Hepatocellular Carcinoma Patient Samples Research Articles

Low levels of tumor suppressor candidate 3 predict poor prognosis of patients with hepatocellular carcinoma.

PurposeThe tumor suppressor candidate 3 (TUSC3) has been considered to be closely associated with the occurrence, development and invasion of various malignant tumors. However, the expression of TUSC3 in hepatocellular carcinoma (HCC) tissues remains ambiguous. The purpose of this research was to investigate the expression of TUSC3 in HCC tissues and analyze the relationship between TUSC3 levels and clinicopathological characteristics and prognosis of HCC patients.Materials and methodsImmunohistochemistry was used to detect the expression of TUSC3 in HCC and the corresponding para-cancerous tissues from 92 samples of HCC patients. mRNA and protein expression levels of TUSC3 were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays in 25 paired HCC and corresponding adjacent nontumor tissues. Furthermore, statistical analysis was applied to evaluate the correlation between TUSC3 level and the clinicopathological features and prognosis of HCC patients.ResultsImmunohistochemical assay indicated that the expression of TUSC3 was significantly lower in HCC tissues when compared with the corresponding para-cancerous tissues (χ2=11.512, P=0.001). The analysis of clinicopathological characteristics showed that low expression of TUSC3 in HCC tissues was significantly associated with Edmondson grade, Barcelona Clinic Liver Cancer stage and tumor size (P=0.008, 0.009 and 0.020, respectively). Univariate analysis showed that the expression of TUSC3 was strongly correlated with overall survival (OS) and disease-free survival (DFS) after radical surgery in HCC patients (P<0.001, P<0.001, respectively). Multivariate analysis revealed that the TUSC3 level was an independent risk factor for OS and DFS in HCC patients (P=0.001, P<0.001, respectively). Results of qRT-PCR and Western blot assays indicated that the level of TUSC3 in HCC tissues was significantly lower than that in the corresponding adjacent noncancerous tissues (P<0.01, P<0.001).ConclusionThe expression of TUSC3 in HCC was significantly downregulated and was correlated with tumor progression and prognosis, which could be used as an independent predictor of prognosis in HCC patients.

Effects of Lidocaine-Mediated CPEB3 Upregulation in Human Hepatocellular Carcinoma Cell Proliferation In Vitro.

Lidocaine displays antitumor activity by inducing apoptosis and suppressing tumor growth in human hepatocellular carcinoma (HepG2) cells in vitro. However, the molecular mechanism underlying lidocaine-mediated antitumor activity is unclear. In this study, HepG2 cells were treated with lidocaine, and cell proliferation and colony-forming ability were assessed. The expression level of cytoplasmic polyadenylation element binding protein 3 (CPEB3) was detected by real-time quantitative PCR and western blot. Lidocaine treatment resulted in decreased HepG2 cell viability and colony formation in a dose-dependent manner. In hepatocellular carcinoma patient samples, CPEB3 was downregulated and was associated with poor prognosis and high-grade malignancy. Additionally, CPEB3 was a critical mediator of lidocaine-induced repression of HepG2 cell proliferation. These results demonstrated that lidocaine decreased cell viability and colony-forming ability of HepG2 cells by upregulating CPEB3 expression.

Serum and exosome long non coding RNAs as potential biomarkers for hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the most aggressive cancers, with limited new diagnostic and therapeutic measures. This study aimed to investigate the utility of specific serum and exosome lncRNAs as biomarkers for early diagnosis of HCC.Methods: The relative expression levels of eight selected lncRNAs in serum were evaluated by quantitative reverse transcription-PCR (qRT-PCR) in the training and validation sets of HCC patients and matched healthy controls. Additionally, the stability, specificity and diagnostic efficiency of these lncRNAs were evaluated to determine their potential as biomarkers. The levels of the final validated lncRNAs in exosome and urine samples of 15 HCC patients and 15 healthy controls were examined for source and path analysis.Results: LINC00161 was significantly upregulated in serum samples of HCC patients and showed excellent stability and specificity (P< 0.001, fold change=2.85). The area under the receiver operating characteristic (ROC) curve of the validated lncRNA signature was 0.794 (95% CI, 0.712-0.877). LINC00161 expression was detected in serum exosome, exosome-free, and urine samples, and its levels in serum exosome were upregulated in patients with HCC as compared to controls (P= 0.011, fold change=4.27).Conclusions: Our results indicated that circulating exosomal LINC00161 in serum may be a novel biomarker for HCC. LINC00161 is derived from exosomes into serum and may at least be partly metabolized through urine.

Oncogenic dependency on β-catenin in liver cancer cell lines correlates with pathway activation.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/β-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (β-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of β-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on β-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on β-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of β-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant β-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Serum fibronectin distinguishes the early stages of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death, necessitating the discovery of serum markers for its early detection. In this study, a total of 180 serum samples from liver cirrhosis (LC), hepatocellular carcinoma (HCC) patients and paired samples of HCC patients who recovered (Recovery) were analyzed by multiple reaction monitoring-mass spectrometry (MRM-MS) to verify biomarkers. The three-fold crossvalidation was repeated 100 times in the training and test sets to evaluate statistical significance of 124 candidate proteins. This step resulted in 2 proteins that had an area under the receiver operating curve (AUROC) values ≥0.800 in the training (n = 90) and test sets (n = 90). Specifically, fibronectin (FN1, WCGTTQNYDADQK), distinguished HCC from LC patients, with an AUROC value of 0.926 by logistic regression. A FN1 protein was selected for validation in an independent sample (n = 60) using enzyme-linked immunosorbent assay (ELISA). The combination of alpha-fetoprotein (AFP) and FN1 improved the diagnostic performance and differentiated HCC patients with normal AFP levels. Our study has examined candidate markers for the benign disease state and malignancy and has followed up on the consequent recovery. Thus, improvement in the early detection of HCC by a 2-marker panel (AFP + FN1) might benefit HCC patients.

HIF-1\u03b1 promoted vasculogenic mimicry formation in hepatocellular carcinoma through LOXL2 up-regulation in hypoxic tumor microenvironment

BackgroundThe incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in recent years. A hypoxic microenvironment is one of the most important characteristics of solid tumors which has been shown to promote tumor metastasis, epithelial-mesenchymal transition and angiogenesis. Epithelial-mesenchymal transition and vasculogenic mimicry have been regarded as crucial contributing factors to cancer progression. HIF-1α functions as a master transcriptional regulator in the adaptive response to hypoxia. Lysyl oxidases like 2 (LOXL2) is a member of the lysyl oxidase family, which main function is to catalyze the covalent cross-linkages of collagen and elastin in the extracellular matrix. Recent work has demonstrated that HIF-1α promotes the expression of LOXL2, which is believed to amplify tumor aggressiveness. LOXL2 has shown to promote metastasis and is correlated with poor prognosis in hepatocellular carcinoma. The purpose of our study is to explore the role of HIF-1α in progression and metastasis of hepatocellular carcinoma by promoting the expression of LOXL2 as well as the potential regulatory mechanism.MethodsHIF-1α, LOXL2 expression and CD31/periodic acid-Schiff double staining in HCC patient samples were examined by immunohistochemical staining. shRNA plasmids against HIF-1α was used to determine whether LOXL2 been increased by HIF-1α. We monitored a series of rescue assays to demonstrate our hypothesis that LOXL2 is required and sufficient for HIF-1α induced EMT and VM formation, which mediates cellular transformation and takes effect in cellular invasion. Then we performed GeneChip® Human Transcriptome Array (HTA) 2.0 in HepG2 cells, HepG2 cells overexpressed LOXL2 and HepG2 cells treated with CoCl2.ResultsIn clinical HCC tissues, it confirmed a positive relationship between HIF-1α and LOXL2 protein. Importantly, HIF-1α and LOXL2 high expression and the presence of vasculogenic mimicry were correlated to poor prognosis. HIF-1α was found to induce EMT, HCC cell migration, invasion and VM formation by regulating LOXL2. The results of microarray assays were analyzed.ConclusionHIF-1α plays an important role in the development of HCC by promoting HCC metastasis, EMT and VM through up-regulating LOXL2. This study highlights the potential therapeutic value of targeting LOXL2 for suppression of HCC metastasis and progression.

Whole genome sequencing of matched tumor, adjacent non-tumor tissues and corresponding normal blood samples of hepatocellular carcinoma patients revealed dynamic changes of the mutations profiles during hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) has become the third most deadly disease worldwide and HBV is the major factor in Asia and Africa. We conducted 9 WGS (whole genome sequencing) analyses for matched samples of tumor, adjacent non-tumor tissues and normal blood samples of HCC patients from three HBV positive patients. We then validated the mutations identified in a larger cohort of 177 HCC patients. We found that the number of the unique somatic mutations (average of 59,136) in tumor samples is significantly less than that in adjacent non-tumor tissues (average 83, 633). We discovered that the TP53 R249S mutation occurred in 7.7% of the HCC patients, and it was significantly associated with poor diagnosis. In addition, we found that the L104P mutation in the VCX gene (Variable charge, X-linked) was absent in white blood cell samples, but present at 11.1% frequency in the adjacent tissues and increased to 14.6% in HCC tissues, suggesting that this mutation might be a tumor driver gene driving HCC carcinogenesis. Finally, we identified a TK1-RNU7 fusion, which would result in a deletion of 103 amino acids from its C-terminal. The frequencies of this fusion event decreased from the adjacent tissues (29.2%) to the tumors (16.7%), suggesting that a truncated thymidine Kinase1 (TK1) caused by the fusion event might be deleterious and be selected against during tumor progression. The three-way comparisons allow the identification of potential driver mutations of carcinogenesis. Furthermore, our dataset provides the research community a valuable dataset for identifying dynamic changes of mutation profiles and driver mutations for HCC.

Transforming growth factor-β-induced plasticity causes a migratory stemness phenotype in hepatocellular carcinoma

As part of its potential pro-tumorigenic actions, Transforming Growth Factor-(TGF)-β induces epithelial–mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells. Whether EMT induces changes in tumor cell plasticity has not been fully explored yet. Here, we analyze the effects of TGF-β on the EMT and stem-related properties of HCC cells and the potential correlation among those processes. The translational aim of the study was to propose a TGF-β/EMT/stem gene signature that would help in recognizing HCC patients as good candidates for anti-TGF-β therapy. Results indicate that when TGF-β induces EMT in HCC cells, a switch in the expression of stem genes is observed and their stemness potential and migratory/invasive capacity are enhanced. However, TGF-β may induce a partial EMT in some epithelial HCC cells, increasing the expression of mesenchymal genes and CD44, but maintaining epithelial gene expression. Epithelial cells show higher stemness potential than the mesenchymal ones, but respond to TGF-β increasing their migratory and invasive capacity. In HCC patient samples, TGFB1 expression most frequently correlates with a partial EMT, increase in mesenchymal genes and CD44 expression, as well as maintenance or over-expression of epithelial-related genes.

Field deployable platform for prognostic hepatic cancer screening.

267 Background: Hepatocellular carcinoma (HCC) incidence rates are increasing in many parts of the world particularly in low- and middle-income countries (LMICs). Although treatable if identified in the early stages in many LMICs patients are identified later in disease. A low cost point of care system that could identify patients at a higher risk of having HCC could be used as a screening tool to identify patients at an earlier stage. We are currently developing a low cost point of care hand held platform that can simultaneously evaluate the blood levels of common HCC markers. We present our preliminary findings on the limit of detection (LOD) of our system. Methods: Our device combines the rapid but qualitative results of a vertical flow assay with the quantitative capabilities of surface enhanced Raman scattering spectroscopy. Our final product will be able to simultaneously measure the blood levels of the five most proven markers of HCC: alphafetoprotein (AFP), lens-culinaris agglutinin binding alphafetoprotein (AFP-L3), des-gamma-carboxy prothrombin (DCP), core antibodies to hepatitis B virus (HBV), and core antibodies to hepatitis C virus (HCV). In this pilot study we initially evaluated the LOD of our system by evaluating known concentrations of AFP in human serum samples. We then evaluated serum levels of AFP in HCC patient samples (n = 5) with our device and compared with values obtained using a standard ELISA. Results: The LOD of our system for the detection of AFP in spiked serum samples was 1 ng/mL. Our quantitative lateral flow assay measured AFP within 10% of the values of obtained by standard ELISA, with serum values ranging from 10-900 ng/mL. The quantitative vertical flow assay was able to provide results within 5 minutes at a cost of approximately $2/sample. Conclusions: Our inexpensive, portable and rapid test was able to measure serum levels of AFP at values comparable to standard clinical methods. Importantly, one blood marker would be insufficient to identify high risk patients, so further research will be required to expand the number of markers within the panel. The end goal will be to evaluate the ability of our point of care system to screen high risk population in LMICs, thereby identifying at risk patients earlier in disease progression.

Serum microRNA panel for early diagnosis of the onset of hepatocellular carcinoma.

Unique change of circulating microRNAs (miRNAs) was recognized to occur in early oncogenesis, which conferred it the potential as biomarkers for early detection of cancer. However, its diagnostic capability for hepatocellular carcinoma (HCC) has not been fully understood. In this study, microarray analysis was applied to screen the initial candidate miRNA from both the supernatants of anoikis-resistant cellular models and the sera samples of HCC patients. The selected differentially expressed miRNAs were further verified in 115 HCC patients and 40 health controls by qRT-PCR. Among these, 4 miRNAs (miR-16-2-3p, 92a-3p, 107, and 3126-5p) were significantly changed in HCC patients compared with controls. Logistic regression analysis identified a 3-miRNA panel (miR-92-3p, miR-107, and miR-3126-5p) as valuable diagnostic marker for HCC, especially for early stage patients (AUC = 0.975) and for low-level AFP HCC patients (AUC = 0.971). In addition, the combination of 3-miRNA panel and AFP was even more effective for discriminating the early stage HCC patients (AUC = 0.988) and low-level AFP HCC patients (AUC = 0.989) from control. In conclusion, diagnostic efficacy of the combination of 3-miRNA panel and AFP was powerful for HCC diagnosis, especially in early tumor screening and low-level AFP patients.

Down-regulation of β-arrestin2 promotes tumour invasion and indicates poor prognosis of hepatocellular carcinoma.

β-arrestins, including β-arrestin1 and β-arrestin2, are multifunctional adaptor proteins. β-arrestins have recently been found to play new roles in regulating intracellular signalling networks associated with malignant cell functions. Altered β-arrestin expression has been reported in many cancers, but its role in hepatocellular carcinoma (HCC) is not clear. We therefore examined the roles of β-arrestins in HCC using an animal model of progressive HCC, HCC patient samples and HCC cell lines with stepwise metastatic potential. We demonstrated that β-arrestin2 level, but not β-arrestin1 level, decreased in conjunction with liver tumourigenesis in a mouse diethylnitrosamine-induced liver tumour model. Furthermore, β-arrestin2 expression was reduced in HCC tissues compared with noncancerous tissues in HCC patients. β-arrestin2 down-regulation in HCC was significantly associated with poor patient prognoses and aggressive pathologic features. In addition, our in vitro study showed that β-arrestin2 overexpression significantly reduced cell migration and invasion in cultured HCC cells. Furthermore, β-arrestin2 overexpression up-regulated E-cadherin expression and inhibited vimentin expression and Akt activation. These results suggest that β-arrestin2 down-regulation increases HCC cell migration and invasion ability. Low β-arrestin2 expression may be indicative of a poor prognosis or early cancer recurrence in patients who have undergone surgery for HCC.

Hepatitis B virus X induces inflammation and cancer in mice liver through dysregulation of cytoskeletal remodeling and lipid metabolism.

Hepatitis B virus X protein (HBx) participates in the occurrence and development processes of hepatocellular carcinoma (HCC) as a multifunctional regulation factor. However, the underlying molecular mechanism remains obscure. Here, we describe the use of p21HBx/+ mouse and SILAM (Stable Isotope Labeling in Mammals) strategy to define the pathological mechanisms for the occurrence and development of HBx induced liver cancer. We systematically compared a series of proteome samples from regular mice, 12- and 24-month old p21HBx/+ mice representing the inflammation and HCC stages of liver disease respectively and their nontransgenic wild-type (WT) littermates. Totally we identified 22 and 97 differentially expressed proteins out of a total of 2473 quantified proteins. Bioinformatics analysis suggested that the lipid metabolism and CDC42-induced cytoskeleton remodeling pathways were strongly activated by the HBx transgene. Interestingly, the protein-protein interaction MS study revealed that HBx directly interacted with multiple proteins in these two pathways. The same effect of up-regulation of cytoskeleton and lipid metabolism related proteins, including CDC42, CFL1, PPARγ and ADFP, was also observed in the Huh-7 cells transfected with HBx. More importantly, CFL1 and ADFP were specifically accumulated in HBV-associated HCC (HBV-HCC) patient samples, and their expression levels were positively correlated with the severity of HBV-related liver disease. These results provide evidence that HBx induces the dysregulation of cytoskeleton remodeling and lipid metabolism and leads to the occurrence and development of liver cancer. The CFL1 and ADFP might be served as potential biomarkers for prognosis and diagnosis of HBV-HCC.

Regulation of glycosylphosphatidylinositol-anchored proteins and GPI-phospholipase D in a c-Myc transgenic mouse model of hepatocellular carcinoma and human HCC.

Recent research implicated glycosylphosphatidylinositol-anchored proteins (GPI-AP) and GPI-specific phospholipase D (GPI-PLD) in the pathogenesis of fatty liver disease and hepatocellular carcinoma (HCC). Given that c-Myc is frequently amplified in HCC, we investigated their regulation in a c-Myc transgenic disease model of liver cancer and HCC patient samples. Whole genome scans defined 54 significantly regulated genes coding for GPI-AP of which 29 and 14 were repressed in expression in transgenic tumors and steatotic human hepatocyte cultures, respectively, to influence lipid-mediated signal transduction, extracellular matrix and immunity pathways. Analysis of gene specific promoter revealed >95% to carry c-Myc binding sites thus establishing a link between c-Myc activity and transcriptional response. Alike, serum GPI-PLD activity was increased 4-fold in transgenic mice; however its tissue activity was reduced by 70%. The associated repression of the serine/threonine phosphatase 2A (PP2A), i.e. a key player of c-Myc proteolysis, indicates co-ordinate responses aimed at impairing tissue GPI-PLD anti-proliferative activities. Translational research identified >4-fold increased GPI-PLD serum protein expression though enzyme activities were repressed by 60% in NASH and HCC patients. Taken collectively, c-Myc influences GPI-AP signaling transcriptionally and posttranslational and represses GPI-AP anti-proliferative signaling in tumors. The findings broaden the perspective of molecular targeted therapies and disease monitoring.

Hepatitis B e antigen and its precursors promote the progress of hepatocellular carcinoma by interacting with NUMB and decreasing p53 activity.

Hepatitis B viral infection is one of the leading causes of hepatocellular carcinoma (HCC) worldwide. Although several viral factors have been identified that may increase the risk for HCC development, the molecular mechanisms leading to the transformation of normal hepatocytes into cancer cells remain elusive. In this study, we demonstrated that the intracellular hepatitis B e antigen (HBeAg) and its precore precursors, but not their homologous core protein, could associate with NUMB and thereby impair the stability and transcriptional activity of tumor suppressor p53. HBeAg and its precursors could disrupt p53-NUMB and HDM2-NUMB interactions and tricomplex p53-HDM2-NUMB formation, inhibit the acetylation and translocation of p53 from cytosol to the nucleus, promote HDM2-mediated ubiquitination and degradation of p53, and suppress p53-dependent apoptosis. A xenograft tumorigenicity assay showed that expression of HBeAg and its precursors promoted carcinogenesis in a mouse model. Immunohistochemical analysis of the bioptic liver samples of HCC patients revealed that HBeAg positivity was associated with reduced transcriptional activity of p53. Taken together, the results suggest a role of intracellular HBeAg and its precursors in HCC development. HBeAg and its precursors promote HDM2-mediated degradation and impair transcriptional activity of p53 by interacting with NUMB, consequently contributing to HCC development. (Hepatology 2016;64:390-404).

Dynamic regulation of uncoupling protein 2 expression by microRNA-214 in hepatocellular carcinoma.

Gemcitabine (GEM), a commonly used chemotherapeutic agent in hepatocellular carcinoma (HCC) patients, uses oxidative stress induction as a common effector pathway. However, GEM alone or in combination with oxaliplatin hardly renders any survival benefits to HCC patients. We have recently shown that this is part due to the overexpression of the mitochondrial uncoupling protein 2 (UCP2) that in turn mediates resistance to GEM in HCC patients. However, not much is known about regulatory mechanisms underlying UCP2 overexpression in HCC. Differential protein expression in HCC cell lines did not show a concomitant change in UCP2 transcript level, indicating post-transcriptional or post-translational regulatory mechanism. In situ analysis revealed that UCP2 is a putative target of miR-214 miR-214 expression is significantly down-regulated in HCC patient samples as compared with normal adjacent tissues and in cell line, human hepatoblastoma cells (HuH6), with high UCP2 protein expression. We demonstrated using miR-214 mimic and antagomir that the miRNA targeted UCP2 expression by directly targeting the wild-type, but not a miR-214 seed mutant, 3' UTR of UCP2 Overexpression of miR-214 significantly attenuated cell proliferation. Finally, analysis in 20 HCC patients revealed an inverse correlation in expression of UCP2 and miR-214 (Pearson's correlation coefficient, r=-0.9792). Cumulatively, our data indicate that in the context of HCC, miR-214 acts as a putative tumour suppressor by targeting UCP2 and defines a novel mechanism of regulation of UCP2.

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.

MiR-133b Regulation of Connective Tissue Growth Factor: A Novel Mechanism in Liver Pathology

miRNAs are involved in liver regeneration, and their expression is dysregulated in hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF), a direct target of miR-133b, is crucial in the ductular reaction (DR)/oval cell (OC) response for generating new hepatocyte lineages during liver injury in the context of hepatotoxin-inhibited hepatocyte proliferation. Herein, we investigate whether miR-133b regulation of CTGF influences HCC cell proliferation and migration, and DR/OC response. We analyzed miR-133b expression and found it to be down-regulated in HCC patient samples and induced in the rat DR/OC activation model of 2-acetylaminofluorene with partial hepatectomy. Furthermore, overexpression of miR-133b via adenoviral system invitro led to decreased CTGF expression and reduced proliferation and Transwell migration of both HepG2 HCC cells and WBF-344 rat OCs. Invivo, overexpression of miR-133b in DR/OC activation models of 2-acetylaminofluorene with partial hepatectomy in rats, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine in mice, led to down-regulation of CTGF expression and OC proliferation. Collectively, these results show that miR-133b regulation of CTGF is a novel mechanism critical forthe proliferation and migration of HCC cells and OC response.

Tripartite motif 16 inhibits hepatocellular carcinoma cell migration and invasion.

Tripartite motif 16 (TRIM16), a member of the RING B-box coiled-coil (RBCC)/tripartite totif (TRIM) protein family, has been demonstrated to have significant effects on tumor migration by previous studies, but its specific contribution to hepatocellular carcinoma (HCC) is currently unknown. The aim of this study was to evaluate the prognostic value of TRIM16 and investigate its functional roles in HCC. The expression of TRIM16 in HCC patient samples were examined using qRT-PCR and western blotting. HCC cell lines with either TRIM16 overexpression or knockdown were established. The effect of TRIM16 on HCC cell migration and invasion was investigated using these cells. Compared with paired normal liver tissues in clinical cancer samples, we found that the expression of TRIM16 was significantly downregulated in HCC lesions. We also found knockdown of TRIM16 promoted epithelial-mesenchymal transition (EMT) in a manner associated with HCC metastasis in vitro and in vivo. Mechanistically, TRIM16 inhibited ZEB2 expression, which in turn inhibited transcription of the pivotal ZEB2 target gene E-cadherin. RNA interference-mediated silencing of ZEB2 attenuated shTRIM16-enhanced cell migration and invasion. In conclusion, our findings define TRIM16 as an inhibitor of EMT and metastasis in HCC that predicts poor clinical outcomes.

Human MicroRNA-602 Inhibits Hepatitis C Virus Genotype 1b Infection and Promotes Tumor Suppressor Gene Expression in a Hepatoma Cell Line

Objective: Hepatitis C virus (HCV) infects 200 million people worldwide, inducing cirrhosis and hepatocellular carcinoma. Liverspecific microRNA (miR) miR-122 facilitates HCV replication, and Locked Nucleic Acid (LNA)-anti-miR-122 was used successfully as a mono-therapy in HCV infected patients. Studies show that anti-miR-122 may negatively impact hepatocyte metabolism. Previously, we showed that human (hsa) miR-602 relative intracellular expression was up-regulated in microarray profiling when transfected with HCV genotype 1b, and hsa-miR-602 lowered HCV accumulation in infected Huh-7.5 cells. This study compares the effectiveness of hsa-miR-602 and LNA-anti-miR-122 on inhibiting HCV replication and regulating the expression of tumor suppressor genes p53, p73, and RASSF1A. Methods: Huh-7.5 cells were transfected with HCV genotype 1b and treated with hsa-miR-602, LNA-anti-miR-122 or both. Cytopathic effect (CPE) and cell aggregation were observed regularly after transfection. HCV load and localization in the un-transfected, transfected, and treated cells were detected by qRT-PCR and immunostaining, respectively. qRT-PCR detected the expression of p53, p73, and RASSF1A. The Cancer Cell Lines Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) were used to correlate gene copy number and expression for TP53, TP73 and RASSF1A in normal and hepatocellular carcinoma (HCC) patient tissues and HCC cell lines. The survival curve for patients with MIR- 602 expression was done using TCGA HCC clinical data sets. Results: HCV load decreased in all treated cells but was more pronounced with hsa-miR-602. HCV transfected Huh-7.5 cells treated with hsa-miR-602 showed no morphological changes whereas cells treated with LNA-anti-miR-122 or LNA-anti-miR-122 and hsa-miR-602 together showed morphological changes. p53, p73, RASSF1A were down-regulated in HCV transfected cells and all treatments restored expression to near normal levels. Similar down-regulated expression was seen in the HCC patient samples and cell lines. Conclusion: Hsa-miR-602 effectively reduces HCV load and restores p53, p73, and RASSF1A expression in HCV transfected cells.

Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation

Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p < 0.01). Additionally, the change of NA3Fb was not influenced by Hepatitis B virus (HBV)and cirrhosis. Furthermore, the mRNA and protein expression of N-acetylglucosaminyltransferase IVa (GnT-IVa), which was related to the synthesis of the NA3Fb, was substantially increased in HCC tissues. Knockdown of GnT-IVa leads to a decreased level of NA3Fb and decreased ability of invasion and migration in HCC cells. NA3Fb can be regarded as a specific cell surface N-glycan of HCC. The high expression of GnT-IVa is the cause of the abnormal increase of NA3Fb on the HCC cell surface, which regulates cell migration. This study demonstrated the specific N-glycans of the cell surface and the mechanisms of altered glycoform related with HCC. These findings lead to better understanding of the function of glycan and glycosyltransferase in the tumorigenesis, progression and metastasis of HCC.