The Effect Of GPC-3 On The Growth And Invasion Of Hepatocellular Carcinoma

Background and ObjectivesHCC(Hepatocellular carcinoma)is the most common primary malignant hepatic carcinoma and accounts for around 90 percent of liver cancer.HCC is now the second leading cause of cancer in the world,and the incidence is increasing.GPC3 is a functional mutant gene that is commonly found in patients with Simpson Golabi Behmel Syndrome(SGBS)and is a member of the heparan sulfate(HS)proteoglycan family.GPC-3 is anchored to the cell membrane by a glycosylphosphatidylinositol(GPI)anchor.And it can be combined with corresponding ligands through co-receptor,such as growth factors,cell-adhesion-molecules,which participate in the regulating of biological functions of cells.GPC-3 specifically overexpressed in HCC,however,low expressed or no expressed in heathy people and benign liver disorder,which indicates that GPC3 is a promoting factor for the occurrence of HCC.GPC-3 is a potential marker for the diagnosis of HCC,and a new target for immunotherapy of liver cancer.To date,the structure and function relationships of GPC-3 is still not fully understood.Based on the background above,this study aims to investigate the role of GPC-3 in HCC by overexpressing and silencing GPC-3 expression in hepatoma cells.Methods1 The expression level of GPC-3 was detected by western-blot and Q-PCR in multiple HCC cell lines.2 The overexpression GPC-3 plasmid was constructed based on the molecular techenology.3 The knock out(K.O.)GPC-3 plasmid base on CRISPR/Cas9 was constructed.4 The lentivirus were packaging by 3rd-generation lentiviral packaging system for GPC-3 overexpression or GPC-3 K.O..5 Stable GPC3 overexpressed/knockout cell lines were identified by Western-blot and Q-PCR.6 Biological behaviors of the stable cells were proformed,including cell apoptosis,proliferation,migration and invasion.7 The tumorigenic ability of GPC3-K.O.cells were analyzed with subcutaneous tumor-cell-transplantation of nude mice in vivo.Result1 The GPC-3 gene-expression profile were: Hep G2 > Hep3 B > Huh-7 > LM3 >MHCC97H>MHCC97L.Meanwhile,GPC-3 was hardly expressed in SK-hep-1.2 The high GPC-3 protein-expression level were detected in Hep G2,Hep3 B and Huh-7 by Western-blot,while,GPC3 were not detected in the others.3 GPC-3 overexpression/knockout vectors were successfully constructed.4 The MHCC97 L and SK-hep-1 cells with overexpression GPC-3 and the Huh-7 with GPC-3 K.O.were obtained.Successful overexpression/knockdown of GPC-3 were confirmed by Western-blot and Q-PCR5 The proliferation decreased in the stable SK-hep-1,while,it was not significantly changed in the stable MHCC97 L cells.Moreover,the proliferation decreased in the stable Huh-7.6 The migration and invasion increased in the stable SK-hep-1 and MHCC97 L.Moreover,The migration and invasion decreased in the stable Huh-7.7 The apoptosis was no significantly different in all cells.8 The tumorigenesis of GPC3-K.O.cells was lower than that of Mock cells in vivo.ConclusionsGPC-3 can obviously promote the migration and invasion of HCC cells in vitro,but a particular HCC cell type can result in different effect on proliferation in vitro and in vivo,and GPC-3 is not closely related to the apoptosis of HCC cells.The above results suggest that the inhibition of GPC-3 expression in HCC patients can be used as an effective treatment for HCC.