAc Glycosylation On Migration And Invasion Of Hepatocellular Carcinoma Cells And Its Mechanisms

Hepatocellular carcinoma(HCC)is a malignant tumor in the liver and is the fifth most common malignant tumor worldwide.The mortality rate is second and sixth in men and women.According to statistics,in 2012 alone,there were 782500 new cases of liver cancer in the world,while the number of liver cancer deaths was as high as 745500,of which only China accounted for about 50%of the new cases and deaths.In recent years,with the development of computer tomography(CT)or magnetic resonance imaging(MRI)used in the early detection of HCC,survival rates of HCC patients have been prolonged after surgery,liver transplantation,percutaneous ablation or transarterial chemotherapy.However,the mortality rate of HCC patients is very high,due to the invasion and metastasis of HCC.Thus,exploring novel treatments to prevent the invasion of HCC is needed for improving clinical outcome of this fatal disease.Lectins,a group of non-immune origin proteins,selectively recognize and reversibly bind to specific glycan.They can be used for the detection of abnormal glycosylation in the process of cancer occurrence and development,and also for the treatment of cancer.Based on this,we treated hepatocellular carcinoma cells with different metastatic potential as the research objects,used lectin micraoarray to screen candidate lectin specificly binding to metastasis-associated HCC cells,detected the effect of candidate lectin on biological behavior of metastasis-associated HCC cells and elucidated the related mechanisms.Further,the glycosylation of the main target molecule binding to candidate lectin was investigated and the function of glycosylation of the main target molecule binding to candidate lectin was presented as well.The main work content and related research results include the following two aspects:1.Lectin BS-I inhibits cell migration and invasion via AKT/GSK-3p/p-catenin pathway in hepatocellular carcinomaWe identified lectin BS-I bind to metastasis-associated HCC cells surface glycans by a lectin microarray.The migration and invasion capability of MHCC97L and HCCLM3 were inhibited by 1 ?g/mL or 4 p,g/mL BS-I.These effects of BS-I were mediated by inhibiting the activation of AKT/GSK-3?/?-catenin pathway and depended on specificity of lectin BS-I binding to GalNAc.GSK3? inhibitors rescued BS-I-mediated inhibition of migration and invasion of HCC cell.Further,we identified that lectin BS-I interacted with sGrp78,affected membrane localization of sGrp78 and attenuated the binding of sGrp78 and p85,which resulted in inhibiting the activation of AKT/GSK-3?/?-catenin pathway.Overexpression of Grp78 or p85 rescued BS-I-mediated inhibition of migration and invasion of HCC cell.These findings demonstrated for the first time that BS-I can inhibit the migration and invasion of HCC cells,which provided a new idea for the research and development of drugs for antimetastatic therapy of hepatocellular carcinoma.2.Grp78-promoted migration and invasion of hepatocellular carcinoma cells depends on the O-GalNAc glycosylation at S637 residueWe found that there is no N-glycosylation of cell membrane Grp78 by N-glycosylation site prediction and PNGase F digestion assay.Further,cell membrane Grp78 protein was isolated and recovered from SDS-PAGE and detected by lectin microarray,O-glycosylation was found in membrane Grp78 protein and we speculated the O-glycan of membrane Grp78 protein may contain Gal,GalNAc,GlcNAc,Mannose and Sia according to specificity of lectins to glycan.Next,O-GalNAc glycosylation sites mutation plasmids were constructed and lectin immunoprecipitation assay were preformed to identify the O-GalNAc glycosylation site.The result showed that the S637 residue exist O-GalNAc glycosylation.In addition,we found that overexpression of Grp78 promoted cell migration,invasion and EMT via AKT/GSK-3?/?-catenin pathway,however,the promoting roles was disappeared with mutation at S637 residue.Moreover,distribution of membrane Grp78 depended on the O-GalNAc glycosylation at S637 residue and the formation of the Grp78-p85 complex was attenuated after the O-GalNAc glycosylation site was mutated.These results demonstrated that the O-GalNAc glycosylation of Grp78 at 637 residue affects its function,which provided a potential therapeutic target for antimetastatic therapy of hepatocellular carcinoma.