Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor. Emerging studies showed that long noncoding RNAs (lncRNAs) have crucial regulatory roles in cancer biology. In the current study, differentially expressed lncRNAs between HCC and paired non-tumor tissues were identified using microarrays. The effects of a specific differentially expressed lncRNA (termed ZEB1-AS1) on tumor progression were investigated in vitro and in vivo. We found that ZEB1-AS1 is frequently upregulated in HCC samples, especially in metastatic tumor tissues. DNA methylation analysis shows a tumor-specific ZEB1-AS1 promoter hypomethylation. Aberrant methylation is tightly correlated with overexpression of ZEB1-AS1 in HCC. Patients with ZEB1-AS1 hypomethylation or with high ZEB1-AS1 expression have poor recurrence-free survival. Functionally, ZEB1-AS1 promotes tumor growth and metastasis, acts as an oncogene in HCC. The ZEB1-AS1 gene is located in physical contiguity with ZEB1 and positively regulates the ZEB1 expression. ZEB1 inhibition partially abrogates ZEB1-AS1-induced epithelial to mesenchymal transition (EMT) and cancer metastasis. Our results provide novel insights into the function of lncRNA-driven hepatocarcinogenesis, highlight the important role of ZEB1-AS1 and ZEB1 in HCC progression, and indicate that ZEB1-AS1 may be served as a valuable prognostic biomarker for HCC.

Despite progress in the diagnostics and treatment of hepatocellular carcinoma (HCC), its prognosis remains poor. In this study, we globally assessed long noncoding RNAs (lncRNA) for contributions to HCC using publicly available microarray data, in vitro and in vivo assays. Here, we report that ZFAS1, encoding a lncRNA that is frequently amplified in HCC, is associated with intrahepatic and extrahepatic metastasis and poor prognosis of HCC. ZFAS1 functions as an oncogene in HCC progression by binding miR-150 and abrogating its tumor-suppressive function in this setting. miR-150 repressed HCC cell invasion by inhibiting ZEB1 and the matrix metalloproteinases MMP14 and MMP16. Conversely, ZFAS1 activated ZEB1, MMP14, and MMP16 expression, inhibiting these effects of miR-150. Our results establish a function for ZFAS1 in metastatic progression and suggest its candidacy as a new prognostic biomarker and target for clinical management of HCC. Cancer Res; 75(15); 3181-91. Ó2015 AACR.

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BackgroundSimvastatin exerts pleiotropic effects on cardiovascular system. However, its effect on non-alcoholic fatty liver disease, especially the liver fibrosis, remains obscure. We aimed to clarify the relationship between simvastatin and liver fibrosis both in vivo and in vitro.MethodsA High-fat diet was given to establish rat models with non-alcoholic steatohepatitis (NASH)-related liver fibrosis and simvastatin (4mg·kg-1·d-1) was administrated intragastrically until hepatic histological findings confirmed the appearance of fibrosis. Human hepatic stellate cell (HSC) line lx-2 cells were cultured in an adipogenic differentiating mixture (ADM) and then were treated with transforming growth factorβ1 (TGF-β1), served as a positive control, simvastatin, TGF-β1 plus simvastatin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, a inhibitor of nitric oxide synthase), and L-NAME plus simvastatin, respectively. The expressions of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and Collagen І as well as cellular α-smooth muscle actin (α-SMA) were measured by real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot in liver tissue and HSC.ResultsWith the progress of NASH-related fibrosis, hepatic mRNA and protein expressions of iNOS, α-SMA, and Collagen І were increased while those of eNOS were decreased. Compared with model rats in 24th week group, rats in simvastatin group had less expressions of iNOS, α-SMA, and Collagen І and more expressions of eNOS. In vitro, LX-2 cells acquired quiescent phenotype when cultured in ADM, and TGF-β1 could activate the quiescent HSC. Simvastatin inhibited LX-2 cells activation due to TGF-β1 or L-NAME by increasing the expression of eNOS and decreasing the expression of iNOS.ConclusionsSimvastatin improves the prognosis of NASH-related fibrosis by increasing the expression of eNOS, decreasing the expression of iNOS, and inhibiting the activation of HSC.

Virus-like particles encapsulating HBV-RNA represent a serum biomarker for assessing viral replication activity in clinical practice. However, baseline levels of serum HBV-RNA and their associations with viral replicative intermediates and liver disease in phases of chronic hepatitis B remain unknown. In this cross-sectional study, 102 patients were categorized into immune-tolerant (IT), HBeAg-positive immune active (HBeAg+IA), inactive carrier (IC) and HBeAg-negative immune active (HBeAg-IA) phases. HBV-RNA in serum samples and in 66 paired liver biopsies were quantified and correlated with serum ALT levels, histopathological scores and the levels of other viral replicative intermediates. Mean levels of serum HBV-RNA differed among phases, with the highest levels among IT (6.78 ± 0.83 log copies mL ) patients, followed by HBeAg+IA (5.73 ± 1.16 log copies mL ), HBeAg-IA (4.52 ± 1.25 log copies mL ) and IC (2.96 ± 0.40 log copies mL ) patients. Serum HBV-RNA levels correlated with HBV DNA in all phases, although correlations with other viral replicative intermediates weakened or disappeared when cases were stratified into phases. Distinct compositions of viral products were found among phases: the ratio of HBsAg to serum HBV-RNA was highest in IC patients, while the ratio of serum HBV-RNA to intrahepatic HBV-RNA and the ratio of intrahepatic HBV-DNA to intrahepatic HBV-RNA were significantly higher in IT patients. In conclusion, baseline levels of HBV-RNA and the composition of viral replicative intermediates differ significantly across the natural course of chronic HBV infection. These findings shed light on the nature of viral replication and pathogenesis of disease among different phases of chronic HBV infection.

BackgroundNonalcoholic fatty liver disease (NAFLD) is one of the critical public health problems in China. The full spectrum of the disease ranges from simple steatosis and nonalcoholic steatohepatitis (NASH) to cirrhosis and hepatocellular carcinoma(HCC). The infiltration of inflammatory cells characterizes NASH. This characteristic contributes to the progression of hepatitis, fibrosis, cirrhosis, and HCC. Therefore, distinguishing NASH from NAFLD is crucial.Objective and MethodsNinety-five patients with NAFLD, 44 with NASH, and 51 with non-NASH were included in the study to develop a new scoring system for differentiating NASH from NAFLD. Data on clinical and biological characteristics, as well as blood information, were obtained. Cytokeratin-18 (CK-18) fragments levels were measured using an enzyme-linked immunosorbant assay.ResultsSeveral indexes show significant differences between the two groups, which include body mass index (BMI), waist-on-hip ratio (WHR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), platelets, uric acid (UA), hs-C-reactive protein (hs-CRP), triglycerides (TG), albumin (ALB), and CK-18 fragments (all P < 0.05). The CK-18 fragment levels showed a significant positive correlation with steatosis severity, ballooning, lobular inflammation, and fibrosis stage (all P < 0.05). Therefore, a new model that combines ALT, platelets, CK-18 fragments, and TG was established by logistic regression among NAFLD patients. The AUROC curve in predicting NASH was 0.920 (95% CI: 0.866 - 0.974, cutoff value = 0.361, sensitivity = 89%, specificity = 86%, positive predictive value = 89%, negative predictive value = 89%). ConclusionThe novel scoring system may be considered as a useful model in predicting the presence of NASH in NAFLD patients.

BackgroundThe relationship between resistin and non-alcoholic steatohepatitis (NASH) is not clear, some studies claimed that serum resistin levels were associated with neither the presence of NASH nor its severity, others declared that serum resistin was related with inflammation and fibrosis in NASH. Our animal study verified that the distribution of resistin in the liver is correlated with inflammation in NASH. However, there is no pertinent study in humans.MethodsThirty patients with NASH, 28 simple steatosis, and 43 controls were recruited. Blood was collected for resistin, liver chemistries, fasting insulin and some metabolic parameters. Liver histology was scored according to NAFLD activity scoring system. Hepatic resistin expression was examined by real-time polymerase chain reaction, immunohistochemistry. Resistin protein expression was confirmed by western blotting in 13 patients with concomitant NAFLD and gallstone.ResultsSerum resistin was significantly elevated in both NASH and simple steatotic subjects compared with controls (all P < 0.05). Hepatic resistin was significantly increased in NASH patients in both mRNA and protein levels than those in simple steatosis and control subjects (all P < 0.05). Both serum and hepatic resistin had a correlation with obesity, but not with insulin resistance. The distribution of resistin positive cells was predominantly in perisinusoidal cells (such as Kupffer cells and hepatic stellate cells) in human NASH. Multivariate analysis revealed that waist-hip ratio, higher serum triglyceride, and hyperresistinemia were independent factors related to higher grade of steatosis; whereas hepatic resistin and serum cytokeratin predict NASH and severity of liver fibrosis.ConclusionsHepatic resistin overexpression in NASH patients is associated with the severity of liver inflammation and fibrosis. Liver-derived resistin may be involved in the pathogenesis of human NASH.

BackgroundInsulin-like growth factor-1 receptor (IGF-1R) is a well-studied oncogenic factor that promotes cell proliferation and energy metabolism and is overexpressed in numerous cancers including hepatocellular carcinoma (HCC). Aerobic glycolysis is a hallmark of cancer, and drugs targeting its regulators, including IGF-1R, are being developed. However, the mechanisms of IGF-1R inhibition and the physiological significance of the IGF-1R inhibitors in cancer cells are unclear.Materials and methodsCell proliferation was evaluated by cell counting Kit-8 and colony formation assay. Western blot and real-time PCR were accordingly used to detect the relevant proteins, miRNA and gene expression. Luciferase reporter assays were used to illustrate the interaction between miR-342-3p and IGF-1R. The effect of miR-342-3p on glycolysis was determined by glucose uptake, ATP concentration, lactate generation, extracellular acidification rate and oxygen consumption rate assays. In vivo, subcutaneous tumor formation assay and PET were performed in nude mice.ResultsIn this study, we demonstrate that by directly targeting the 3′-UTR (3′-untranslated regions) of IGF-1R, microRNA-342-3p (miR-342-3p) suppresses IGF-1R-mediated PI3K/AKT/GLUT1 signaling pathway both in vitro and in vivo. Through suppression of IGF-1R, miR-342-3p dampens glycolysis by decreasing glucose uptake, lactate generation, ATP production, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) in hepatoma cells. Importantly, glycolysis regulated by miR-342-3p is critical for its regulating HCC growth both in vitro and in vivo.ConclusionOur findings provide clues regarding the role of miR-342-3p as a tumor suppressor in liver cancer mainly through the inhibition of IGF-1R. Targeting IGF-1R by miR-342-3p could be a potential therapeutic strategy in liver cancer.