Noncancerous Liver Tissues Research Articles

An ancestry informative marker panel design for individual ancestry estimation of Hispanic population using whole exome sequencing data

BackgroundEuropeans and American Indians were major genetic ancestry of Hispanics in the U.S. These ancestral groups have markedly different incidence rates and outcomes in many types of cancers. Therefore, the genetic admixture may cause biased genetic association study with cancer susceptibility variants specifically in Hispanics. For example, the incidence rate of liver cancer has been shown with substantial disparity between Hispanic, Asian and non-Hispanic white populations. Currently, ancestry informative marker (AIM) panels have been widely utilized with up to a few hundred ancestry-informative single nucleotide polymorphisms (SNPs) to infer ancestry admixture. Notably, current available AIMs are predominantly located in intron and intergenic regions, while the whole exome sequencing (WES) protocols commonly used in translational research and clinical practice do not cover these markers. Thus, it remains challenging to accurately determine a patient’s admixture proportion without additional DNA testing.ResultsIn this study we designed an unique AIM panel that infers 3-way genetic admixture from three distinct and selective continental populations (African (AFR), European (EUR), and East Asian (EAS)) within evolutionarily conserved exonic regions. Initially, about 1 million exonic SNPs from selective three populations in the 1000 Genomes Project were trimmed by their linkage disequilibrium (LD), restricted to biallelic variants, and finally we optimized to an AIM panel with 250 SNP markers, or the UT-AIM250 panel, using their ancestral informativeness statistics. Comparing to published AIM panels, UT-AIM250 performed better accuracy when we tested with three ancestral populations (accuracy: 0.995 ± 0.012 for AFR, 0.997 ± 0.007 for EUR, and 0.994 ± 0.012 for EAS). We further demonstrated the performance of the UT-AIM250 panel to admixed American (AMR) samples of the 1000 Genomes Project and obtained similar results (AFR, 0.085 ± 0.098; EUR, 0.665 ± 0.182; and EAS, 0.250 ± 0.205) to previously published AIM panels (Phillips-AIM34: AFR, 0.096 ± 0.127, EUR, 0.575 ± 0.290, and EAS, 0.330 ± 0.315; Wei-AIM278: AFR, 0.070 ± 0.096, EUR, 0.537 ± 0.267, and EAS, 0.393 ± 0.300). Subsequently, we applied the UT-AIM250 panel to a clinical dataset of 26 self-reported Hispanic patients in South Texas with hepatocellular carcinoma (HCC). We estimated the admixture proportions using WES data of adjacent non-cancer liver tissues (AFR, 0.065 ± 0.043; EUR, 0.594 ± 0.150; and EAS, 0.341 ± 0.160). Similar admixture proportions were identified from corresponding tumor tissues. In addition, we estimated admixture proportions of The Cancer Genome Atlas (TCGA) collection of hepatocellular carcinoma (TCGA-LIHC) samples (376 patients) using the UT-AIM250 panel. The panel obtained consistent admixture proportions from tumor and matched normal tissues, identified 3 possible incorrectly reported race/ethnicity, and/or provided race/ethnicity determination if necessary.ConclusionsHere we demonstrated the feasibility of using evolutionarily conserved exonic regions to infer admixture proportions and provided a robust and reliable control for sample collection or patient stratification for genetic analysis. R implementation of UT-AIM250 is available at https://github.com/chenlabgccri/UT-AIM250.

MEIS2C and MEIS2D promote tumor progression via Wnt/\u03b2-catenin and hippo/YAP signaling in hepatocellular carcinoma

BackgroundMEIS2 has been identified as one of the key transcription factors in the gene regulatory network in the development and pathogenesis of human cancers. Our study aims to identify the regulatory mechanisms of MEIS2 in hepatocellular carcinoma (HCC), which could be targeted to develop new therapeutic strategies.MethodsThe variation of MEIS2 levels were assayed in a cohort of HCC patients. The proliferation, clone-formation, migration, and invasion abilities of HCC cells were measured to analyze the effects of MEIS2C and MEIS2D (MEIS2C/D) knockdown with small hairpin RNAs in vitro and in vivo. Chromatin immunoprecipitation (ChIP) was performed to identify MEIS2 binding site. Immunoprecipitation and immunofluorescence assays were employed to detect proteins regulated by MEIS2.ResultsThe expression of MEIS2C/D was increased in the HCC specimens when compared with the adjacent noncancerous liver (ANL) tissues. Moreover, MEIS2C/D expression negatively correlated with the prognosis of HCC patients. On the other hand, knockdown of MEIS2C/D could inhibit proliferation and diminish migration and invasion of hepatoma cells in vitro and in vivo. Mechanistically, MESI2C activated Wnt/β-catenin pathway in cooperation with Parafibromin (CDC73), while MEIS2D suppressed Hippo pathway by promoting YAP nuclear translocation via miR-1307-3p/LATS1 axis. Notably, CDC73 could directly either interact with MEIS2C/β-catenin or MEIS2D/YAP complex, depending on its tyrosine-phosphorylation status.ConclusionsOur studies indicate that MEISC/D promote HCC development via Wnt/β-catenin and Hippo/YAP signaling pathways, highlighting the complex molecular network of MEIS2C/D in HCC pathogenesis. These results suggest that MEISC/D may serve as a potential novel therapeutic target for HCC.

Histological changes in patients who developed hepatocellular carcinoma after hepatitis C virus eradication by interferon-based therapy.

Although the incidence of hepatocellular carcinoma (HCC) occurring after hepatitis C virus (HCV) eradication has decreased, there are still reports of hepatocarcinogenesis. The present study investigated the histological changes of non-cancerous liver tissue obtained prior to interferon (IFN) therapy and after HCC development. A total of 669 HCV-infected Japanese patients who achieved sustained virological response (SVR) by IFN-based therapy were retrospectively enrolled. Of these, the present study investigated 18 patients who developed HCC after IFN-based SVR. Specimens from 9 of 18 patients were available for histological comparisons prior to IFN therapy and following HCC development. Of these 9 patients, the specimens of 5 individuals were compared via immunohistochemical staining [CD3, CD4, CD8, CD20, forkhead box P3 (FOXP3), transforming growth factor-β1 and granzyme B]. The current study included 6 control patients with HCV-associated chronic liver disease who subsequently developed HCC (non-SVR-HCC group). Mann-Whitney and Wilcoxon tests were used to compare groups. Bonferroni correction was used for multiple comparisons. P<0.05 was used as a critical P-value, and following Bonferroni's correction, P<0.017 was considered to indicate a statistically significant difference. In the 9 patients examined, continuous inflammation and fibrosis were observed after HCC development. There was also a significant decrease in the positive rate of FOXP3 in all 5 patients at the time of HCC development compared with that prior to IFN therapy (P=0.0084). Additionally, there was a significant difference in the positive rate of FOXP3 between the 5 patients after HCC development and the control individuals (P=0.0022). In patients who developed HCC after IFN-based SVR, the frequency of FOXP3 decreased, but inflammation and fibrosis remained. The extent of the reduction of FOXP3 differed in patients who developed HCC in the presence of HCV. Inflammation and fibrosis remained for a long duration after SVR, which may be associated with hepatocarcinogenesis.

Circ_0015756 promotes proliferation, invasion and migration by microRNA-7-dependent inhibition of FAK in hepatocellular carcinoma

ABSTRACT Background: Hepatocellular carcinoma (HCC) afflicts more than half a million people each year worldwide. It was reported that circ_0015756 was up-regulated in HCC, but the mechanism did not extensively studied. Methods: we collected 24 paired cancerous and noncancerous liver tissues surgically resected from HCC patients. HCC cell proliferation, invasion, migration and apoptosis in vitro were evaluated using MTT assay, Transwell assay, scratch test and Annexin-V/PI staining respectively. Interactions between circ_0015756 and miR-7, miR-7 and FAK were further validated by the luciferase reporter assay. Tumor xenografts of HCC cells with circ_0015756 knockdown were established in nude mice. Results: The expression level of circ_0015756 was increased and the expression level of miR-7 was diminished in cancerous liver tissues relative to noncancerous liver tissues. Circ_0015756 knockdown was shown to increase the expression of miR-7, reduce the proliferation, invasion, migration and resistance to apoptosis, and down-regulate the expression of FAK in HCC. We found miR-7 impaired expression of FAK to inhibit HCC cells, suggesting that miR-7 is responsible for the dysfunction of FAK. Importantly, we showed circ_0015756 could up-regulate FAK via targeting miR-7. These in vitro findings were reproduced in vivo that circ_0015756 knockdown decreased HCC xenograft growth. Conclusion: Our present study reveals a model of HCC development that is composed of circ_0015756, miR-7 and FAK. Modulation of their levels exhibits a promise in the treatment of HCC. Abbreviations: HCC: hepatocellular carcinoma; circRNAs: circular RNAs; miRNA/miR: microRNA; miR-7: microRNA-7; FAK: focal adhesion kinase; KLF-4: kruppel like factor 4; DKK1: dickkopf WNT signaling pathway inhibitor 1; ccRCC: clear cell renal cell carcinoma; PI3K: phosphoinositide 3-kinase; Ct: comparative threshold cycle; RPMI: Roswell Park Memorial Institute; FBS: fetal bovine serum; RT: reverse transcription; qPCR: quantitative polymerase chain reaction; RIPA: radioimmunoprecipitation assay; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF: polyvinylidene difluoride; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO: dimethyl sulfoxide; DMEM: Dulbecco’s modified Eagle’s medium; PI: propidium iodide; SPF: specific pathogen-free; SD: standard deviation; p-Akt: phosphorylated-Akt; shRNAs: small hairpin RNAs; 3’UTR: 3’-untranslated regions

MiR-106b-5p promotes aggressive progression of hepatocellular carcinoma via targeting RUNX3.

Background and ObjectivesThe roles of microRNA(miR)‐106b‐5p in hepatocellular carcinoma (HCC) remain unclear. We aimed here to investigate the clinical significance of miR‐106b‐5p expression in HCC and its underlying mechanisms.MethodsExpression levels of miR‐106b‐5p in 108 HCC clinical samples by quantitative real‐time reverse transcription PCR. Associations of miR‐106b‐5p expression with various clinicopathological features and patients' prognosis were evaluated by Chi‐square test, Kaplan‐Meier, and Cox proportional regression analyses, respectively. The target gene of miR‐106b‐5p and their functions in HCC cells were investigated by luciferase reporter, CCK‐8, and Transwell Matrigel invasion assays.ResultsmiR‐106b‐5p expression was markedly higher in HCC tissues than in noncancerous adjacent liver tissues (P < .001). miR‐106b‐5p upregulation was significantly associated with advanced TNM stage (P = .02), short recurrence‐free (P = .005), and overall (P = .001) survivals. Importantly, miR‐106b‐5p expression was an independent predictor of poor prognosis (P < .05). RUNX3 was identified as a direct target gene of miR‐106b‐5p in HCC cells. Functionally, miR‐106b‐5p upregulation promoted the viability and invasion of HCC cells, while enforced RUNX3 expression reversed the oncogenic effects of miR‐106b‐5p overexpression.ConclusionsmiR‐106b‐5p may serve as a potent prognostic marker for tumor recurrence and survival of HCC patients. miR‐106b‐5p may exert an oncogenic role in HCC via regulating its target gene RUNX3.

Hepatic copper and other trace mineral concentrations in dogs with hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is the most common primary liver tumor in dogs. Abnormalities in hepatic copper, iron, zinc, and selenium concentrations increase risk for HCC development in other species, but trace mineral concentrations have not been evaluated in dogs with HCC.ObjectivesTo investigate hepatic trace mineral concentrations in dogs with HCC.AnimalsArchived liver specimens from 85 dogs with HCC and 85 control dogs.MethodsRetrospective case‐control study. A histopathology database was searched to identify dogs with HCC (test population) and an age‐matched control population. Demographic information was retrieved, and H&E and rhodanine stained slides were reviewed for all cases. Copper, iron, zinc, and selenium concentrations were determined in noncancerous liver tissues (test and control population) and in HCC tissues (test population) using inductively coupled plasma mass spectrometry.ResultsHepatic copper concentrations (non‐neoplastic hepatic tissue) were greater in test population dogs (median, IQR; 294.9 μg/g, 233.5‐475.9 μg/g) than in control dogs (202.8 μg/g, 135.0‐295.3 μg/g; P < .001). Hepatic zinc concentrations in test (132.1 μg/g,108.6‐163.2 μg/g) and control dogs (151.5 μg/g, 117.1‐184.5 μg/g) also were different (P = .03). Within test population dogs, all trace mineral concentrations were decreased in the HCC tissue as compared to the non‐neoplastic hepatic tissue (all P < .001).Conclusions and Clinical ImportanceHepatic copper accumulation and other abnormalities in hepatic trace mineral concentrations could be involved in the pathogenesis of HCC in some dogs.

MicroRNA-769-5p contributes to the proliferation, migration and invasion of hepatocellular carcinoma cells by attenuating RYBP

Hepatocellular carcinoma (HCC) is the commonest primary liver cancer with highly aggressive features. MicroRNAs (miRNAs) are demonstrated to play important roles in the tumorigenesis and progression of HCC. miR-769-5p is a recently identified cancer-associated miRNA. But, the expression level of miR-769-5p and its function in HCC are unexplored. In this study, we found that miR-769-5p expression was obviously increased in HCC samples compared to adjacent noncancerous liver tissues. Additionally, we revealed that miR-769-5p was over-expressed in HCC cells as compared with LO2 cells. Notably, HCC tissues from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages showed higher levels of miR-769-5p compared to those from corresponding controls. Interestingly, our data indicated that HCC patients highly expressing miR-769-5p had significant shorter survivals. Next, functional experiments verified that miR-769-5p knockdown markedly suppressed HCC cell proliferation, migration and invasion. Conversely, ectopic expression of miR-769-5p promoted these biological behaviors of Hep3B cells. Furthermore, depletion of miR-769-5p repressed the growth and metastasis of HCCLM3 cells in vivo. Importantly, miR-769-5p inversely modulated RING1 and YY1 binding protein (RYBP) by directly binding to 3’ untranslated region (UTR) in HCC cells. The expression of RYBP mRNA was down-regulated in HCC tissues and negatively correlated with miR-769-5p level. RYBP overexpression remarkably inhibited the proliferation, migration and invasion of HCCLM3 cells. Accordingly, knockdown of RYBP partially abolished miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells. In summary, our study revealed the up-regulated expression of miR-769-5p, which contributed to HCC progression possibly by targeting RYBP.

Relationship of telomere length in colorectal cancer patients with cancer phenotype and patient prognosis

BackgroundTelomeres, repetitive DNA capping ends of eukaryotic chromosomes, are important in the maintenance of genomic integrity. Perturbed telomeres are common features of many human malignancies, including colorectal cancer.MethodsTelomere length (TL), measured by a Monochrome Multiplex Real-Time qPCR, was investigated in tumour tissues, adjacent mucosa, and blood from patients with colorectal cancer with different clinicopathological features and its impact on patient survival. TL was also measured in a limited number of liver metastases, non-cancerous liver tissues or corresponding tissues from the same patients.ResultsTL in tumour tissues was shorter than in the adjacent mucosa (P < 0.0001). Shorter TL was observed in tumours with lower stage than in those with advanced stages (P = 0.001). TL was shorter in tumours at the proximal than at the distal sites of the colon (P < 0.0001). Shorter TL was also associated with microsatellite instability (P = 0.001) and mucinous tumour histology (P < 0.0001). Patients with a smaller TL ratio between tumour tissues and the adjacent mucosa were associated with increased overall survival (P = 0.022). Metastasised tumours had shorter telomeres than the adjacent non-cancerous liver tissues (P = 0.0005).ConclusionsOverall, the results demonstrate differences in TL between tumours and the adjacent mucosa, between tumours located at different sites and association with patient survival.

Abstract 3183: Initial safety of AFP SPEAR T-cells in patients with advanced hepatocellular carcinoma

Abstract Background: Genetically engineered affinity-enhanced autologous SPEAR T-cells (AFPc332T-cells) directed towards the HLA-A*02-restricted AFP peptide FMNKFIYEI are being tested in an ongoing Phase 1 trial to evaluate safety and antitumor activity in patients with hepatocellular carcinoma (HCC) (NCT03132792). Methods: This is a first-in-human study in HCC patients not amenable to transplant, resection, or loco-regional therapy and failed/intolerant/refused standard of care treatment. Patients must be HLA-A*02:01+ or 02:642+. Patients must have AFP expression by immunohistochemistry at ≥1+ in ≥20% HCC tumor cells or serum AFP ≥400 ng/ml and ≤5% IHC AFP in non-cancerous liver tissue. Up to 24 patients will be enrolled using a modified 3+3 design. Lymphodepletion is with fludarabine 20 mg/m2/day and cyclophosphamide 500 mg/m2/day on days -7 to -5. The initial transduced cell dose is 0.1×109 cells; additional doses are 1×109 and 5×109. Cohort expansion will occur at maximum tolerated dose and may allow doses up to 10×109 transduced cells. Dose-limiting toxicities (DLTs) are adjudicated by a Safety Review Committee. Results: As of 21Sep18, 2 patients were treated with 0.1×109 AFP SPEAR T-cells. Both had cytopenias related to lymphodepleting chemotherapy. Neither experienced cytokine release syndrome or SAEs during initial hospitalization. Liver chemistries show no AFPc332T-related hepatotoxicity. AFPc332T-cells were detected in both patients. One patient had grade 1 cognitive disturbance on day 8. This patient had SAEs of biliary obstruction at week 9 treated with stenting, and abdominal pain at week 12; neither was considered related to AFPc332T. Post-treatment imaging shows stable disease at week 12 by RECIST v1.1. Serum AFP was 12665 ng/ml at baseline, 29616 ng/ml at week 2, and 16,489 at week 12. Week 8 tumor biopsy showed diffuse tissue necrosis with cholestasis suspicious for necrotic tumor cells. No viable tissue was present. Immunostaining for CD3 showed numerous T-cells and T-cell aggregates within the necrotic tissue. The 2nd patient had no SAEs reported; post-treatment imaging is pending. Conclusions: AFP SPEAR T-cells at the 0.1×109 cell dose show no evidence of on target or off target toxicity in the first 2 patients. No protocol defined DLTs were reported. Current data support continued investigation of AFPc332T-cells. Updated data will be presented. Citation Format: Lipika Goyal, Matthew Frigault, Tim Meyer, Lynn G. Feun, Jordi Bruix, Anthony El-Khoueiry, Petr Hausner, Bruno Sangro, Theodore T. Pierce, Elliot Norry, Sulabha Ranganathan, Rafael G. Amado, Richard S. Finn. Initial safety of AFP SPEAR T-cells in patients with advanced hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3183.

Evaluation of miR-302b-5p expression and molecular mechanism in hepatocellular carcinoma: Findings based on RT-qPCR and in silico analysis

Background and aimExtensive research has revealed that microRNAs (miRNAs) play a principle role in cancer, and miRNAs associated with specific cancers have also been identified. The role of microRNA (miR)-302b-5p, which is one of the miRNAs reported in association with cancer, in hepatocellular carcinoma (HCC) is still unclear. Thus, the present study aimed to reveal the expression and potential molecule mechanism of miR-302b-5p in HCC. MethodsAn extensive meta-analysis of data from real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR), Gene Expression Omnibus and ArrayExpress microarrays was used to determine the expression of miR-302b-5p in HCC tissue samples and non-cancerous liver tissue samples. The sensitivity and specificity of miR-302b-5p as an indicator of HCC was estimated by plotting the receiver operating characteristic (ROC) and summarized ROC (sROC). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were employed to unravel the molecular mechanisms and biological functions of miR-302b-5p in HCC. Further, the putative target genes of miR-302b-5p were harvested based on the predicted genes and differentially expressed genes in HCC. Finally, the protein-protein interaction (PPI) network was built to determine the hub genes. ResultsAccording to the RT-qPCR results, the expression of miR-302b-5p was pronouncedly decreased in 39 HCC tissue samples as compared to 39 non-cancerous liver tissue samples. The standard mean difference (SMD) values of all the samples used in the meta-analysis also indicated lower miR-302b-5p expression in the 558 HCC tissue samples than in the 286 non-cancerous liver tissue samples. ROC and sROC analyses showed that miR-302b-5p had good specificity and sensitivity for distinguishing HCC tissue from non-cancerous liver tissue. Bioinformatics analyses identified 227 putative genes, and these genes were evidently enriched in the processes of organelle fission, chromosome and chromatin binding and were centralized in a “lysosome” pathway. The PPI network indicated that DNA topoisomerase II alpha (TOP2 A) was the most prominent hub gene of miR-302b-5p in HCC. Interestingly, according to the TCGA and Genotype-Tissue Expression databases, the mRNA and protein expression of TOP2 A were both elevated in HCC tissue samples as compared to non-cancerous liver tissue samples, and the overall survival and disease-free survival revealed that a high level of TOP2 A might reflect poor HCC outcome. ConclusionsOur findings indicate that miR-302b-5p might suppress HCC progression, and TOP2 A might be a potential target of miR-302b-5p in HCC. However, in-depth in vivo and in vitro experiments are required to verify these findings and explore the mechanisms involved.

C14orf166 Is a Biomarker for Predicting Hepatocellular Carcinoma Recurrence

Aim: Chromosome 14 open reading frame 166 (C14orf166) acts as a transcriptional repressor and is correlated with centrosome architecture manipulation. Nevertheless, the function of C14orf166 in hepatocellular carcinoma (HCC) progression remains unclear. We aimed to investigate the role C14orf166 plays in HCC and further compared the prognostic value of C14orf166 with that of clinicopathological features. Methods: C14orf166 expression was evaluated in a human liver cell line, HCC cell lines, HCC tissues and adjacent noncancerous liver tissues with qRT-PCR, western blot and immunohistochemistry. Patients were divided into two different groups according to C14orf166 level. The relationship between C14orf166 expression and clinicopathological features was assessed by Pearson chi-squared test and receiver operating characteristic curves. Cumulative disease-free survival (DFS) and overall survival (OS) curves were evaluated using the Kaplan–Meier method. Results: C14orf166 mRNA and protein expression is upregulated in HCC cell lines and tissues. The level of C14orf166 was correlated with serum alpha-fetoprotein level, lymph node metastasis, tumor size and recurrence, with high C14orf166 expression correlating with high HCC recurrence risk. The poor OS and DFS of HCC patients are partly due to the persistently high HCC recurrence risk. When combined with serum alpha-fetoprotein level, the predictive accuracy of C14orf166 for HCC recurrence was enhanced (AUC = 0.712, 95% CI 0.603–0.821; p = 0.001). Conclusions: This study demonstrated that C14orf166 is a high-risk biomarker and predictive factor for HCC recurrence, providing information for the selection of appropriate treatment strategies.

Overexpression of miR-21 Is Associated With Recurrence in Patients With Hepatitis B Virus–Mediated Hepatocellular Carcinoma Undergoing Liver Transplantation

Liver transplantation (LT) is the best treatment option for hepatitis B virus (HBV)–mediated hepatocellular carcinoma (HCC). Nevertheless, recurrence is the most important issue after LT. The aims of the present study were to evaluate the relation of dysregulated expression of microRNAs (miRNAs) in recurrence formation in HBV-mediated HCC cases. A total of 42 HBV-mediated HCC patients were evaluated in this study.Among 21 miRNAs, the expression level of miR-106a and miR-21 were higher and miR-143 and miR145 were lower in patients with HCC compared with noncancerous liver tissues (P = .0388, P = .0214, P = .0321, and P = .002, respectively). Compared with nonrecurrent patients, the expression level of miR-21 was 3.54-fold higher and miR-145 was 2.42-fold lower in patients with recurrence during the 5-year follow-up (P = .004 and P = .032; respectively). In addition, according to multivariate Cox regression analysis, the overexpression of miR-21 was found to be a prognostic indicator in HBV-mediated HCC patients (P = .002).In conclusion, we show a significant association between high expression of miR-21 and recurrence in HBV-mediated HCC. Therefore, up-regulation of miR-21 could serve as a promising prognostic marker for HCC.

Chloride intracellular channel protein 2 in cancer and non-cancer human tissues: relationship with tight junctions

ABSTRACTChloride intracellular channel protein 2 (CLIC2) belongs to the CLIC family of conserved metazoan proteins. Although CLICs have been identified as chloride channels, they are currently considered multifunctional proteins. CLIC2 is the least studied family member. We investigated CLIC2 expression and localization in human hepatocellular carcinoma, metastatic colorectal cancer in the liver, and colorectal cancer. Significant expression of mRNAs encoding CLIC1, 2, 4, and 5 were found in the human tissues, but only CLIC2 was predominantly expressed in non-cancer tissues surrounding cancer masses. Fibrotic or dysfunctional (aspartate aminotransferase ≥40) non-cancer liver tissues and advanced stage HCC tissues expressed low levels of CLIC2. Endothelial cells lining blood vessels but not lymphatic vessels in non-cancer tissues expressed CLIC2 as well as high levels of the tight junction proteins claudins 1 and 5, occludin, and ZO-1. Most endothelial cells in blood vessels in cancer tissues had very low expressions of CLIC2 and tight junction proteins. CD31+/CD45− endothelial cells isolated from non-cancer tissues expressed mRNAs encoding CLIC2, claudin 1, occludin and ZO-1, while similar cell fractions from cancer tissues had very low expressions of these molecules. Knockdown of CLIC2 expression in human umbilical vein endothelial cells (HUVECs) allowed human cancer cells to transmigrate through a HUVEC monolayer. These results suggest that CLIC2 may be involved in the formation and/or maintenance of tight junctions and that cancer tissue vasculature lacks CLIC2 and tight junctions, which allows the intravasation of cancer cells necessary for hematogenous metastasis.

Comment on: LncRNA SBF2-AS1 promotes hepatocellular carcinoma metastasis by regulating EMT and predicts unfavorable prognosis.

Objective Recent studies have furthered our understanding of the function of long noncoding RNAs (lncRNAs) in numerous biological processes, including cancer. The present study aimed to investigate the expression of lncRNA SBF2-AS1 (SBF2-AS1) in patients with hepatocellular carcinoma (HCC) and to investigate its effect on HCC cells. Patients and methods Using quantitative reverse transcription-polymerase chain reaction, we detected SBF2-AS1 expression in HCC cell lines and primary tumor tissues. The associations between SBF2-AS1 expression and the clinicopathological factors and outcome of HCC patients were statistically analyzed. MTT assay and transwell assay were performed to determine the proliferation, migration and invasion, respectively. In addition, we evaluated the activation of Mesenchymal-epithelial transition (EMT) pathway by Western blot. Results We found that SBF2-AS1 expression levels were significantly up-regulated in HCC tissues and cell lines compared with the corresponding noncancerous liver tissues and normal hepatic cell line. In addition, high SBF2-AS1 expression levels were correlated with vein invasion (p = 0.008) and TNM stage (p = 0.013). Furthermore, Kaplan-Meier survival analysis indicated that high expressions of SBF2-AS1 were correlated with shorter overall survival of HCC patients. Univariate and multivariate analysis identified high SBF2-AS1 expression as an unfavorable prognostic factor for overall survival. Further functional analysis demonstrated that knockdown of SBF2-AS1 significantly inhibited HCC cells proliferation, migration and invasion. Mechanistically, we found that SBF2-AS1 could promote the activation of EMT pathway, which was demonstrated by measuring the expression levels of EMT-related markers. Conclusions SBF2-AS1 might be considered as a novel molecule involved in HCC development, which provides a potential therapeutic target for HCC.

Survey of the translation shifts in hepatocellular carcinoma with ribosome profiling.

Despite the critical position of translation in the multilevel gene expression regulation program, high-resolution and genome-wide view of the landscape of RNA translation in solid tumors is still limited.Methods: With a ribosome profiling procedure optimized for solid tissue samples, we profiled the translatomes of liver tumors and their adjacent noncancerous normal liver tissues from 10 patients with hepatocellular carcinoma (HCC). A set of bioinformatics tools was then applied to these data for the mining of novel insights into the translation shifts in HCC.Results: This is the first translatome data resource for dissecting dysregulated translation in HCC at the sub-codon resolution. Based on our data, quantitative comparisons of mRNA translation rates yielded the genes and processes that were subjected to patient specific or universal dysregulations of translation efficiencies in tumors. For example, multiple proteins involved in extracellular matrix organization exhibited significant translational upregulation in tumors. We then experimentally validated the tumor-promoting functions of two such genes as examples: AGRN and VWA1. In addition, the data was also used for de novo annotation of the translatomes in tumors and normal tissues, including multiple types of novel non-canonical small ORFs, which would be a resource for further functional studies.Conclusions: The present study generates the first survey of the HCC translatome with ribosome profiling, which is an insightful data resource for dissecting the translatome shift in liver cancer, at sub-codon resolution.

Upregulation of Spondin-2 protein expression correlates with poor prognosis in hepatocellular carcinoma.

ObjectiveThe aim of this study was to measure the extracellular matrix protein Spondin-2 (SPON2) in hepatocellular carcinoma (HCC) tissues and to determine its potential value as a prognostic indicator by assessing its correlation with clinicopathological variables and survival.MethodsSPON2 mRNA expression was assessed in 20 matched pairs of HCC and non-cancerous liver tissues by quantitative reverse transcription-polymerase chain reaction analysis. SPON2 protein expression was determined in 107 matched pairs of HCC and normal liver tissue by immunohistochemical staining of tissue microarrays.ResultsAnalysis of patient tissues and Oncomine datasets showed that SPON2 mRNA and SPON2 protein expression were both significantly upregulated in HCC tissues, compared with non-cancerous liver tissue; moreover, both correlated significantly with tumor size. Kaplan-Meier analysis revealed that HCC patients who showed high levels of cytoplasmic SPON2 protein had poorer survival following curative resection, compared with HCC patients who exhibited low protein expression levels. Multivariate Cox regression analysis showed that tumor thrombus and SPON2 protein expression both independently correlated with reduced survival in HCC patients.ConclusionUpregulated expression of SPON2 protein in tumor tissue could be an effective prognostic indicator for patients with HCC.

LncRNA SBF2-AS1 promotes hepatocellular carcinoma metastasis by regulating EMT and predicts unfavorable prognosis.

Recent studies have furthered our understanding of the function of long noncoding RNAs (lncRNAs) in numerous biological processes, including cancer. The present study aimed to investigate the expression of lncRNA SBF2-AS1 (SBF2-AS1) in patients with hepatocellular carcinoma (HCC) and to investigate its effect on HCC cells. Using quantitative reverse transcription-polymerase chain reaction, we detected SBF2-AS1 expression in HCC cell lines and primary tumor tissues. The associations between SBF2-AS1 expression and the clinicopathological factors and outcome of HCC patients were statistically analyzed. MTT assay and transwell assay were performed to determine the proliferation, migration and invasion, respectively. In addition, we evaluated the activation of Mesenchymal-epithelial transition (EMT) pathway by Western blot. We found that SBF2-AS1 expression levels were significantly up-regulated in HCC tissues and cell lines compared with the corresponding noncancerous liver tissues and normal hepatic cell line. In addition, high SBF2-AS1 expression levels were correlated with vein invasion (p = 0.008) and TNM stage (p = 0.013). Furthermore, Kaplan-Meier survival analysis indicated that high expressions of SBF2-AS1 were correlated with shorter overall survival of HCC patients. Univariate and multivariate analysis identified high SBF2-AS1 expression as an unfavorable prognostic factor for overall survival. Further functional analysis demonstrated that knockdown of SBF2-AS1 significantly inhibited HCC cells proliferation, migration and invasion. Mechanistically, we found that SBF2-AS1 could promote the activation of EMT pathway, which was demonstrated by measuring the expression levels of EMT-related markers. SBF2-AS1 might be considered as a novel molecule involved in HCC development, which provides a potential therapeutic target for HCC.

Expression of Allograft Inflammatory Factor-1 (AIF-1) in Hepatocellular Carcinoma.

BackgroundAllograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein cloned from activated macrophages in human and rat allografts. AIF-1 has been identified as a modulator of inflammatory response, and recently published studies have shown its increased expression in carcinogenesis. However, there are still limited data on the potential functional role of AIF-1 in hepatocellular carcinoma (HCC).Material/MethodsWe evaluated the expression of AIF-1 in 104 cases of paired HCC and adjacent non-cancerous liver tissues using immunohistochemistry, Western blotting, and qPCR analysis, and sought to determine whether its expression was correlated with clinicopathological features. In vitro assays, including cell proliferation and migration assays, were used to study the effects of AIF-1 knockdown in L02 human hepatocyte, and Huh7 and SMMC7721 liver cancer cell lines.ResultsExpression of AIF-1 was increased in HCC compared to adjacent normal liver tissues and was positively correlated with median tumor size (p=0.046), number of tumor deposits (p=0.009), the Barcelona Clinic Liver Cancer (BCLC) stage (p=0.004), and portal vein tumor thrombus (PVTT) (p<0.001). Huh7 and SMMC7721 human HCC cells demonstrated upregulated AIF-1 expression compared to normal hepatocytes. Small interfering RNA (siRNA)-mediated silencing of AIF-1 expression resulted in a reduction in cell proliferation and migration in human HCC cells.ConclusionsThese findings suggest AIF-1 may have roles as a diagnostic or prognostic biomarker and a promising therapeutic target in HCC.

Overexpression of long noncoding RNA LINC00882 is associated with poor prognosis in hepatocellular carcinoma.

IntroductionHepatocellular carcinoma (HCC) is the third major cause of malignant tumor-related death worldwide because it is initially diagnosed in the advanced stage and its therapeutic outcomes are usually poor. Based on this, it is urgent to identify effective early diagnosis biomarkers and new therapeutic targets to promote HCC treatment. Long noncoding RNAs (lncRNAs) have been reported as promising biomarkers for tumor diagnosis and treatment.Materials and methodsIn this study, we profiled expression patterns and dysregulation of lncRNAs in HCC tissues by analyzing two datasets GSE55191 and GSE64631 from Gene Expression Omnibus database firstly, each of which contains expression profiles of 3 primary HCC tissues and 3 normal liver tissues, respectively.ResultsLncRNA 882 (LINC00882) is one of the lncRNAs that was significantly upregulated in HCC tissues compared with normal liver tissues. We verified the upregulation of LINC00882 in HCC by using two separate cohorts that contained 85 HCC tissues and paired adjacent noncancerous tissues and 86 HCC tissues and 89 independent noncancerous liver tissues, respectively.DiscussionWe found that upregulation of LINC00882 is correlated with poorer prognosis of HCC patients. In vitro cell experiments demonstrated that knockdown of LINC00882 inhibits proliferation, migration and invasion of HCC cell lines.ConclusionThese results indicated that LINC00882 promotes HCC progression and could be a potential prognostic biomarker and therapeutic target for HCC.

Prognostic value of ubiquitin-conjugating enzyme E2 S overexpression in hepatocellular carcinoma

Previous study has shown that ubiquitin-conjugating enzyme E2 S (UBE2S) is highly expressed in various human cancers. In order to study the clinical value and potential function of UBE2S in hepatocellular carcinoma (HCC), three datasets from the Oncomine database and RNA-seq data from The Cancer Genome Atlas (TCGA) were analyzed. UBE2S expression was found to be significantly higher in HCC samples, which was supported with qPCR validation. Both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that UBE2S co-expressed genes were involved in cell cycle and DNA replication. Survival analysis showed a significant reduction in overall survival of patients with high UBE2S expression from both the GSE14520 cohort and TCGA Liver hepatocellular carcinoma (LIHC) cohort. Furthermore, Gene set enrichment analysis (GSEA) revealed that high UBE2S expression in HCC patients is associated with increased expression in gene sets associated with decreased survival, increased metastasis and increased recurrence. Finally, qPCR results showed that UBE2S overexpression has diagnostic value in distinguishing between HCC and non-cancerous liver tissue, as the area under the curve (AUC) was 0.8095, and overexpression of UBE2S was significantly associated with decreased overall survival and disease-free survival in HCC patients. In conclusion, UBE2S may hold prognostic value in the treatment of HCC.