Loss-of-function mutations of microRNA-142-3p promote ASH1L expression to induce immune evasion and hepatocellular carcinoma progression

BACKGROUND Hepatocellular carcinoma(HCC)has been a pervasive malignancy throughout the world with elevated mortality.Efficient therapeutic targets are beneficial to treat and predict the disease.Currently,the exact molecular mechanisms leading to the progression of HCC are still unclear.Research has shown that the microRNA-142-3p level decreases in HCC,whereas bioinformatics analysis of the cancer genome atlas database shows the ASH1L expression increased among liver tumor tissues.In this paper,we will explore the effects and mechanisms of microRNA-142-3p and ASH1L affect the prognosis of HCC patients and HCC cell bioactivity,and the association between them.AIM To investigate the effects and mechanisms of microRNA-142-3p and ASH1L on the HCC cell bioactivity and prognosis of HCC patients.METHODS In this study,we grouped HCC patients according to their immunohistochemistry results of ASH1L with pathological tissues,and retrospectively analyzed the prognosis of HCC patients.Furthermore,explored the roles and mechanisms of microRNA-142-3p and ASH1L by cellular and animal experiments,which involved the following experimental methods:Immunohistochemical staining,western blot,quantitative real-time-polymerase chain reaction,flow cytometric analysis,tumor xenografts in nude mice,etc.The statistical methods involved in this study contained t-test,one-way analysis of variance,theχ^(2)test,the Kaplan-Meier approach and the log-rank test.RESULTS In this study,we found that HCC patients with high expression of ASH1L possess a more recurrence rate as well as a decreased overall survival rate.ASH1L promotes the tumorigenicity of HCC and microRNA-142-3p exhibits reduced expression in HCC tissues and interacts with ASH1L through targeting the ASH1L 3′untranslated region.Furthermore,microRNA-142-3p promotes apoptosis and inhibits proliferation,invasion,and migration of HCC cell lines in vitro via ASH1L.For the exploration mechanism,we found ASH1L may promote an immunosuppressive microenvironment in HCC and ASH1L affects the expression of the cell junction protein zonula occludens-1,which is potentially relevant to the immune system.CONCLUSION Loss function of microRNA-142-3p induces cancer progression and immune evasion through upregulation of ASH1L in HCC.Both microRNA-142-3p and ASH1L can feature as new biomarker for HCC in the future.

Expression of the Glypican-3 Gene in α-fetoprotein-negative Human Hepatocellular Carcinoma

Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocelhllar carcinoma （HCC）, especially for AFP-negative ones. Methods： Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results： Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC （73.17%）. In contrast, Glypican-3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors （〉5 cm） （79.31%） and in small tumors （〈5 cm） （41.67%） （P〈0.01）. Gypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones （76.47% vs. 42.86%, P〈0.05）. The Glypican-3 expression was not correlated with age. sex. ttbsAg seropositivity, fibroeapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion： Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocareinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.

Lecithin-cholesterol acyltransferase is a potential tumor suppressor and predictive marker for hepatocellular carcinoma metastasis

BACKGROUND Hepatocellular carcinoma(HCC)is a major cause of cancer mortality worldwide,and metastasis is the main cause of early recurrence and poor prognosis.However,the mechanism of metastasis remains poorly understood.AIM To determine the possible mechanism affecting HCC metastasis and provide a possible theoretical basis for HCC treatment.METHODS The candidate molecule lecithin-cholesterol acyltransferase(LCAT)was screened by gene microarray and bioinformatics analysis.The expression levels of LCAT in clinical cohort samples was detected by quantitative realtime polymerase chain reaction and western blotting.The proliferation,migration,invasion and tumor-forming ability were measured by Cell Counting Kit-8,Transwell cell migration,invasion,and clonal formation assays,respectively.Tumor formation was detected in nude mice after LCAT gene knockdown or overexpression.The immunohistochemistry for Ki67,E-cadherin,N-cadherin,matrix metalloproteinase 9 and vascular endothelial growth factor were performed in liver tissues to assess the effect of LCAT on HCC.Gene set enrichment analysis(GSEA)on various gene signatures were analyzed with GSEA version 3.0.Three machine-learning algorithms(random forest,support vector machine,and logistic regression)were applied to predict HCC metastasis in The Cancer Genome Atlas and GEO databases.RESULTS LCAT was identified as a novel gene relating to HCC metastasis by using gene microarray in HCC tissues.LCAT was significantly downregulated in HCC tissues,which is correlated with recurrence,metastasis and poor outcome of HCC patients.Functional analysis indicated that LCAT inhibited HCC cell proliferation,migration and invasion both in vitro and in vivo.Clinicopathological data showed that LCAT was negatively associated with HCC size and metastasis(HCC size≤3 cm vs 3-9 cm,P<0.001;3-9 cm vs>9 cm,P<0.01;metastatic-free HCC vs extrahepatic metastatic HCC,P<0.05).LCAT suppressed the growth,migration and invasion of HCC cell lines via PI3K/AKT/mTOR signaling.Our results indicated that the logistic regression model based on LCAT,TNM stage and the serum level of α-fetoprotein in HCC patients could effectively predict high metastatic risk HCC patients.CONCLUSION LCAT is downregulated at translational and protein levels in HCC and might inhibit tumor metastasis via attenuating PI3K/AKT/mTOR signaling.LCAT is a prognostic marker and potential therapeutic target for HCC.

Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma

Glypican-3(GPC3)is a cell surface oncofetal proteoglycan that is anchored by glycosylphosphatidylinositol.Whereas GPC3 is abundant in fetal liver,its expression is hardly detectable in adult liver.Importantly,GPC3is overexpressed in hepatocellular carcinoma(HCC),and several immunohistochemical studies reported that overexpression predicts a poorer prognosis for HCC patients.Therefore,GPC3 would serve as a useful molecular marker for HCC diagnosis and also as a target for therapeutic intervention in HCC.Indeed,some immunotherapy protocols targeting GPC3 are under investigations;those include humanized anti-GPC3cytotoxic antibody,peptide vaccine and immunotoxin therapies.When considering the clinical requirements for GPC3-targeting therapy,companion diagnostics to select the appropriate HCC patients are critical,and both immunohistochemical analysis of tissue sections and measurement of serum GPC3 level have been suggested for this purpose.This review summarizes current knowledge regarding the clinical implication of GPC3detection and targeting in the management of patients with HCC.

Diagnostic value of glypican-3 in serum and liver for primary hepatocellular carcinoma

AIM:To evaluate the diagnostic value of glypican-3(GPC3) in serum and liver for primary hepatocellular carcinoma(HCC).METHODS:Serum levels of GPC3 and α-fetoprotein(AFP) were measured in 75 patients with primary HCC and 32 patients with liver cirrhosis.Expression of GPC3 and AFP in 58 HCC and 12 cirrhotic specimens was detected with immunohistochemical staining.RESULTS:When the cut-off value of serum GPC3 was set at 300 ng/L,its sensitivity and specificity for HCC were 47.0% and 93.5%,respectively.Among the 14 patients with HCC at stage according to the Barcelona Clinic Liver Cancer staging system,the serum GPC3 level was higher than 300 ng/L in 50%(7/14) patients,the serum AFP level was not ≥ 400 μg/L in any patient.Combined serum AFP and GPC3 significantly increased the sensitivity to the diagnosis of HCC.The GPC3 expression was detected in cytoplasm of HCC cells but not in hepatocytes and bile ducts of benign tumors.Among the 58 HCC patients,the GPC3 was expressed in 100%(28/28) patients with their serum AFP level ≥ 400 μg/L,and in 90%(27/30) patients with their AFP level < 400 μg/L,respectively.The GPC3 was weakly or negatively expressed in all paracarcinomatous and cirrhotic tissue samples.AFP positive HCC cells were only found in 1 out of the 58 HCC patients.CONCLUSION:GPC3 protein is a sensitive and specific serum marker for diagnosis of early HCC.Its expression in liver tissues can be used to discriminate tumor cells from benign hepatic cells.

Oncofetal antigen glypican-3 as a promising early diagnostic marker for hepatocellular carcinoma

BACKGROUND:Hepatocellular carcinoma (HCC) is characterized by a multi-cause,multi-stage and multi-focus process of tumor progression.Its prognosis is poor and early diagnosis is of utmost importance.This study was undertaken to investigate the dynamic expression of oncofetal antigen glypican-3 (GPC-3) and GPC-3 mRNA in hepatocarcinogenesis and to explore their early diagnostic value for HCC.METHODS:A hepatoma model was induced in male Sprague-Dawley rats with 0.05% 2-fluorenylacetamide and confirmed by hematoxylin and eosin staining and gamma-glutamyltransferase (GGT) expression.Total RNA was purified and transcribed into cDNA by reverse transcription.Fragments of the GPC-3 gene were amplified by nested RT-PCR,and confirmed by sequencing.GPC-3 was analyzed by immunohistochemistry,Western blotting or ELISA.RESULTS:Positive GPC-3 expression showed as brown granule-like staining localized in the cytoplasm.Histological examination of hepatocytes revealed three morphological stages of granule-like degeneration,atypical hyperplasia (precancerous),and cancer formation,with a progressive increase of liver total RNA and GGT expression.The incidence of liver GPC-3 mRNA and GPC-3,and serum GPC-3 was 100%,100% and 77.8% in the HCC group,100%,100%,and 66.7% in the precancerous group,83.3%,83.3%,and 38.9% in the degeneration group,and no expression in the liver or blood of the control group,respectively.There was a positive correlation between liver GPC-3 mRNA and total RNA level (r=0.475,P<0.05) or liver GPC-3 (r=1.0,P<0.001) or serum GPC-3 (r= 0.994,P<0.001).CONCLUSION:Abnormal oncofetal antigen GPC-3 and GPC-3 mRNA expression in hepatocarcinogenesis may be promising molecular markers for early diagnosis of HCC.

Association of glypican-3 expression with growth signaling molecules in hepatocellular carcinoma

AIM:To clarify the association of glypican-3(GPC3)expression with Wnt and other growth signaling molecules in hepatocellular carcinoma(HCC). METHODS:Expression of GPC3,Wnt,matrix metalloproteinases(MMPs),sulfatase(SULF)1,SULF2,and other growth signaling molecules was analyzed in HCC cell lines and tissue samples by real-time reverse transcriptionpolymerase chain reaction,immunoblotting,and/or immunostaining.Expression of various genes in GPC3 siRNA-transfected HCC cells was analyzed. RESULTS:GPC3 was overexpressed in most HCCs at mRNA and protein levels and its serum levels weresignificantly higher in patients with HCC than in non- HCC subjects(P<0.05).Altered expressions of various MMPs and growth signaling molecules,some of which were correlated with GPC3 expression,were observed in HCCs.Down-regulation of GPC3 expression by siRNA in GPC3-overexpressing HCC cell lines resulted in a significant decrease in expressions of MMP2,MMP14,fibroblast growth factor receptor 1,insulin-like growth factor 1 receptor.GPC3 expression was significantly correlated with nuclear/cytoplasmic localization ofβ-catenin. CONCLUSION:These results suggest that GPC3,in conjunction with MMPs and growth signaling molecules, might play an important role in the progression of HCC.

Prognostic and clinicopathological significance of glypican-3 overexpression in hepatocellular carcinoma: A meta-analysis

AIM: To investigate the prognostic and clinicopathological significance of glypican-3 (GPC3) overexpression in hepatocellular carcinoma (HCC). METHODS: Publications were searched using PubMed, EMBASE, the Cochrane Library and the Chinese Biomedical Literature Database up to March 2013. Inclusion and exclusion criteria were established to screen eligible studies for meta-analysis. The hazard ratios (HRs) of the eligible studies were pooled using RevMan 5.2 software to evaluate the impact of GPC3 overexpression on overall survival (OS) and disease-free survival (DFS) in HCC patients. The correlation between GPC3 expression and clinicopathological parameters of HCC was also analyzed. RESULTS: A total of five studies with 493 patients were included in the meta-analysis. The combined HRs indicated that GPC3 overexpression can predict poor OS (n = 362 in 3 studies, HR = 2.18, 95%CI: 1.47-3.24, Z = 3.86, P = 0.0001) and DFS (n = 325 in 3 studies, HR = 2.05, 95%CI: 1.43-2.93, Z = 3.94, P < 0.0001) in HCC patients without heterogeneity. Egger's and Begg's tests were applied to detect publication bias, and the results showed that there was no evidence of publication bias detected in the OS studies (the P value for Egger's test was 0.216) or DFS studies (the P value for Egger's test was 0.488). The combined odds ratios (ORs) suggested that GPC3 expression tends to be associated with tumor vascular invasion (OR = 2.74, 95%CI: 1.15-6.52, P = 0.02), hepatic cirrhosis (OR = 2.10, 95%CI: 1.31-3.36, P = 0.002), poor tumor differentiation (OR = 0.22, 95%CI: 0.13-0.40, P < 0.00001) and advanced TNM stage (OR = 0.31, 95%CI: 0.18-0.51, P < 0.00001). CONCLUSION: From this study, we conclude that GPC3 overexpression tends to be associated with a poor prognosis (poor OS or DFS) in HCC. (C) 2014 Baishideng Publishing Group Inc. All rights reserved.

Glypican-3 is a biomarker and a therapeutic target of hepatocellular carcinoma

BACKGROUND： The carcinogenesis of hepatocellular car- cinoma （HCC） is a multi-factorial, multi-step and complex process. Early diagnosis and effective treatments are of utmost importance. This review summarized the recent studies of on- cofetal glypican-3 （GPC-3）, a membrane-associated heparan sulfate proteoglycan, in the diagnosis and treatment of HCC. DATA SOURCES： English-language reports published from Iune 2001 to September 2014 were searched from MEDLINE. The key words searched included： GPC-3, biomarker, target and HCC. The sensitivity, specificity, positive and negative predictive values were extracted, and the effect of GPC-3 tar- geted therapy on HCC was also evaluated. RESULTS： GPC-3 plays a crucial role in HCC cell prolifera- tion and metastasis. It mediates oncogenesis involving signal- ing pathways during hepatocyte malignant transformation. GPC-3 expression is increased in atypical hyperplasia and cancerous tissues. GPC-3 levels in HCC patients are related to HBV infection, TNM stage, periportal cancerous embolus, and extrahepatic metastasis. The diagnostic accuracy of the combination of serum GPC-3 and alpha-fetoprotein in HCC is up to 94.3%. Down-regulation of GPC-3 with specific siRNA or anti-GPC-3 antibody alters cell migration, metastasis and invasion behaviors. The nude mice xenograft tumor growth is inhibited by silencing GPC-3 gene transcription.CONCLUSION： Oncofetal GPC-3 is a highly specific biomark- er for the diagnosis of HCC and a promising target molecule for HCC gene therapy.

Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism

AIM: To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma (HCC) tumor endothelial cells (TECs).

Prognostic value of glypican-3 in patients with HBV-associated hepatocellular carcinoma after liver transplantation

BACKGROUND：Glypican-3（GPC-3）is frequently overexpressed in hepatocellular carcinoma（HCC）.Recent studies have shown that GPC-3 is a highly efficient diagnostic biomarker of HCC and an indicator of poor prognosis in HCC patients who have undergone hepatectomy.However,its prognostic value in patients with HBV-associated HCC after liver transplantation（LT）is not clear.The present study is to evaluate the prognostic value of GPC-3 in patients with HBV-associated HCC after LT.METHODS： A cohort of 104 HCC patients with HBV-associ- ated cirrhosis who had undergone LT at our hospital between 2002 and 2011 were enrolled in this study. Samples of HCC were taken from these patients. GPC-3 protein expression was detected in paraffin-embedded specimens using immunohis- tochemistry. All related clinical data were obtained from the China Liver Transplant Registry. The relationship between GPC-3 expression and clinicopathological parameters was analyzed. Univariate and multivariate Cox-regression analyses were used to identify risk factors for poor prognosis. RESULTS： GPC-3 was expressed in samples from 74 （71.2%） of the 104 patients. GPC-3 was expressed only in HCC cells. Positive staining was correlated with tumor size （P=0.004）, encapsulation （P=0.018）, pathological stage （P--0.027）, portal vein invasion （P=0.043）, tumor differentiation （P=0.002） and the Milan criteria （P=0.016）. The 5-year survival rate and dis- ease-free survival rate of patients with GPC-3-positive were lower than those （38.2% vs 75.4%, P〈0.001; 30.8% vs 69.7%, P=0.001） of patients with GPC-3-negative. Multivariate Coxregression analysis revealed that GPC-3 was an independent risk factor for 5-year survival rate （P=0.031） and disease-free survival rate （P=0.047）, together with tumor differentiation, Milan criteria and pre-operative alpha-fetoprotein.CONCLUSION： GPC-3 is a potential biomarker for poor prognosis after LT in HCC patients with HBV-associated cirrhosis.

Role of Serum Glypican-3 in the Diagnosis of Hepatocellular Carcinoma in the Upper Egypt

Background and Aim: Early diagnosis of hepatocellular carcinoma (HCC) is essential for achieving good prognosis. Glypican 3 (GPC3) has been reported to be raised in HCC in comparison with non-neoplastic lesions. This work aimed to study the role of GPC3 in the early diagnosis of HCC in post-chronic hepatitis C (CHC) cirrhotic patients. Patients and Methods: A comparative study included 60 patients, 40 patients with HCC (HCC group) and 20 patients of CHC without HCC (control group). Diagnosis of HCC was based on abdominal ultrasound and triphasic CT, while biopsy was performed in debating cases. Serum samples for measurement of GPC3 and AFP levels were obtained from all participants. Results: The median levels of both AFP and GPC3 were significantly higher among HCC cases compared to controls. Analysis of the ROC curve showed that both AFP and GPC3 could be used to differentiate HCC cases from controls. AUROCs of GPC3 and AFP were 0.928 and 0.727 respectively, and both were statistically significant with p-values Conclusion: Serum GLP-3 is highly sensitive and specific for detecting HCC, more than AFP for the early detection of HCC, and the combination of both yielded improved sensitivity.

Glypican-3-specific cytotoxic T lymphocytes induced by human leucocyte antigen-A*0201-restricted peptide effectively kill hepatocellular carcinoma cells in vitro

Objective: To investigate potential human leucocyte antigen(HLA)-A2-restricted epitope peptides of glypican-3(GPC3) and determine the cytotoxicity of peptidespecific cytotoxic T lymphocytes(CTLs) against hepatocellular carcinoma(HCC) cells.Methods: The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3+SMMC 7721 and Hep G2 cells was detected using IFN-g based enzymelinked immunospot and lactate dehydrogenase release assays in vitro.Results: A total of six peptides were identified for bindings to HAL-A2 and the GPC3522–530 and GPC3 229–237 peptides with HLA-A*0201 molecules displayed high binding affinity and stability. The CTLs induced by the GPC3 522–530 or positive control GPC3 144–152 peptide responded to the peptide by producing IFN-g, which were abrogated by treatment with anti-HLA-A2 antibody. The GPC3 522–530-specific CTLs responded to and killed SMMC 7721 and Hep G2 cells, instead of GPC3-silenced SMMC7721 or Hep G2 cells. GPC3 522–530-specific CTLs response to HCC cells was blocked by anti-HLA-A2 antibody.Conclusions: The GPC3 522–530 peptide contains antigen-determinant and its specific CTLs can effectively kill HCC in a HLA-A2-restricted and peptide-dependent manner. Our findings suggest that this peptide may be valuable for development of therapeutic vaccine.

Glypican-3 versus alpha-fetoprotein as a biomarker for hepatocellular carcinoma: a diagnostic meta-analysis

Objective:To assess the diagnostic accuracy of serum GPC3 versus alpha-fetoprotein(AFP)for HCC by using the method of system review.Methods:PubMed and EMBASE were searched from its inception to 20,April 2014 for studies that compared diagnostic accuracy of serum GPC3 with AFP for HCC.Sensitivity,specificity and other measures were pooled using random-effects models.Summary receiver operating characteristic curve analysis was used to summarize the overall test performance.Results:Fourteen studies were included in this meta-analysis.Summary estimates for serum GPC3 and AFP in diagnosing HCC were as follows:sensitivity,69%(95%confidence interval(CI),56-80%)vs.60%(95%CI,50-69%);specificity,91%(95%CI,76-97%)vs.92%(95%CI,84-98%);diagnostic odds ratio(DOR),22(95%CI,6-83)vs.18(95%CI,8-41);and area under sROC,0.85(95%CI,0.81-0.88)vs.0.80(95%CI,0.76-0.83).The pooled sensitivity and specificity for(GPC3+AFP)and AFP were:sensitivity 80%(95%CI,75-85%)vs.64%(95%CI,53-73%)and specificity 86%(95%CI,74-93%)vs.96%(95%CI,86-99%).A significant heterogeneity was found among the ten studies,and meta-regression and subgroup meta-analysis suggested that race and assay type were probably responsible for the heterogeneity.Conclusions:Serum GPC3 may be a promising diagnostic marker of HCC and it was helpful for early detection of primary hepatocellular carcinoma when combined with AFP.More studies for specific race of patients,and using certain methods for detecting GPC3 are required to further confirm the diagnostic value of GPC3 for HCC.

Value of miR-1271 and glypican-3 in evaluating the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

BACKGROUND Hepatocellular carcinoma(HCC)is the third leading cause of cancer death,causing about 750000 deaths worldwide every year.Patients with advanced hepatocellular carcinoma will often only receive transcatheter arterial chemoembolization(TACE).Glypican-3(GPC3)is one of the most promising serum markers for HCC.Abnormal expression of miRNAs may be involved in the occurrence and development of tumor.AIM To explore the value of miR-1271 and GPC3 in evaluating the prognosis of patients with HCC after TACE.METHODS From January 2016 to December 2018,162 patients with advanced HCC who received TACE in our hospital were selected into the cancer group,and 162 patients who underwent physical examination during the same period were selected into the health group.The patients in the HCC group were treated with TACE.The changes of serum GPC3 and circulating miR-1271 in the HCC before and after TACE were analyzed.The expression of serum GPC3 was detected by enzyme-linked immunosorbent assay,and the expression of circulating miR-1271 was detected by real-time quantitative polymerase chain reaction.The methodological results of sensitivity,specificity,and accuracy of miR-1271 and GPC3 alone and joint detection of HCC were also evaluated.RESULTSThe level of serum GPC3 in patients with HCC was significantly higher than that in healthy controls.GPC3 levels were increased in both HCC patients and those treated with TACE compared with healthy controls.After TACE,the level of serum GPC3 was significantly lower than that before treatment(P<0.05),and the level of circulating miR-1271 was significantly higher than that before treatment(P<0.05).There were 112 cases(69.14%)with remission(complete remission+complete remission+stable disease)and 50 cases(30.86%)with relapse disease progression in HCC patients.After TACE,the miR-1271 level in patients with remission and relapse was lower than that in the healthy group,and the GPC3 level was higher than that in the healthy group,the differences were statistically significant(P<0.05).The miR-1271 of relapsed patients was lower than that of remission patients,and the level of GPC3 was higher than that of remission patients,and the difference was statistically significant(P<0.05).The sensitivity of combined detection of miR-1271 and GPC3 was significantly higher than that of single detection,and the difference was statistically significant(P<0.05);while the specificity of the two combined detections was lower than that of the single detection;and the accuracy was slightly higher than that of single detection,but the difference was not statistically significant.CONCLUSION The level of miR-1271 in patients with HCC was significantly increased and the level of GPC3 was decreased after TACE.Monitoring the levels of serum GPC3 and circulating miR-1271 has important clinical reference value for evaluating the prognosis of patients with HCC.The levels of serum GPC3 and circulating miR-1271 may help to determine tumor recurrence,evaluate survival status,and guide the next step of treatment.

New Immunological Approaches in Hepatocellular Carcinoma: Glypican-3 (GPC-3) Opportunities and Challenges

Immunotherapy is one of the strategies to boost natural defenses to fight cancer. Immuno-oncology is an artificial stimulation of the human immune system to recognize and kill selectively neoplastic cells at different stage of transformation. Cancer cells have tumor antigens and the antibody of the immune system, binding them, can detect molecules on their extracellular side of cell membrane. Among these proteins, it is rising in interest and used for early detection of hepatocellular carcinoma (HCC) Glypican-3 (GPC-3) protein. It is a heparan sulfate proteoglycan (HSPG), anchored to the cell membrane of transformed hepatocytes. We investigated its function as key regulator of hepatocytes neoplastic transformation. Noteworthy, GPC-3 protein has been implicated in different pathways from cell growth to cell motility and migration. More recently, GPC-3 has been evaluated as a useful marker for HCC due to its increased expression in the liver during tumorigenesis and its absence in normal liver. Immunotherapy that targets GPC-3 domains and its connected proteins are currently under investigation. These new biomarkers may hold potential for the detection and treatment of HCC and other diseases in which GPC-3 may be overexpressed and/or play a crucial role. This review will summarize the current knowledge regarding the active immunotherapy developed to treat HCC and it will evaluate aspects of GPC-3 (structure and biology) as advantages and potential pitfalls for considering it as a valuable immunotherapeutic target. We also elaborated the current literature with the aim to better understand its biological interactions at a molecular and cellular level to identify alternative or combined targets, due to the existing gap in the literature surrounding GPC-3. The role GPC-3 plays in the hepatocellular carcinoma phenotype can be targeted for a novel immunotherapy strategy that can specify cell-mediated destruction of neoplastic cell that spares normal liver tissue, and it can be exploited as a new serum marker to trend for diagnosis and disease progression measurements. We believe further investigation of its functions and structure, including alternative cellular localizations, is necessary to evaluate GPC-3 as valuable target to cure this cancer.

FOXP3 expression and clinical characteristics of hepatocellular carcinoma

AIM: To study the biological and clinical characteristics of transcription factor forkhead box protein 3 (FOXP3) in hepatocellular carcinoma (HCC). METHODS: We analyzed the expression and localization of FOXP3 in HCC tissues and cell lines to evaluate its biological features. The relationship between FOXP3 staining and clinical risk factors of HCC was assessedto identify the clinical characteristics of FOXP3 in HCC. RESULTS: The mRNA and protein expression of FOXP3 were found in some hepatoma cell lines. Immunohistochemical (IHC) analysis of HCC sections revealed that 48% of HCC displayed FOXP3 staining, but we did not find any FOXP3 staining in normal liver tissues and para-tumor tissues. IHC and Confocal analysis showed that the expressions of FOXP3 were mainly present in the nucleus and cytoplasm of tumor cells in tissues or cell lines. In HCC, the distribution of FOXP3 was similar to that of the cirrhosis, but not to the hepatitis B virus. Those findings implicate that FOXP3 staining seems to be associated with the high risk of HCC. CONCLUSION: The clinical characteristics of FOXP3 in HCC warrants further studies to explore its functions and roles in the cirrhosis and development of HCC.

Values of circulating GPC-3 mRNA and alpha-fetoprotein in detecting patients with hepatocellular carcinoma

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative realtime PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P【0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (χ2 =6.318, P=0.012) or HBV infection (χ2 =23.362, P【0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P【0.001). The combination ofcirculating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.

Auphen and dibutyryl cAMP suppress growth of hepatocellular carcinoma by regulating expression of aquaporins 3 and 9 in vivo

AIM: To investigate whether the regulation of aquaporin 3 (AQP3) and AQP9 induced by Auphen and dibutyryl cAMP (dbcAMP) inhibits hepatic tumorigenesis.METHODS: Expression of AQP3 and AQP9 was detected by Western blot, immunohistochemistry (IHC), and RT-PCR in HCC samples and paired non-cancerous liver tissue samples from 30 hepatocellular carcinoma (HCC) patients. A xenograft tumor model was used in vivo. Nine nude mice were divided into control, Auphen-treated, and dbcAMP-treated groups (n = 3 for each group). AQP3 and AQP9 protein expression after induction of xenograft tumors was detected by IHC and mRNA by RT-PCR analysis. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and histological evaluation were used to detect apoptosis of tumor cells, and the concentration of serum &#x003b1;-fetoprotein (AFP) was measured using RT-PCR and an ELISA kit.RESULTS: The volumes and weights of tumors decreased significantly in the Auphen- and dbcAMP-treated mice compared with the control mice (P &#x0003c; 0.01). The levels of AQP3 were significantly lower in the Auphen treatment group, and levels of AQP9 were significantly higher in thedbcAMP treatment mice than in the control mice (P &#x0003c; 0.01). The reduction of AQP3 by Auphen and increase of AQP9 by dbcAMP in nude mice suppressed tumor growth of HCC, which resulted in reduced AFP levels in serum and tissues, and apoptosis of tumor cells in the Auphen- and dbcAMP-treated mice, when compared with control mice (P &#x0003c; 0.01). Compared with para-carcinoma tissues, AQP3 expression increased in tumor tissues whereas the expression of AQP9 decreased. By correlating clinicopathological and expression levels, we demonstrated that the expression of AQP3 and AQP9 was correlated with clinical progression of HCC and disease outcomes.CONCLUSION: AQP3 increases in HCC while AQP9 decreases. Regulation of AQP3 and AQP9 expression by Auphen and dbcAMP inhibits the development and growth of HCC.

Low glucose metabolism in hepatocellular carcinoma with GPC3 expression

AIM To investigate the relationship between glucose metabolism and glypican-3(GPc3)expression in hepatocellular carcinoma(Hcc).METHODSImmunohistochemical staining of pathological samples for GPc3 and glucose transporter 1(GLUT1),and whole-body ^(18)F-FDG PET/c T for measuring tumour glucose uptake were performed in 55 newly diagnosed Hcc patients.The maximum standard uptake value(s UVmax)and tumour-to-non-tumourous liver uptake(T/NT)ratio were used to quantify ^(18)F-FDG uptake.In vitro ^(18)F-FDG uptake assay of GPc3-expressing Hep G2 and non-GPc3-expressing RH7777 cel ls was used to examine the effect of GPc3 in cellular glucose metabolism.The relationships between GPc3 expression and ^(18)F-FDG uptake,GLUT1 expression,tumour differentiation,and other clinical indicators were analysed using spearman rank correlation,univariateand multiple logistic regression analyses.RESULTSPositive GPc3 expression was observed in 67.3%of Hcc patients,including 75.0%of those with well or moderately differentiated Hcc and 36.4%of those with poorly differentiated Hcc.There was an inverse relationship between GPc3 expression and s UVmax(Spearman correlation coefficient=-0.281,P=0.038)and a positive relationship between GLUT1 expression and sU Vmax(Spearman correlation coefficient=0.681,P<0.001)in patients with Hcc.Univariate analysis showed that two glucose metabolic parameters(sU Vmax and T/NT ratio),tumour differentiation,lymph node metastasis,and TNM stage were all significantly associated with GPc3 expression(P<0.05),whereas GLUT1 expression,sex,age,tumour size,intrahepatic lesion number,and distant metastasis showed no statistical association(P>0.05).Further multivariate analysis revealed that only the T/N ratio was significantly correlated with GPC3 expression in patients with Hcc(P<0.05).In vitro assay revealed that the uptake of ^(18)F-FDG in GPc3-expressing HepG2 cells was significantly lower than that of non-GPc3-expressing RH7777 cells(t=-20.352,P<0.001).CONCLUSIONThe present study demonstrated that GPc3 expression is inversely associated with glucose metabolism,suggesting that GPc3 may play a role in regulating glucose metabolism in Hcc.