Studies Of Gene GPC3in The Invasion And Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is among the most common malignancies worldwide, with a high prevalence in Asia and Africa.An increasing trend in the incidence of HCC in the Unite State and Canada. China is among the high incidence area of HCC, with The number of HCC patients is increasing by300,000a year which is the third highest incidence in common tumors. In addition, HCC’s mortality ranked the second in cities and first in rural areas. Surgical resection remains the standard choice of treatment for patients in the early stage of HCC. However, even with radical resection,60-70%of patients develop metastasis and recurrence within5years of surgery (3). Although several clinicopathological features, including a poorly differentiated phenotype, large-sized tumor, and portal venous invasion have been found to contribute to the poor prognosis in HCC patients before operation, the underlying molecular mechanisms of the development of HCC remain unclear. Thus, it is urgent to study the pathogenesis of HCC.Heparan sulfate proteoglycans(HSPGs) are proteins that carry one or more heparin sulfate(HS) chains. These chains consist of long linear polymers of glucuronic acid/N-aceltylglucosamine repeating disaccharide unit. The length of the chains is highly variable, and a significant proportion of the disaccharides are modified by N-deacetylation/N-sulfation, epimerization and O-sulfation. The sulfation confers a highly negative charge to HS chains that allows HSPGs to interact in a rather promiscuous way with proteins that display positively charged domains, including the so called "heparin-binding growth factor" such as fibroblast growth factors (FGFs), Wnts, Hedgehogs(Hhs) and bone morphogenetic proteins(BMPs). Glypicans are a family of HSPGs whose members are bound to the cell surface by a glycosylphosphatidylinositol(GPI) anchor. The mammalian genome contains six members of glypican family (GPC1to GPC3),and two members of this family have been found in Drosophila(dally and dlp).In1996Pilia et al reported that the Glypican-3gene (GPC3) displays loss of function mutations in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre-and post-natal overgrowth, a broad spectrum of visceral and skeletal abnormalities, and an increased risk for the development of embryonic tumors. Subsequent research indicated that mutation of GPC3resulted in disequilibrium of cell proliferation and apoptosis, and caused tumorigenesis. GPC3also could bind to adhesion factor, component part of matrix, growth factor, enzyme, engage in the regulation of cell proliferation, differentiation, adhesion and migration. GPC3is highly expressed in HCC, colon cancer, melanoma, Wilms’ tumor, neuroblastoma, hepatoblastoma cells. On the other hand, GPC3is silenced in breast cancer, mesothelioma, epithelial ovarian cancer and lung adenocarcinoma, which suggested that GPC3played an important role in tumor development.In our previous study, GPC3protein expression was detected in the human common tumor and human normal tissue by using tissue chip. The result indicated that GPC3is high expressed in the tissues of HCC, lung squamous carcinoma, chronic superficial gastritis. On the other hand, GPC3is silenced in gastric carcinoma, breast cancer, epithelial ovarian cancer, pancreatic carcinoma, cervical carcinoma, and cholangiocarcinoma. In addition, expression of GPC3is higher in HCC than that in adjacent tissue. GPC3protein expression was not related with age, gender, pathological grade of HCC patients. Expression of GPC3and its relationship with clinicopathological factors were determined by immunohistochemical analysis in61primary HCC patients. The potential prognostic value of GPC3was investigated by comparing the survival time between HCC patients with high and low GPC3expression. The results demonstrated that GPC3expression was closely related with metastasis/recurrence in an HCC patient who can receive the operation. The risk of metastasis/recurrence after surgery in an HCC patient with high GPC3expression was increased to3.214as compared to that of an HCC patient with low GPC3expression. Survival analysis showed that HCC patients with high GPC3expression had a significantly shorter overall survival time than HCC patients with low GPC3expression. Further, multivariate analysis showed that GPC3expression was a significant, independent prognostic parameter for HCC patients. At the same time, we also found that GPC3level of serum is high in HCC patients than that in liver benign tumor patients. The sensitivity and specificity for HCC diagnosis were:GPC350%and92%, GPC3+AFP73.3%and95%. Furthermore, we found that miR-219-5p was significantly downregulated in83HCC tissues and three HCC cell lines, compared to their non-tumor counterparts. MiR-219-5p expression correlated with tumor size, histological differentiation, and overall survival time in HCC patients. We also found that miR-219-5p could inhibit cell proliferation in vitro and arrest cell cycle at the Gl to S transition. Further research demonstrated that miR-219-5p reduced both the mRNA and protein levels of GPC3. These findings indicated that miR-219-5p exerts tumor-suppressive effects in hepatic carcinogenesis through negative regulation of GPC3expression. Above all, GPC3is a promising HCC tumor marker, which involved in tumor metastasis. On the basis of previous work, RNA interference (RNAi) with a GPC3small hairpin RNA (GPC3shRNA) and pEGFP-Cl/GPC3Plasmid were used to identify the effects of GPC3on the regulation of malignant behaviors of HCC. MHCC97-H and MHCC97-L, had similar genetic background and were thus selected as a cell model for in vitro and in vivo experiments. This article shows a research and analysis that whether GPC3can be used as a new target for early HCC diagnostic marker and targeted therapy.Methods1. Detection of GPC3expression in MHCC-97H and MHCC-97LWestern blot and qPCR were used to examined the GPC3expression of MHCC-97H and MHCC-97L.2. Effect of GPC3overexpression on the biological behaviors of MHCC-97LMHCC97-L cells were grown to60-70%confluency and transfected with different vectors (p-EGFP-C3-GPC3, p-EGFP-C3) using Lipofectamine2000(Invitrogen). Stable transfectants, named MHCC97-L/GPC3and MHCC97-L/neo, were selected in a medium containing400μg/ml G418for14days and later maintained in the medium with100μg/ml G418. qRT-PCR and Western blotting were applied to analyze GPC3mRNA and protein levels, respectively. The biological behaviors of tranfectant were investigated by plate colony formation assay, MTT assay, transwell assay in vitro and subcutaneous tumor model in nude mice assay in vivo.3. Effect of GPC3knockdown on the biological behaviors of MHCC-97LAccording to the manufacturer’s protocol, MHCC97-H cells were grown to60-70%confluency and transfected with different vectors (pGenesil-1-GPC3-shRNA1,2,3,4, pGenesil-1-negative) using Lipofectamine2000(Invitrogen). Stable transfectants, named MHCC97-H/GPC3-shRNA1,2,3,4and MHCC97-H/negative-shRNA, were selected in a medium containing400μg/ml G418for14days and later maintained in the medium with200μg/ml G418. qRT-PCR and Western blotting were applied to analyze GPC3mRNA and protein levels, respectively. One group that transfected GPC3-shRNA would be chosen for further comparison of biological behaviors.4. The mechanism that GPC3affect the biological behaviors of HCCWe analyzed the activation of Akt pathway by studying the levels of pAkt phosphorylation in cells growing both in presence or absence of serum. We detected Akt expression using western blot and qPCR.5. Statistical analysisSPSS19.0software (Abbott Laboratories, North Chicago, IL) was used for statistical analysis. Results of qRT-PCR, western blot analysis, plate clone formation assay and the Transwell assay were assessed using one-way ANOVA. MTT assay, subcutaneous tumo model in nude mice were analyzed by Factorial analysis of variance square analyze Differences were considered statistically significant when P<0.05.Result1. GPC3expression of HCC cell lines MHCC-97H and MHCC-97LCompared with normal liver cell, GPC3was more strongly expressed in the highly metastatic MHCC97-H cells, and more weakly expressed in the non-metastatic, MHCC97-L cell line.2. The effect of overexpression of GPC3on the biological behaviors of HCCThe subclone HCC cancer cell line MHCC-97L, stable expressing GPC3+EGFP and neo+EGFP respectively, were successfully selected, named as MHCC-97L/GPC3, MHCC-97L/con. Compared with the MHCC-97L/neo and MHCC-97L, the GPC3expression level of MHCC-97L/GPC3was significantly increased (P=0.000). We examined cell proliferation in vitro using MTT and plate clone formation assay. The results indicated that the growth of MHCC-97L/GPC3cells in vitro was markedly increased after the transfection of GPC3+EGFP (P=0.000). This indicates a positive relation between the expression of GPC3and the rate of hepatocellular cancer cell growth. In addition, MHCC-97L/GPC3cells, compared with MHCC-97L and MHCC-97L/con cells, had a significant enhanced in their ability to form colonies, and their ability to form colonies correlated with GPC3expression (P=0.000).We examined cell migration and invasion activity in vitro using transwell migration and invasion assay. MHCC-97L/GPC3displayed remarkable increase in migration ability as compared with either MHCC-97L/con or MHCC-97L (P<0.05, for both). The migration cells of MHCC-97L/GPC3was15.480±1.561, while those of the MHCC-97L and MHCC-97L/con groups were5.103±1.994and4.174±0.898, respectively. Reexpression of GPC3cause significantly enhanced migration of MHCC-97L cells. Invasion assays were carried out using Matrigel-coated Transwell culture chambers. After24h invading cells were counted using image analysis. MHCC-97L/GPC3showed much higher invasion activities than either MHCC-97L or MHCC-97L/con (P=0.000, respectively). The invasion cells of the MHCC-97L/GPC3,MHCC-97L, MHCC-97L/con groups were12.521±1.620,4.707±1.131and4.569±1.502, respectively. Reexpression of GPC3led to a significant increase in the invasion of MHCC-97L cells.The effect of GPC3on in vivo tumor growth was assessed by the subcutaneous injection of MHCC-97L/GPC3, MHCC-97L or MHCC-97L/con cells for21days. The results indicated that remarkable increase of tumor size was observed in the MHCC-97L/GPC3group as compared to that of the control group (P=0.000). By day21after cell injection, the average tumor weight (n=4) of the MHCC-97L/GPC3, MHCC-97L and MHCC-97L/con groups was2.987±0.168g,1.801±0.015g and1.758±0.040g, respectively (P=0.000), indicating that reexpression of GPC3in hepatocellular cancer cells enhanced their tumorigenic potential.3. The effect of silence of GPC3on the biological behaviors of HCCWe successfully construct Stable transfectants, named MHCC97-H/GPC3-shRNA1,2,3,4and MHCC97-H/con-shRNA. The qPCR and western blotting were applied to analyze GPC3mRNA and protein levels, respectively. The results indicated that cells transfected with pGenesil-l-GPC3shRNAl showed a significantly reduced transcription of GPC3mRNA when compared with that of vector control and negative transfectants, respectively. The reduction in mRNA transcription was not detectable in cells transfected with pGenesil-l-GPC3-shRNA3and pGenesil-l-GPC3-shRNA4. In addition, Western blot analysis showed a remarkable reduction of GPC3protein levels in the MHCC97-H cell lines transfected with pGenesil-l-GPC3-shRNAl.Thus, MHCC97-H/GPC3-shRNA1and MHCC97-H/con-shRNA were chosen for further comparison of biological behaviors.We examined cell proliferation in vitro using MTT and plate clone formation assay. The results indicated that the growth of MHCC97-H/GPC3-shRNA1cells in vitro was markedly decreased (P=0.000). This indicates a positive relation between the expression of GPC3and the rate of hepatocellular cancer cell growth. In addition, MHCC97-H/GPC3-shRNAl cells, compared with MHCC97-H/con-shRNA and MHCC97-H cells, had a significant reduction in their ability to form colonies, and their ability to form colonies correlated with GPC3expression (P=0.000).We examined cell migration and invasion activity in vitro using transwell migration and invasion assay. MHCC97-H/GPC3-shRNA1displayed remarkable decrease in migration ability as compared with either MHCC97-H/con-shRNA or MHCC-97H (P=0.000, for both). The migration cells of MHCC97-H/GPC3-shRNAl were4.421±1.536, while those of the MHCC-97H and MHCC97-H/con-shRNA groups were14.374±2.015and15.902±1.450, respectively. Downregulation of GPC3cause significantly attenuated migration of MHCC-97H cells. Invasion assays were carried out using Matrigel-coated Transwell culture chambers. After24h invading cells were counted using image analysis. MHCC97-H/GPC3-shRNAl showed much lower invasion activities than either MHCC-97H or MHCC97-H/con-shRNA (P=0.000, respectively). The invasion rates of the MHCC-97H, MHCC97-H/con-shRNA and MHCC97-H/GPC3-shRNAl groups werel5.770±2.025,14.774±1.112, and4.647±1.319, respectively. Silence of GPC3led to a significant decrease in the invasion of MHCC-97H cells.The effect of GPC3on in vivo tumor growth was assessed by the subcutaneous injection of MHCC97-H/GPC3-shRNA1, MHCC-97H or MHCC97-H/con-shRNA cells for21days. The results indicated that remarkable decrease of tumor size was observed in the MHCC97-H/GPC3-shRNAl group as compared to that of the control group (P=0.000). By day21after cell injection, the average tumor weight (n=4) of the MHCC97-H/GPC3-shRNA1, MHCC-97H or MHCC97-H/con-shRNA groups was1.541±0.096g,2.424±0.095g and2.402±0.080g, respectively (P=0.000), indicating that downregulation of GPC3in hepatocellular cancer cells attenuated their tumorigenic potential.4. GPC3overexpression (or silence) promoted(or inhibited) the activation of Akt signal pathwayWe found that whereas this pathway was constitutively active in control cells even in serum withdrawal conditions, GPC3silence induced decrease of phospho-Akt basal levels. In the other hand, compared to the control cells, GPC3reexpression promoted the incease of phospho-Akt. Conclusions1. We established cell line MHCC-97L/GPC3that stable transfected GPC3through using the plasmid transfection mediated by liposomal transfection reagent and found that GPC3could enhance HCC cell proliferation, invasion and migration.2. We established GPC3downregulation cells MHCC-97H through using the plasmid transfection mediated by liposomal transfection reagent and found that knockdown of GPC3could decrease HCC cell proliferation, invasion and migration.3. GPC3reexpression (or downregulation) induces and promotion (or inhibition) of the pro-survival Akt signaling pathway.Novelty1. In this study, we determined that GPC3expression plays an important role on the biological behaviors of HCC cells. Our results provide new clues regarding the mechanism involved in the modulation induced by GPC3of HCC cells growth and survival.2. Now we demonstrate that GPC3is able to enhance the Akt over-activation detected in HCC cells. Thus, GPC3may promote HCC invasion and growth by modulating Akt signaling in part.