Hepatocellular Carcinoma Liver Tissues Research Articles

Integrator complex subunit 6 (INTS6) inhibits hepatocellular carcinoma growth by Wnt pathway and serve as a prognostic marker

BackgroundIntegrator complex subunit 6 (INTS6) was found to play a tumour suppressing role in certain types of solid tumours. In this study, we wanted to determine the expression level of INTS6 in hepatocellular carcinoma (HCC) and evaluate its clinical characteristics and mechanisms in HCC patients (Lui and Lu, European Journal of Cancer, 51:S94, 2015).MethodsFirst, we used a microarray analysis to explore the mRNA expression levels in HCC and paired normal liver tissues; second, we used qRT-PCR to measure the INTS6 mRNA levels in a cohort of 50 HCC tissues and adjacent normal liver tissues; third, we used Western blot analyses to detect the INTS6 protein levels in 20 paired HCC and normal liver tissues; fourth, we used immunohistochemistry to determine the INTS6 expression levels in 70 archived paraffin-embedded HCC samples. Finally, we investigated the suppressive function of INTS6 in the Wnt pathway.ResultsHerein, according to the microarray data analysis, the expression levels of INTS6 were dramatically down-regulated in HCC tissues vs. those in normal liver tissues (p<0.05). qRT-PCR and Western blot analyses showed that the INTS6 mRNA and protein expression was significantly down-regulated in tumour tissues compared to the adjacent normal liver tissues (p<0.05). Immunohistochemical assays revealed that decreased INTS6 expression was present in 62.9% (44/70) of HCC patients. Correlation analyses showed that INTS6 expression was significantly correlated with serum alpha-fetoprotein levels (AFP, p =0.004), pathology grade (p =0.005), and tumour recurrence (p =0.04). Kaplan-Meier analysis revealed that patients with low INTS6 expression levels had shorter overall and disease-free survival rates than patients with high INTS6 expression levels (p =0.001 and p =0.001). Multivariate regression analysis indicated that INTS6 was an independent predictor of overall survival and disease-free survival rates. Mechanistically, INTS6 increased WIF-1 expression and then inhibited the Wnt/β-catenin signalling pathway.ConclusionThe results of our study show that down-regulated INTS6 expression is associated with a poorer prognosis in HCC patients. This newly identified INTS6/WIF-1 axis indicates the molecular mechanism of HCC and may represent a therapeutic target in HCC patients.

Identification of Potential Gene Network Associated with HCV-Related Hepatocellular Carcinoma Using Microarray Analysis.

In order to identify potential specific gene networks of Hepatitis C virus (HCV) related hepatocellular carcinoma (HCC), weighted gene co-expression network analysis (WGCNA) was performed, which may provide an insight into the potential mechanism of the HCC development. HCV-related HCC and normal sample data were downloaded from GEO, T test of limma package was used to screen different expression genes (DEGs); KEGG pathway was used to analyze related biochemical pathways, and WGCNA was used to construct clustering trees and screen hub genes in the HCC-specific modules. A total of 1151 DEGs were authenticated between the HCC and normal liver tissue samples, including 433 upregulated and 718 downregulated genes. Among these genes, three specific modules of HCC were constructed, including Tan, Yellow and Cyan, but only Yellow module had a significant enrichment score in substance combination module with three hub genes: SLA2547, EFNA4 and MME. Although Tan and Cyan separately had four and three hub genes, but the bio-functions of them did not have significant enrichment scores (score<2). SLA2547, EFNA4 and MME may play important roles in the substance combination of HCV-related HCC, so studying the function of this gene network may provide us a deeper understanding of HCV-related HCC.

Expression and clinical value of NRP-1 in hepatocellular carcinoma

Objective To determine the expression and clinical value of neuropilin-1 (NRP-1) in hepatocellular carcinoma (HCC). Methods One hundred and fifty-one cases of HCC tissues and 89 cases of healthy liver tissues were chosen to compare the expression of NRP-1 by immunohistochemistry. Then the relationships between different clinical factors and the expression of NRP-1 were analyzed by univariate and multivariate statistical analysis. Moreover, the survival rates were compared by survival analysis between different expressions of NRP-1 in HCC patients. Results Eleven cases were lost to follow-up or died for non HCC disease, and the effective cases in the final study were 140 cases. The positive expression rates of NRP-1 in HCC and normal liver tissues were 65.00% and 35.96% respectively, and the difference was statistically significant (χ2=18.843, P<0.001). According to the expression level of NRP-1, 140 patients with HCC were divided into negative expression group (n=49) and positive expression group (n=91). Univariate analysis showed that the expression of NRP-1 in HCC was correlated with tumor number (χ2=8.025, P=0.005), TNM stage (χ2=26.467, P<0.001), differentiation degree (χ2=15.296, P<0.001), portal vein invasion (χ2=9.054, P=0.003) and hepatic vein invasion (χ2=5.928, P=0.015). Multivariate statistical analysis showed that TNM stage (OR=1.392, 95%CI: 1.121-1.730, χ2=8.950, P=0.003), differentiation degree (OR=1.469, 95%CI: 1.102-1.958, χ2=6.862, P=0.009), portal vein invasion (OR=1.829, 95%CI: 1.157-2.893, χ2=6.665, P=0.010) and hepatic vein invasion (OR=2.161, 95%CI: 1.172-3.987, χ2=6.084, P=0.014) were important factors for NRP-1 expression. The median survival time of NRP-1 negative HCC patients was significantly longer than that of positive group (44 months vs. 23 months), and the difference was statistically significant (χ2=21.922, P<0.001). Conclusion NRP-1 is over-expression in HCC tissue and related to the malignant progress of HCC, and this suggests poor prognosis in patients with HCC. Key words: Carcinoma, hepatocellular; Neuropilin-1; Survival rate; Gene expression

Regulator of Calcineurin 1 Gene Isoform 4, Down-regulated in Hepatocellular Carcinoma, Prevents Proliferation, Migration, and Invasive Activity of Cancer Cells and Metastasis of Orthotopic Tumors by Inhibiting Nuclear Translocation of NFAT1

Individuals with Down syndrome have a low risk for many solid tumors, prompting the search for tumor suppressor genes on human chromosome 21 (HSA21). We aimed to identify and explore potential mechanisms of tumor suppressors on HSA21 in hepatocellular carcinoma (HCC). We compared expression of HSA21 genes in 14 pairs of primary HCC and adjacent noncancer liver tissues using the Affymetrix HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). HCC tissues and adjacent normal liver tissues were collected from 108 patients at a hospital in China for real-time polymerase chain reaction and immunohistochemical analyses; expression levels of regulator of calcineurin 1 (RCAN1) isoform 4 (RCAN1.4) were associated with clinical features. We overexpressed RCAN1.4 from lentiviral vectors in MHCC97H and HCCLM3 cells and knocked expression down using small interfering RNAs in SMMC7721 and Huh7 cells. Cells were analyzed in proliferation, migration, and invasion assays. HCC cells that overexpressed RCAN1.4 or with RCAN1.4 knockdown were injected into livers or tail veins of nude mice; tumor growth and numbers of lung metastases were quantified. We performed bisulfite pyrosequencing and methylation-specific polymerase chain reaction analyses to analyze CpG island methylation. We measured phosphatase activity of calcineurin in HCC cells. RCAN1.4 mRNA and protein levels were significantly decreased in primary HCC compared with adjacent noncancer liver tissues. Reduced levels of RCAN1.4 mRNA were significantly associated with advanced tumor stages, poor differentiation, larger tumor size, and vascular invasion. Kaplan-Meier survival analysis showed that patients with HCCs with lower levels of RCAN1.4 mRNA had shorter time of overall survival and time to recurrence than patients whose tumors had high levels of RCAN1.4 mRNA. In HCC cell lines, expression of RCAN1.4 significantly reduced proliferation, migration, and invasive activity. HCC cells that overexpressed RCAN1.4 formed smaller xenograft tumors, with fewer metastases and blood vessels, than control HCC cells. In HCC cells, RCAN1.4 inhibited expression of insulin-like growth factor 1 and vascular endothelial growth factor A by reducing calcineurin activity and blocking nuclear translocation of nuclear factor of activated Tcells (NFAT1). HCC cells incubated with the calcineurin inhibitor cyclosporin A had decreased nuclear level of NFAT1. HCC cells had hypermethylation of a CpG island in the 5' regulatory region of RCAN1.4, which reduced its expression. RCAN1.4 is down-regulated in HCC tissues, compared with non-tumor liver tissues. RCAN1.4 prevents cell proliferation, migration, and invasion invitro; overexpressed RCAN1.4 in HCC cells prevents growth, angiogenesis, and metastases of xenograft tumors by inhibiting calcineurin activity and nuclear translocation of NFAT1.

Epigenome-wide association study in hepatocellular carcinoma: Identification of stochastic epigenetic mutations through an innovative statistical approach.

Hepatocellular carcinoma (HCC) results from accumulation of both genetic and epigenetic alterations. We investigated the genome-wide DNA methylation profile in 69 pairs of HCC and adjacent non-cancerous liver tissues using the Infinium HumanMethylation 450K BeadChip array. An innovative analytical approach has been adopted to identify Stochastic Epigenetic Mutations (SEMs) in HCC.HCC and peritumoral tissues showed a different epigenetic profile, mainly characterized by loss of DNA methylation in HCC. Total number of SEMs was significantly higher in HCC tumor (median: 77,370) than in peritumoral (median: 5,656) tissues and correlated with tumor grade. A significant positive association emerged between SEMs measured in peritumoral tissue and hepatitis B and/or C virus infection status. A restricted number of SEMs resulted to be shared by more than 90% of HCC tumor samples and never present in peritumoral tissue. This analysis allowed the identification of four epigenetically regulated candidate genes (AJAP1, ADARB2, PTPRN2, SDK1), potentially involved in the pathogenesis of HCC.In conclusion, HCC showed a methylation profile completely deregulated and very far from adjacent non-cancerous liver tissues. The SEM analysis provided valuable clues for further investigations in understanding the process of tumorigenesis in HCC.

Fatty acid-binding protein 5 function in hepatocellular carcinoma through induction of epithelial-mesenchymal transition.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with poor prognosis. The correlation between overexpression of fatty acid‐binding protein 5 (FABP5) and malignant potential of tumor growth and metastasis in several cancers has been previously reported. However, the correlation between FABP5 expression and HCC malignant behavior remains unknown. We compared FABP5 expression and patient characteristics in paired HCC and adjacent noncancerous liver tissues from 243 patients who underwent surgical resection of primary HCC. Cell proliferation, invasion, and migration assays were performed in HCC cell lines overexpressing FABP5 or downregulated for FABP5. Tumor growths were monitored in xenograft model, and liver and lung metastasis models were established. In the 243 HCC patients, FABP5‐positive staining (n = 139/243, 57.2%) was associated with poor prognosis and recurrence (P < 0.0001) and showed positive correlation with distant metastasis, tumor size and vascular invasion (P < 0.05). Cell proliferation, invasion, and migration in vitro were enhanced by upregulation of FABP5 and decreased by downregulation of FABP5 in HCC cell lines. Similar results in tumor formation and metastasis were obtained through in vivo analyses. PCR array results revealed upregulation of SNAI1 in FABP5‐overexpressing HepG2 cells. Western blot analysis showed significantly increased expression of E‐cadherin and ZO‐1 and decreased SNAI1 expression and nuclear translocation of β‐catenin by knockdown of FABP5. We revealed a significant role for FABP5 in HCC progression and metastasis through the induction of epithelial‐to‐mesenchymal transition. FABP5 may be a potential novel prognostic biomarker and new therapeutic target for HCC.

An imaging target TGF-β1 for hepatocellular carcinoma in mice.

The TGF-β1 antibody was obtained from Bioss Inc. The Huh-7 cell line (Liver Cancer Institute, Zhongshan Hospital, Fudan University) is a HCC cell line with high metastatic potential. Each mouse was subcutaneously injected with 5×106/0.1mL Huh-7 cells in the right upper flank region for the establishment of a subcutaneous xenograft model. The Iodogen method was used to label TGF-β1 antibody with 125I. In this experiment, 100μL of 125I- TGF-β1 antibody solution, which contained approximately 18,5MBq of 125I-liraglutide, was injected into the tail veins of each of three nude mice with Huh-7 HCC. Micro SPET/CT imaging was performed for each mouse using a nano SPET/CT. The average percentage of injected dose per gram of tissue (ID%) was 1,3% and 2,4%. The tumor was strongly positive for TGF-β1. This pilot study provides an experimental basis for further exploration of the feasibility of TGF-β1 receptor as a target in HCC imaging and in other cancers.

The influence of TNF-α and Ang II on the proliferation, migration and invasion of HepG2 cells by regulating the expression of GRK2.

Hepatocellular carcinoma (HCC) is a common digestive system malignancy that is associated with a poor prognosis. This study researched the interaction of tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) in HCC cells proliferation, migration and invasion and examined their influence on the expression of G protein-coupled receptor kinase 2 (GRK2) and relevant receptors. Cell Counting Kit-8 and Transwell assays were performed to evaluate the effects of TNF-α and Ang II on HepG2 cells proliferation, migration and invasion. Flow cytometry was used to investigate the expression of tumor necrosis factor receptor 1 (TNFR1), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R) on the surface of HepG2 cells. Additionally, Western blot was performed to assess the modulation of GRK2 expression by TNF-α and Ang II in HepG2 cells. Meanwhile, GRK2 siRNA-transfected HepG2 cells were used to confirm the effects of GRK2, TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HCC cells. Finally, the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues were tested by immunohistochemistry. The data demonstrated that TNF-α and Ang II can enhance the proliferation, migration and invasion of HepG2 cells through suppressing GRK2 expression but that the two reagents combined did not have synergistic effects. Moreover,overexpression of TNFR1 and AT1R perhaps promoted the formation and progression of HCC, while high AT2R expression had the opposite effect. This study provides new ideas for the prevention and treatment of HCC by researching the interaction and probable mechanism of different bioactive factors associated with HCC.

FOXM1 regulates glycolysis in hepatocellular carcinoma by transactivating glucose transporter 1 expression.

The Forkhead box M1 (FOXM1) transcription factor plays crucial roles in the initiation and progression of various malignancies, including hepatocellular carcinoma (HCC). However, the mechanism by which FOXM1 regulates cancer metabolism remains unclear. In the present study, overexpression and RNA interference (RNAi) approaches were used to investigate the role of FOXM1 in the regulation of glycolysis invitro. Luciferase reporter assays were used to explore the transcriptional regulation of the glucose transporter1 (GLUT1) promoter by FOXM1. Then, immunohistochemical staining was used to examine the expression of FOXM1 and GLUT1 in 100paired HCC and adjacent non-cancerous liver tissues. Chi-square test and logistic regression analysis were performed to evaluate the association between FOXM1 and GLUT1 expression with clinicopathological characteristics. Our data showed that FOXM1 promoted glycolysis in the HCC cells. FOXM1 knockdown significantly reduced the expression of GLUT1 among key glycolysis-related molecules in the different HCC cell lines. Glucose uptake and lactate production assay showed that FOXM1 positively regulated glycolysis based on GLUT1 expression. Moreover, FOXM1 overexpression increased and knockdown decreased GLUT1 expression. Luciferase reporter assays showed that the -206 to -199bp region of the GLUT1 promoter is important for FOXM1 to enhance GLUT1 promoter activity. The results of the IHC analysis showed that the protein expression of FOXM1 and GLUT1 was closely related to the tumor histological grade and TNM stage. In addition, GLUT1 expression was also related to microvascular invasion. In conclusion, overexpression of FOXM1 and GLUT1 may play critical roles in HCC. FOXM1 promotes HCC glycolysis by transactivating GLUT1 expression.

NEK2 serves as a prognostic biomarker for hepatocellular carcinoma.

Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is a microtubule-associated protein that regulates spindle assembly in human cells and is overexpressed in various malignancies. However, the role of NEK2 in hepatocellular carcinoma (HCC) remains undetermined. We performed RNA-seq of the HCC cell line SMMC-7721 and the normal liver cell line HL-7702 using the Ion Proton System. NEK2 expression was detected using quantitative reverse transcription polymerase chain reaction in two cell lines and 5 matched HCC and adjacent non-tumorous liver tissues. The correlation between survival and NEK2 expression was analyzed in 359 patients with HCC using RNASeqV2 data available from The Cancer Genome Atlas (TCGA) website (https://tcga-data.nci.nih.gov/tcga/). The expression of NEK2, phospho-AKT and MMP-2 was evaluated by immunohistochemistry in 63 cases of HCC and matched adjacent non-tumorous liver tissues. Relationships between protein expression and clinicopathological parameters were assessed, and the correlations between NEK2 with phospho-AKT and MMP-2 expressions were evaluated. A total of 610 differentially expressed genes (DEGs) were revealed in the transcriptome comparison, 297 of which were upregulated and 313 were downregulated in HCC. NEK2, as the most obviously different DEG in cells and tissues from the RNA-seq data, was listed as an HCC candidate biomarker for further verification. NEK2 was overexpressed in HCC cells and tissues (P=0.002, P=0.013) and HCC patients with a high expression of NEK2 had a poor prognosis (P=0.0145). Clinical analysis indicated that the overexpression of NEK2 in HCC was significantly correlated with diolame complete (P<0.001), tumor nodule number (P=0.012) and recurrence (P=0.004). NEK2 expression was positively correlated with the expression of phospho-AKT (r=0.883, P<0.01) and MMP-2 (r=0.781, P<0.01). Overexpression of NEK2 was associated with clinicopathological characteristics and poor patient outcomes, suggesting that NEK2 serves as a prognostic biomarker for HCC. Alteration of NEK2 protein levels may contribute to invasion and metastasis of HCC, which may occur through activation of AKT signaling and promotion of MMP-2 expression.

Molecular alterations in the carcinogenesis and progression of hepatocellular carcinoma: Tumor factors and background liver factors.

Although hepatocellular carcinoma (HCC) is associated with poor prognosis worldwide, the molecular mechanisms underlying the carcinogenesis and progression of this disease remain unclear. Several tumor characteristics have previously been demonstrated to be prognostic factors of survival following hepatic resection, or the recurrence of HCC or other types of cancer. Comparisons of normal tissues and HCC tumor tissues have revealed the presence of numerous molecular alterations in HCC, including genetic and epigenetic mechanisms, particularly mutations in certain genes and DNA methylation in the promoter regions of tumor-suppressor genes. A number of studies have previously used array analysis to detect variations in the expression levels of cancer-associated genes and microRNAs, and in DNA methylation. However, an investigation of HCC tumor tissues may not determine the effect of noncancerous liver tissues (background liver) in patients with HCC. As HCC may recur multicentrically following resection, a damaged or chronically diseased HCC background liver may be considered as a pre-cancerous organ. Therefore, the influence of the background liver on HCC requires further study. Detailed studies regarding the background liver may be essential for the improved understanding of the carcinogenesis and progression of this malignancy; however only a few studies have investigated the microenvironment of the HCC background liver. The present review discusses prior molecular studies of hepatocarcinogenesis that focus on HCC and background liver tissues.

BCAT1 promotes tumor cell migration and invasion in hepatocellular carcinoma.

Branched-chain amino acid transaminase 1 (BCAT1) has been associated with numerous types of tumors; however, few previous studies have evaluated the expression and role of BCAT1 in hepatocellular carcinoma (HCC). In the present study, the expression of BCAT1 was detected by reverse transcription-quantitative polymerase chain reaction and immunoblotting in six HCC cell lines and 74 pairs of HCC and adjacent non-cancerous liver tissues. In addition, the correlation between the expression levels of c-Myc and BCAT1 was analyzed using immunohistochemistry. Furthermore, RNA silencing was performed using c-Myc-specific or BCAT1-specific small interfering RNA, after which wound healing and Transwell cell invasion assays were performed. Finally, the clinicopathological characteristics of BCAT1 in patients with HCC were analyzed. It was shown that the expression of BCAT1 was significantly higher in HCC tissues compared with adjacent non-tumor tissues (P<0.001), and in HCC cell lines compared within the L-02 hepatic cell line (P<0.001). In addition, immunohistochemical analyses indicated that the expression of BCAT1 was positively correlated with c-Myc (r=0.706, P<0.001). BCAT1 expression was shown to be downregulated in c-Myc-knockdown cells, and silencing of BCAT1 expression reduced the invasion and migration of HCC cells. Furthermore, a clinical analysis indicated that BCAT1 expression in HCC tissues was significantly associated with the tumor-node-metastasis stage, tumor number and tumor differentiation (all P<0.05), and that BCAT1 was able to predict the 5-year survival and disease-free survival rates of patients with HCC (both P<0.001). The results of the present study suggested that BCAT1 expression is upregulated in patients with HCC, and that BCAT1 may serve as a potential molecular target for the diagnosis and treatment of HCC.

Association of the GLB1 rs4678680 genetic variant with risk of HBV-related hepatocellular carcinoma.

Accumulated evidences demonstrated that GLB1 is involved in cell senescence and cancer development. The GLB1 rs4678680 single nucleotide polymorphism (SNP) has been identified as a hepatocellular carcinoma (HCC) susceptibility polymorphism by a genome-wide association study in Korean population previously. However, little or nothing was known about its involvement and functional significance in hepatitis B viruses (HBV)-related HCC in Chinese. Therefore, we investigated the association between the GLB1 rs4678680 SNP and HBV-related HCC risk as well as its biological function in vivo. Genotypes were determined in two independent case-control sets from two medical centers of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The potential regulation role the rs4678680 genetic variant on GLB1 expression was examined with HCC and normal liver tissues. We found that The rs4678680 G allele was showed to be risk allele; individuals with the TG genotype had an OR of 1.51 (95% CI = 1.10–2.07, P = 0.010, Shandong set) or 1.49 (95% CI = 1.11–1.99, P = 0.008, Jiangsu set) for developing HBV-related HCC, respectively, compared with individuals with the TT genotype. This association was more pronounced in males, individuals aged older than 57 years and drinkers (all P < 0.05). In the genotype-phenotype correlation analyses of fifty-six human liver tissue samples, rs4678680 TG or GG was associated with a statistically significant increase of GLB1 mRNA expression (P < 0.05). Our data indicated that the GLB1 rs4678680 SNP contributes to susceptibility to develop HBV-related HCC, highlighting the involvement of GLB1 and cell senescence in etiology of HCC.

MBD2 as a novel marker associated with poor survival of patients with hepatocellular carcinoma after hepatic resection.

Methyl-CpG binding domain 2 (MBD2) leads to the silencing of methylated genes in cancer cells and was implicated in the activation of prometastatic genes in hepatocellular carcinoma (HCC). The present study aimed to investigate the expression status of MBD2 in HCC and the correlation with surgical outcomes. The correlation between clinical prognostic factors and MBD2 were also evaluated. MBD2 expression was analyzed by western blotting in 20 paired HCC and paratumor liver (PTL) tissues. In addition, immunohistochemistry was performed on the 159 HCC samples following hepatic resection performed between January 2003 and October 2008. The correlation between clinicopathological factors and MBD2 expression was also evaluated by statistical analysis to determine the prognostic value of MBD2 expression in HCC. Postoperative prognostic factors were evaluated using univariate and multivariate analyses. Compared with PTL tissues, MBD2 expression was shown to be upregulated in 10 of the 20 HCC tissues (50%) by western blotting. The immunohistochemistry data indicated significant increase of the MBD2 expression level in 81 cases (50.94%) compared with the PTL tissues (0/159, 0%, P<0.001). The upregulated MBD2 expression in HCC tissues was correlated with BCLC stage B, tumor size >5 cm and microscopic vascular invasion. Multivariate analysis revealed that MBD2 was an independent prognostic factor for overall survival [HR, 2.089; P=0.001] and disease-free survival (HR, 1.601; P=0.022). In conclusion, MBD2 expression was elevated in HCC tissue, which suggesting MBD2 as a candidate prognostic marker of HCC.

NDUFA4L2 Fine-tunes Oxidative Stress in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) lacks effective curative therapy. Hypoxia is commonly found in HCC. Hypoxia elicits a series of protumorigenic responses through hypoxia-inducible factor-1 (HIF1). Better understanding of the metabolic adaptations of HCC cells during hypoxia is essential to the design of new therapeutic regimen. Expressions of genes involved in the electron transport chain (ETC) in HCC cell lines (20% and 1% O2) and human HCC samples were analyzed by transcriptome sequencing. Expression of NDUFA4L2, a less active subunit in complex I of the ETC, in 100 pairs of HCC and nontumorous liver tissues were analyzed by qRT-PCR. Student t test and Kaplan-Meier analyses were used for clinicopathologic correlation and survival studies. Orthotopic HCC implantation model was used to evaluate the efficiency of HIF inhibitor. NDUFA4L2 was drastically overexpressed in human HCC and induced by hypoxia. NDUFA4L2 overexpression was closely associated with tumor microsatellite formation, absence of tumor encapsulation, and poor overall survival in HCC patients. We confirmed that NDUFA4L2 was HIF1-regulated in HCC cells. Inactivation of HIF1/NDUFA4L2 increased mitochondrial activity and oxygen consumption, resulting in ROS accumulation and apoptosis. Knockdown of NDUFA4L2 markedly suppressed HCC growth and metastasis in vivo HIF inhibitor, digoxin, significantly suppressed growth of tumors that expressed high level of NDUFA4L2. Our study has provided the first clinical relevance of NDUFA4L2 in human cancer and suggested that HCC patients with NDUFA4L2 overexpression may be suitable candidates for HIF inhibitor treatment. Clin Cancer Res; 22(12); 3105-17. ©2016 AACR.

Significant change of cytochrome P450s activities in patients with hepatocellular carcinoma.

The lack of information concerning individual variation in drug-metabolizing enzymes is one of the most important obstacles for designing personalized medicine approaches for hepatocellular carcinoma (HCC) patients. To assess cytochrome P450 (CYP) in the metabolism of endogenous and exogenous molecules in an HCC setting, the activity changes of 10 major CYPs in microsomes from 105 normal and 102 HCC liver tissue samples were investigated. We found that CYP activity values expressed as intrinsic clearance (CLint) differed between HCC patients and control subjects. HCC patient samples showed increased CLint for CYP2C9, CYP2D6, and CYP2E1 compared to controls. Meanwhile, CYP1A2, CYP2C8, and CYP2C19 CLint values decreased and CYP2A6, CYP2B6, and CYP3A4/5 activity was unchanged relative to controls. For patients with HCC accompanied by fibrosis or cirrhosis, the same activity changes were seen for the CYP isoforms, except for CYP2D6 which had higher values in HCC patients with cirrhosis. Moreover, CYP2D6*10 (100C>T), CYP2C9*3 (42614 A>C), and CYP3A5*3 (6986A>G) polymorphisms had definite effects on enzyme activities. In the HCC group, the CLint of CYP2D6*10 mutant homozygote was decreased by 95% compared to wild-type samples, and the frequency of this homozygote was 2.8-fold lower than the controls.In conclusion, the activities of CYP isoforms were differentially affected in HCC patients. Genetic polymorphisms of some CYP enzymes, especially CYP2D6*10, could affect enzyme activity. CYP2D6*10 allelic frequency was significantly different between HCC patients and control subjects. These findings may be useful for personalizing the clinical treatment of HCC patients as well as predicting the risk of hepatocarcinogenesis.

Comprehensive Circular RNA Profiling Reveals That hsa_circ_0005075, a New Circular RNA Biomarker, Is Involved in Hepatocellular Crcinoma Development

There is increasing evidence that circular RNAs (circRNAs) are involved in cancer development; however, their role in hepatocellular carcinoma (HCC) remains unclear. Here, we aimed to determine the circRNA expression profile in HCC, and investigate relevant mechanisms for cancer progression. The global circRNA expression profile between HCC (n = 3) and adjacent normal liver (n = 3) tissue was significantly different. Three circRNAs (hsa_circ_0000520, hsa_circ_0005075, and hsa_circ_0066444) showed significantly different expression levels in HCC tissues, which were further validated in 60 matched tissue samples using real-time qRT-PCR. Only hsa_circ_0005075 exhibited significant difference in expression (P <0.001) between HCC and normal tissues. Hsa_circ_0005075 expression correlated with HCC tumor size (P = 0.042), and showed good diagnostic potential (AUROC = 0.94). Finally, we constructed a network of hsa_circ_0005075-targeted miRNA-gene interactions, including miR-23b-5p, miR-93-3p, miR-581, miR-23a-5p, and their corresponding mRNAs. Gene oncology analysis revealed that hsa_circ_0005075 could participate in cell adhesion during HCC development. In summary, we identified hsa_circ_0005075 as a potential HCC biomarker; however, further studies are required to confirm the role of this circRNA, and others, in HCC development.

Hsa_circ_0001649: A circular RNA and potential novel biomarker for hepatocellular carcinoma.

It has been shown that circular RNA (circRNA) is associated with human cancers, however, few studies have been reported in hepatocellular carcinoma (HCC). To estimate clinical values of a circular RNA, Hsa_circ_0001649, in HCC. Expression level of hsa_circ_0001649 was detected in HCC and paired adjacent liver tissues by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Differences in expression level of hsa_circ_0001649 were analyzed using the paired t-test. Tests were performed between clinical information and hsa_circ_0001649 expression level by analysis of variance (ANOVA) or welch t-test and a receiver operating characteristics (ROC) curve was established to estimate the value of hsa_circ_0001649 expression as a biomarker in HCC. hsa_circ_0001649 expression was significantly downregulated in HCC tissues (p = 0.0014) based on an analysis of 89 paired samples of HCC and adjacent liver tissues and the area under the ROC curve (AUC) was 0.63. Furthermore, hsa_circ_0001649 expression was correlated with tumor size (p = 0.045) and the occurrence of tumor embolus (p = 0.017) in HCC. We first found hsa_circ_0001649 was significantly downregulated in HCC. Our findings indicate hsa_circ_0001649 might serve as a novel potential biomarker for HCC and may function in tumorigenesis and metastasis of HCC.

Low metallothionein 1M expression association with poor hepatocellular carcinoma prognosis after curative resection.

According to the typical clinical characteristics of hepatocellular carcinoma (HCC), recurrence and prognosis can differ dramatically between patients. Using RNA sequencing, we identified differential expression of the gene metallothionein 1M (MT1M) by comparing early-recurrence HCC (N = 11), no-recurrence HCC (N = 10), and normal liver tissues (N = 5). Reverse transcription-polymerase chain reaction was employed to test MT1M expression levels in 92 HCC tissue samples from a cohort of patients with whom contact was established for post-operative follow-up. Low MT1M expression correlated with high alpha-fetoprotein levels (P = 0.017) and tumor recurrence within 24 months after surgery (P = 0.029). Recurrence rates in high- and low-MT1M groups were significantly different (MT1M cutoff point = 0.066; P = 0.008). Moreover, the disease-free survival time of patients in the former was longer than that of those in the latter (median of 20.39 vs 14.35 months, respectively; P = 0.002). Among early-stage HCC patients (Barcelona Clinic Liver Cancer stage 0/A), those with reduced MT1M expression exhibited higher recurrence rates (37.5 vs 12.1%; P = 0.023). Low MT1M expression is associated with poor HCC prognosis following curative resection, and this also applies to the early stage of this disease.

Clinical significance of cysteinyl leukotriene receptor expression in primary hepatocellular carcinoma

To investigate expression of the cysteinyl leukotriene receptor (CysLT1R) in hepatocellular carcinoma (HCC) tissues and determine its clinical significance. Cancerous and paraneoplastic liver tissues were collected from 30 patients with HCC and from 12 patients with liver hemangioma patients (controls). Real-time PCR and immunohistochemistry were used to evaluate the expression of CysLT1R in these tissues and assess the relationship with clinical pathological features. T-test,?Fisher's exact test and Kaplan-Meier survival analysis were used for statistical analysis. The expression of CysLT1R in adjacent liver tissues (100%) was higher than that in the HCC (43.33%, P = 0.000) and normal liver tissues (41.67%, P = 0.000). The level of CysLT1R mRNA was also higher in paraneoplastic liver tissues (0.0339+/-0.0221) than in the paired HCC tissues (0.0127+/-0.0116, t = 2.911, P = 0.008) and normal liver tissues (0.0154+/-0.0123, t = -2.310, P = 0.033). There was no difference between the levels in HCC and normal liver tissues (P more than 0.05). Higher level of CysLT1R mRNA, higher level of serum alpha-fetoprotein, and higher tumor stage (III-IV) were associated with poor prognosis (respectively 4.372, P = 0.037; 24.187, P = 0.000; 8.75, P = 0.003). However, no evident relationship between the expression of CysLT1R and other clinical features was observed. Conclusions Overexpression of CysLT1R may contribute to the occurrence and progression of HCC.