Carcinoma, Hepatocellular ; metabolism ; Glypicans ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

Glypican-3 (GPC3) is a key member of Glypican family and plays an important role in the development, angiogenesis and metastasis of hepatocellular carcinoma (HCC). Most HCC overexpresses GPC3, but GPC3 is hardly detected in normal adult liver and benign liver lesions, so it is regarded as a highly specific diagnostic marker and an ideal therapeutic target for HCC. In this study, we cloned the heavy and light chain variable region gene from the monoclonal antibody targeted to GPC3 screened in the previous stage, and linked it with a segment of flexible peptide (Linker) to obtain the single chain antibody against GPC3. The single chain antibody gene was cloned into vector for prokaryotic expression and purified to obtain high purity protein. Detection shows that the single-chain antibody produced by us has the same binding activity with the full-length antibody, and can accurately target the tumor site of Huh7 tumor-bearing model mice after coupling Cy5.5 fluorescence, suggesting that the single-chain antibody has the potential to realize multi-directional liver cancer precise surgical navigation under the guidance of a probe.
Animals ; Antibodies, Monoclonal ; Carcinoma, Hepatocellular/genetics* ; Glypicans/genetics* ; Liver Neoplasms/diagnosis* ; Mice

Sorafenib, as the first approved molecularly targeted agent for hepatocellular carcinoma (HCC), has changed the treatment paradigm for patients with advanced HCC. Although a significant survival advantage has been achieved with sorafenib, the prolongation of survival is modest, even in the cases of Child-Pugh class A. Because of primary resistance and secondary resistance, the anti-tumor effects of sorafenib are limited in a portion of HCC patients. To overcome these limitations of sorafenib, various molecularly targeted therapies have been studied alone or in combination with each other, and also adjuvant to other modalities. The role of sorafenib as an adjuvant or neo-adjuvant therapy needs to be evaluated before and after surgery and locoregional therapies. Because patients with HCC are a highly heterogeneous population in terms of molecular pathogenesis and in terms of the natural course of their disease, development of biomarkers of a response before or during sorafenib treatment and development of other molecularly targeted therapies is imperative for selecting prospective good responders. New agents under development target and block VEGF, VEGFR, PDGFR, FGF, FGFR, EGFR, PI3K/Akt/mTOR, IGFR, MEK, c-MET, glypican-3, JAK2, PD1, CTLA-4, etc. The advent of targeted systemic therapies for advanced HCC may have important implications for the future management of patients with advanced HCC, including a need for improved assessment of disease progression, reliable biomarkers for patient selection, and the use of a multidisciplinary approach.
Biomarkers ; Carcinoma, Hepatocellular* ; Disease Progression ; Drug Therapy* ; General Surgery ; Glypicans ; Humans ; Niacinamide ; Patient Selection ; Phenylurea Compounds ; Vascular Endothelial Growth Factor A

OBJECTIVETo investigate the correlation between the expressions of glypican-3 (GPC3) and Notch1 and their roles in the tumorigenesis and progression of hepatocellular carcinoma (HCC).

METHODSImmunohistochemistry and computerized image analysis were utilized to quantitatively detect the expressions of GPC3 and Notch1 in 30 HCC tissue specimens.

RESULTSIn the 30 HCC specimens, GPC3 expression decreased significantly as the grade of tumor differentiation increased (P<0.05 or P<0.01), while Notch1 expression presented with a reverse pattern of changes (P<0.05 or P<0.01). An obvious negative correlation was found between the expressions of GPC3 and Notch1 in HCC tissues (rp=-0.607, P=0.000; r=-0.692, P=0.000).

CONCLUSIONThe expressions of GPC3 and Notch1 show a negative correlation in HCC, suggesting a possible mechanism for mutual regulation between them to contribute to the tumorigenesis and progression of HCC.

Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Glypicans ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Receptor, Notch1 ; metabolism

Adult ; Asian Continental Ancestry Group ; Case-Control Studies ; Female ; Glypicans ; blood ; Humans ; Male ; Metabolic Syndrome ; blood ; Middle Aged

Objective: To clarify the diagnostic value of magnetic resonance imaging based on liver imaging reporting and data system (LI-RADS) for phosphatidylinositol proteoglycan-3 (GPC3) expression in hepatocellular carcinoma (HCC). Methods: Clinical and pathological data of 95 HCC cases with positive GPC3 expression (+) and 40 HCC cases with negative GPC3 expression (-) were retrospectively analyzed, and their MRI image features based on the 2018 version of LI-RADS were compared. Multivariate logistic regression analysis was used to determine the main predictors of GPC3 expression. Receiver operating characteristic curve was used further to determine the diagnostic efficacy of combined clinical imaging model to predict GPC3 expression. Enumeration data were compared with χ² test or Fisher's exact test. Measurement data were compared using independent samples t-test or Mann-Whitney U-test. Results: There were statistically significant differences between HCC in GPC3 (+) and GPC3(-) group at alpha-fetoprotein (AFP) levels (χ²=31.814, P<0.000 1), and MRI features: capsular enhacement (χ²=4.108, P=0.043), halo type enhancement (χ²=4.847, P=0.028), and lesion apparent dispersion coefficient (ADC) (t=2.552, P=0.011 8). Multivariate regression analysis showed that AFP>20 μg/L (OR=9.358, P<0.000 1) and ADC≤1.404×10^-3 mm²/s (OR=1.003, P=0.017) were independent predictors for GPC3 expression in HCC. The combined model and the area under the curve value for the diagnosis of GPC3(+) in HCC was 0.810, and its diagnostic sensitivity and specificity were 76.8% and 77.5%, respectively. Conclusion: AFP>20 μg/L and ADC≤1.404×10^-3 mm²/s may indicate the expression of GPC3 in HCC, and the combination of the two diagnostic indicators can provide a simple and effective non-invasive diagnostic method for clinical practice.
Biomarkers, Tumor ; Carcinoma, Hepatocellular/pathology* ; Glypicans/metabolism* ; Humans ; Liver Neoplasms/pathology* ; Magnetic Resonance Imaging ; Phosphatidylinositols ; Retrospective Studies ; alpha-Fetoproteins/metabolism*

Objective: To investigate the clinical value of serum glypican-3 (GPC3) detection in predicting recurrence of primary hepatocellular carcinoma (HCC). Methods: Through univariate and multivariate logistic regression analysis, the patients pathologically diagnosed with HCC in our hospital from March 2019 to January 2021 were enrolled as the experimental group (n=113), and patients with follow-up time longer than 6 months were included in the prognosis group(n=64). At the same time,20 healthy individuals and 20 individuals with benign liver disease from the physical examination center were enrolled by simple random sampling as control group (n=40). The serum GPC3 and alpha-fetoprotein (AFP) levels were respectively detected by ELISA and chemiluminescence. Then, the study explored the influential factors of the recurrence in HCC patients and constructed the HCC-GPC3 recurrence predicting model by logistic regression. Results: In the research, the sensitivity of GPC3 for the diagnosis of HCC was 61.95% (70/113) and AFP was 52.21% (59/113), meanwhile, the specificity of GPC3 could reach 87.50% (35/40) and AFP was 90.00% (36/40),respectively; The serum GPC3 levels of HCC patients with progressive stage, tumor size≥3 cm, vascular cancer thrombosis and portal venous thromboembolism were significantly higher than that of HCC patients with early stage, tumor size<3 cm, vascular cancer thrombosis and portal venous thromboembolism (Z=2.677, 2.848, 2.995, 2.252, P<0.05), independent of different ages, presence or absence of ascites, peritoneal metastasis, cirrhosis, intrahepatic metastasis (Z=-1.535, 1.011, 0.963, 0.394, 1.510, P>0.05), respectively. Univariate analysis showed that there were no statistically significant differences between the recurrence group and the non-recurrence group in terms of different age, tumor size, presence or absence of vascular cancer thrombosis, ascites, peritoneal metastasis, cirrhosis and AFP levels (χ²=2.012, 0.119, 2.363, 1.041, 0.318, 0.360, Z=0.748, P>0.05); The ratio of those with the progressive stage, portal venous thromboembolism and intrahepatic metastasis and GPC3 levels were all higher in the recurrence group than in the non-recurrence group (χ²=4.338, 11.90, 4.338, Z=2.805, P<0.05).Including the above risk factors in the logistic regression model, the logistic regression analysis showed that the stage, the presence of portal venous thromboembolism,intrahepatic metastasis and GPC3 levels were correlated with the prognosis recurrence of HCC patients (Wald χ² =4.421, 5.681, 4.995, 4.319, P<0.05), and the HCC-GPC3 recurrence model was obtained as: OcScore=-2.858+1.563×[stage]+1.664×[intrahepatic metastasis]+2.942×[ portal venous thromboembolism]+0.776×[GPC3]. According to the receiver operating characteristic curve(ROC), the area under the curve(AUC)of the HCC-GPC3 prognostic model was 0.862, which was better than that of GPC3 alone (AUC=0.704). The cut-off value of model SCORE was 0.699 (the cut-off value of GPC3 was 0.257 mg/L), furthermore, the total sensitivity and specificity of model were 83.3% and 82.4%, which were better than those of GPC3(60.0% and 79.4%).Kaplan-Meier showed that the median PFS was significantly shorter in HCC patients with high GPC3 levels (≥0.257 mg/L) and high values of the model SCORE (≥0.700) (χ²=12.73, 28.16, P<0.05). Conclusion: Besides diagnosing of HCC, GPC3 can may be an independent risk indicator for the recurrence of HCC and can more efficiently predicting the recurrence of HCC patients when combined with the stage, the presence or absence of intrahepatic metastasis and portal venous thromboembolism.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/diagnosis* ; alpha-Fetoproteins/analysis* ; Biomarkers, Tumor ; Glypicans ; Ascites ; Venous Thromboembolism ; Peritoneal Neoplasms ; Liver Cirrhosis

BACKGROUND/AIMS: alpha-Fetoprotein (AFP) is the biomarker most widely used to detect hepatocellular carcinoma (HCC), despite its suboptimal diagnostic accuracy. Glypican-3 (GPC3) and osteopontin (OPN) are secreted glycoproteins that are reportedly associated with tumorigenesis and metastasis. This study was conducted to evaluate the clinical utility of using plasma GPC3 and OPN as diagnostic biomarkers for HCC. METHODS: We measured the plasma levels of GPC3 and OPN in 120 HCC and 40 chronic liver disease (CLD) patients via an enzyme-linked immunosorbent assay. The diagnostic accuracy of each tumor marker was evaluated using receiver operating characteristic (ROC) curve analysis. RESULTS: The GPC3 levels in the HCC patients (75.8 ng/mL) were significantly higher (p=0.020) than the levels in patients with CLD (66.4 ng/mL). The area under the ROC curve (AUROC) values for GPC3 and OPN were 0.62 and 0.51, respectively. In subgroup analyses, including subgroups of HCC patients with low serum AFP and PIVKA II levels, the AUROC of GPC3 remained relatively high (0.66), and GPC3 showed a high sensitivity (62.1%) for detecting small HCC tumors. CONCLUSIONS: The plasma levels of GPC3 and OPN demonstrated low diagnostic accuracy for HCC. However, GPC3 may have a complementary role in diagnosing HCC in patients with nondiagnostic levels of conventional tumor markers and with small-sized tumors.
Carcinoma, Hepatocellular/*diagnosis ; Enzyme-Linked Immunosorbent Assay ; Female ; Glypicans ; Humans ; Liver Neoplasms/*diagnosis ; Male ; Middle Aged ; Osteopontin/*blood ; ROC Curve ; Tumor Markers, Biological/*blood

PURPOSE: To differentiate the genes that are up-regulated or down-regulated and the cellular morphology in the corneal stromal cells between normal and diabetic rat. METHODS: After isolation with trizol (GIBCO, NY, USA), labeling and purification of complementary DNA(cDNA) probes were carried out using Atlas(TM) Glass Microarrays 1.0. RESULTS: Autoradiographic analysis showed that 35 genes were up-regulated and 8 genes were down-regulated by Atlas Image(TM) Software. Up-regulated genes were as calcium/calmodulin dependent protein kinase I, proteasome 26S subunit 1, prostaglandin F receptor. Down-regulated genes were aquaporin 8, cytochrome c oxidase polypeptide Vb, glypican 1. CONCLUSIONS: There were many differential genes in hormone receptor, voltage gated ion channels, oncogenes and tumor suppressors, G protein between normal and diabetic keratocyte of rat.
Animals ; Cornea* ; Electron Transport Complex IV ; Glass ; Glypicans ; GTP-Binding Proteins ; Ion Channels ; Oncogenes ; Proteasome Endopeptidase Complex ; Protein Kinases ; Rats* ; Stromal Cells

OBJECTIVETo construct glypican-3 (GPC3)-green fluorescent protein eukaryotic expression vector pEGFP-c3-GPC3, and analyze the effect of GPC3 on the proliferation of human hepatoma cell line MHCC-97L.

METHODSThe eukaryotic expression vector pEGFP-c3-GPC3 was constructed with recombinant DNA technique and transfected into MHCC-97L cells via Lipofectamine 2000. The cells stably expressing GPC3 were screened by flow cytometry and G418. The mRNA expression of GPC3 was detected by RT-QPCR method, and the protein expression by Western blotting and fluorescence microscope. The effect of GPC3 gene on the growth of the cells was examined by MTT assay.

RESULTSRestriction endonuclease analysis and DNA sequencing verified correct construction of the recombinant plasmid. The green fluorescence was detected in the transfected MHCC-97L cells under fluorescence microscope. RT-QPCR and Western blotting both confirmed successful expression of GPC3 in MHCC-97L cells. The growth curve showed a significant acceleration of the proliferation of the transfected MHCC97-Lsol;GPC3 cells as compared with MHCC97-L and MHCC97-L/C3 cells (P<0.001).

CONCLUSIONWe have successfully constructed the eukaryotic expression vector pEGFR-c3-GPC3, which allows stable GPC3 expression in MHCC97-L/GPC3 cells. The upregulation of GPC3 expression can stimulate the growth of hepatoma cell line MHCC97-L in vitro.

Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Genetic Vectors ; Glypicans ; pharmacology ; Green Fluorescent Proteins ; genetics ; Humans ; Liver Neoplasms ; pathology ; Plasmids ; Transfection