Paired Noncancerous Liver Tissues Research Articles

Lipidomic study and diagnosis of hepatocellular carcinoma tumor with rapid evaporative ionization mass spectrometry.

Liver cancer is generally considered the leading cause of cancer deaths worldwide, and hepatocellular carcinoma (HCC) contributes to more than 90% of liver cancers. The altered lipid metabolism for rapid cancer cell growth and tumor formation has been frequently proven. In this study, an ambient ionization mass spectrometry technique, rapid evaporative ionization mass spectrometry (REIMS) using a monopolar electric knife, called iKnife, was systematically optimized and employed for ex vivo analysis of 12 human HCC tumor tissue specimens together with the paired paracancerous tissue (PT) and noncancerous liver tissue (NCT) specimens. Nine free fatty acids and 34 phospholipids were tentatively identified according to their extract masses and/or tandem mass spectra. With the help of statistical methods, 7 free fatty acids and 10 phospholipids were distributed differently in 3 types of liver tissue specimens (95% confidence interval). The box plots showed these characterized lipid metabolites varied in PT, HCC, and NCT. Compared with PT and NCT, the upregulations of four common fatty acids FA 18:0, FA 20:4, FA 16:0, and FA 18:1, together with phospholipids PC 36:1, PE 38:3, PE (18:0/20:4), PA (O-36:1), PC (32:1), PC 32:0, PE 34:0, and PC (16:0/18:1), were found in HCC specimens. The sensitivity and specificity of the established statistic model for real-time HCC tumor diagnosis were 100% and 90.5%, respectively. This study demonstrated that the described REIMS technique is a potential method for rapid lipidomic analysis and characterization of HCC tumor tissue.

Novel prognostic implications of YTH domain family 2 in resected hepatocellular carcinoma.

N6-methyladenosine (m6A), the most abundant internal RNA modification, serves a critical role in cancer development. However, the clinical implications of m6A in hepatocellular carcinoma (HCC) remain unclear. The present study sought to reveal the potential roles of m6A readers, which recognize m6A, in HCC. A total of 177 HCC and paired non-cancerous liver tissues from patients who underwent hepatectomy were analysed using quantitative PCR for the expression of m6A readers: YT521-B homology domain family 1 (YTHDF1) and YT521-B homology domain family 2 (YTHDF2). The expression levels of both YTHDF1 and YTHDF2 were not significantly different between tumour and non-cancerous tissues (P=0.93 and P=0.7, respectively). Analysis of the association between clinical features and m6A reader expression revealed that YTHDF1 expression was associated with formation of capsule (P=0.02), whereas low YTHDF2 expression was associated with septal formation (P=0.02). Furthermore, high YTHDF1 expression and high YTHDF2 expression were significantly associated with shorter recurrence-free survival (RFS) [YTHDF1: Mean survival time (MST), 34.0 vs. 19.0 months, P=0.014; YTHDF2: MST, 30.1 vs. 12.9 months, P=0.0032], whereas YTHDF1 and YTHDF2 expression was not significantly associated with overall survival (OS) (YTHDF1: MST, 99.4 vs. 70.2 months, P=0.74; YTHDF2: MST, 98.4 vs. 64.1 months, P=0.28). According to multivariate analysis, serosal invasion [hazard ratio (HR), 2.39; 95% CI 1.30–4.42; P=0.005), portal vein or hepatic vein invasion (HR, 2.82; 95% CI 1.26–6.28; P=0.01) and YTHDF2 expression in HCC tissues (HR, 1.85; 95% CI 1.09–3.15; P=0.02) were identified as significant independent prognostic factors for RFS. α-fetoprotein (HR, 1.79; 95% CI 1.10–2.92; P=0.02), serosal invasion (HR, 1.99; 95% CI 1.17–3.34; P=0.01) and portal vein or hepatic vein invasion (HR, 3.02; 95% CI 1.38–6.61; P=0.006) were identified as significant independent prognostic factors for OS. In conclusion, the present study revealed that high YTHDF2 expression, an m6A reader, in HCC tissues was associated with cancer recurrence.

Novel Prognostic Implications of Methylated RNA and Demethylases in Resected HCC and Background Liver Tissue.

N6-Methyladenosine (m6A), the most abundant internal modification of RNA, plays a critical role in cancer development. However, the clinical implications of m6A in hepatocellular carcinoma (HCC) remain unclear. We analyzed 177 HCC and paired noncancerous liver tissues from patients who underwent hepatectomy according to global m6A quantification and expression of m6A demethylases fat mass and obesity-associated protein (FTO) and alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5). The global m6A quantification revealed no significant difference between HCC and non-cancerous tissue. The expression of m6A demethylases FTO and ALKBH5, was significantly lower in HCC than in non-cancerous tissues (both p<0.001). Furthermore, low ALKBH5 expression in non-cancerous tissues was significantly correlated with worse recurrence-free survival (median of 16.3 vs. 38.9 months, p=0.001). m6A in HCC and its demethylase in surrounding non-cancerous liver tissues might be involved in inherent mechanisms for HCC development and affect malignant potential after HCC resection.

RETRACTED: circRASGRF2 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1224 sponge

Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. However, the contributions and molecular mechanisms of circRNAs to hepatocellular carcinoma (HCC) remain largely unknown. In the present study, we compared the expression of circRNAs between five paired HCC and adjacent noncancerous liver (ANL) tissues by using RNA-sequencing (RNA-seq). circRASGRF2 (a circRNA located on chromosome 5 and derived from RASGRF2, hsa_circ_0073181) was identified and validated by quantitative reverse transcriptase PCR. The role of circRASGRF2 in HCC progression was assessed both in vitro and in vivo. Mechanistically, RNA immunoprecipitation and luciferase reporter assays were performed to confirm the interaction between circRASGRF2 and miR-1224 in HCC. circRASGRF2 was found to be significantly upregulated in HCC tissues and HCC cell lines compared with paired ANL tissues and normal cells. Our in vivo and in vitro data indicated that knockdown of circRASGRF2 inhibits the proliferation and migration of HCC cells. Mechanistically, we found that circRASGRF2 could promote the expression of focal adhesion kinase (FAK) by sponging miR-1224. Our data showed that circRASGRF2 is a central component linking circRNAs to progression of HCC, making it a potential therapeutic target.

Clinical significance of herpes virus entry mediator expression in hepatitis B virus-related hepatocellular carcinoma.

Herpes virus entry mediator (HVEM) is overexpressed in several malignancies, including hepatocellular carcinoma (HCC). However, to the best of our knowledge, the clinical significance of HVEM in hepatitis B virus (HBV)-related HCC remains unclear. Thus, the present study aimed to explore the clinical significance of HVEM in HBV-related HCC. In the present study, HVEM expression was evaluated in HCC cell lines and HCC frozen samples. The prognostic value of HVEM was assessed in a cohort of 221 patients with HBV-related HCC, following radical resection. B- and T-lymphocyte attenuator (BTLA) expression in subsets of CD8+ T cells was determined via flow cytometry analysis. The results demonstrated high HVEM expression in HCC cell lines, and in HCC tissues compared with paired non-cancerous liver tissues. HVEM expression was demonstrated to be significantly associated with tumor encapsulation and vascular invasion. Furthermore, tumor HVEM status was significantly associated with infiltration of regulatory T cells, but not with CD8+ T cells. Notably, high HVEM expression in HCC was determined to be an independent predictor of an unfavorable outcome of patients with HCC following radical resection. Higher BTLA expression (the receptor of HVEM) was observed in both HCC-infiltrating CD8+ effector memory (CCR7- CD45RA-) and CD45RA+ effector memory (CCR7- CD45RA+) T cells in HCC tissues and blood compared with those in paired peritumor tissues or peripheral blood. Taken together, the results of the present study suggest that HVEM may serve a critical role in HBV-related HCC, most likely by promoting tumor progression and tumor immune evasion, thus the HVEM/BLTA signaling pathway may be a potential target in tumor immunotherapy.

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

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.

Circular RNA Hsa_Circ_0091579 Serves as a Diagnostic and Prognostic Marker for Hepatocellular Carcinoma

Background/Aims: An increasing number of studies have suggested that circular RNAs (circRNAs) have vital roles in carcinogenesis and tumor progression. However, the function of circRNAs in hepatocellular carcinoma (HCC) remains poorly characterized. Methods: We investigated the levels of circRNAs in patients with HCC to identify potential diagnostic biomarkers. We examined circRNA expression profiles in liver tumors and paired non-cancerous liver tissues from three HCC patients with cancer thrombus using a circRNA microarray. Bioinformatics analysis was performed to find circRNAs with significantly altered expression levels between tumors and their paired non-tumor tissues. We confirmed our initial findings by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Receiver operating characteristic (ROC) curves were also applied to identify a candidate circRNA with the optimal specificity and sensitivity. Finally, X-tile software was adopted to calculate the most efficient cut-off value for hsa_circ_0091579 expression. Results: Microarray analysis identified 20 unique circRNAs that were differentially expressed between tumor and non-tumor tissues (P < 0.05). The expression of these 20 circRNAs was verified by qRT-PCR. The expression of hsa_circ_16245-1 and hsa_circ_0091579 mRNA was consistent with their levels as tested by the microarray. The ROC curves showed that both hsa_circ_16245-1 and hsa_circ_0091579 had favorable specificity and sensitivity. We further confirmed that hsa_circ_0091579 was significantly upregulated in HCC and its high expression was intimately associated with a worse overall survival in patients with HCC. Conclusion: Hsa_circ_0091579 may play a critical role in HCC progression and serve as a potential biomarker for the prognosis of patients with HCC.

PRMT1 Promoted HCC Growth and Metastasis In Vitro and In Vivo via Activating the STAT3 Signalling Pathway

Background/Aims: Although it has been widely accepted that protein arginine methyltransferase 1 (PRMT1) is a cancer-promoting gene in various cancers, the mechanism of PRMT1 in hepatocellular carcinoma (HCC) requires more exploration. This study aimed to investigate the role of PRMT1 in HCC growth and metastasis. Methods: We compared PRMT1 expression and clinicopathological characteristics using paired HCC and adjacent noncancerous liver tissues from 210 patients and immunohistochemistry analyses. Cell proliferation, colony formation and migration were determined in HCC cell lines with PRMT1 overexpression or downregulation through MTT, crystal violet and Boyden chamber assays. Tumour growth was monitored in a xenograft model, and intrahepatic metastasis models were established. Results: PRMT1 expression was greatly increased in clinical HCC samples and strongly associated with poor prognosis and recurrence; PRMT1 expression was also positively correlated with microvascular invasion (P = 0.024), tumour differentiation (P = 0.014), tumour size (P = 0.002), and portal vein tumour thrombus (PVTT) (P = 0.028). Cell proliferation, colony formation and migration in vitro were enhanced by PRMT1 upregulation and decreased by PRMT1 downregulation in HCC cell lines. Moreover, low PRMT1 expression resulted in slow tumour growth and decreased tumour weight in vivo, as well as tumour metastasis. These phenotypes were associated with STAT3 signalling pathway activation. Cryptotanshinone, a STAT3 inhibitor, inhibited STAT3 phosphorylation and reversed the HCC phenotype of PRMT1 expression. Conclusions: We revealed a significant role for PRMT1 in HCC progression and metastasis in vitro and in vivo via STAT3 signalling pathway activation. PRMT1 may be a potential novel prognostic biomarker and new therapeutic target for HCC.

Prognostic significance of sodium-potassium ATPase regulator, FXYD3, in human hepatocellular carcinoma.

The clinical significance of the sodium-potassium ATPase regulator FXYD domain-containing ion transport regulator 3 (FXYD3) has been demonstrated in a number of types of cancer. However, the role of this protein in human hepatocellular carcinoma (HCC) remains to be elucidated. In the present study, 217 HCC tissue samples were analyzed to evaluate the expression and prognostic significance of FXYD3 in HCC. Reverse transcription-quantitative polymerase chain reaction was used to analyze the mRNA expression of FXYD3 in 80 primary HCC specimens and paired non-cancerous liver tissue samples, while western blotting was used to analyze the protein expression level of FXYD3 in another 24 pairs. These analyses demonstrated that the expression level of FXYD3 was significantly increasedb at the mRNA and protein levels in HCC tumor tissues compared with adjacent non-cancerous tissues. Immunohistochemical analysis of 137 paraffin-embedded HCC tissue samples indicated that the expression of FXYD3 was associated with HCC clinicopathological characteristics. Kaplan-Meier analysis demonstrated that patients with high FXYD3 protein expression (n=60) experienced significantly poorer overall survival time compared with patients with low FXYD3 protein expression (n=77) (P<0.001). Multivariate analysis demonstrated that FYXD3 protein expression level (hazard ratio, 2.137; 95% confidence interval, 1.224–3.732; P=0.008) was an independent prognostic factor in patients with HCC. Overall, the results indicated that FXYD3 expression levels were higher in HCC tumor tissues than in adjacent non-cancerous tissues, and that the FXYD3 protein may serve as a prognostic marker for 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.

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.

KLHL21, a novel gene that contributes to the progression of hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) has very high prevalence and associated-mortality. However, targeted therapies that are currently used in clinical practice for HCC have certain limitations, in part because of the lack of reliable and clinically applicable biomarkers that can be used for diagnosis and prognosis assessments and for the surveillance of treatment effectiveness.MethodsMeta-analysis was used to analyze the integrated microarray data for global identification of a set of robust biomarkers for HCC. Quantitative RT-PCR (qRT-PCR) was performed to validate the expression levels of selected genes. Gene expression was inhibited by siRNA. CellTiter 96® AQueous One Solution Cell Proliferation assays were used to determine cell proliferation, and Transwell assays were used to determine cell migration and invasion potential.ResultsMeta-analysis of the expression data provided a gene expression signature from a total of 1525 patients with HCC, showing 1529 up-regulated genes and 478 down-regulated genes in cancer samples. The expression levels of genes having strong clinical significance were validated by qRT-PCR using primary HCC tissues and the paired adjacent noncancerous liver tissues. Up-regulation of VPS45, WIPI1, TTC1, IGBP1 and KLHL21 genes and down-regulation of FCGRT gene were confirmed in clinical HCC samples. KLHL21 was the most promising gene for potential use as a bioclinical marker in this analysis. Abrogating expression of it significantly inhibited cell proliferation, migration and invasion.ConclusionsOur study suggests that KLHL21 is a potential target for therapeutic intervention. Our findings also provide novel candidate genes on a genome-wide scale, which may have significant impact on the design and execution of effective therapy of HCC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2851-7) contains supplementary material, which is available to authorized users.

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

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

Plasma level of metastasis-associated lung adenocarcinoma transcript 1 is associated with liver damage and predicts development of hepatocellular carcinoma.

Recent studies have shown that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was overexpressed in many human solid cancers, however, its roles in plasma of hepatocellular carcinoma (HCC) patients were unclear. The aim of this study was to investigate the significance of plasma MALAT1 levels in HCC patients. Plasma samples were collected from pre-operative HCC, hepatic disease patients, and healthy controls, and tissue samples from HCC patients and colorectal cancer patients with liver metastasis. Plasma and tissue MALAT1 levels were measured. Plasma MALAT1 levels were progressively and significantly higher in HCC patients than hepatic disease patients, and higher in hepatic disease patients than healthy controls. The expression of MALAT1 in HCC tissue was slightly higher than that in paired non-cancerous liver tissue, but not significant. The expression of MALAT1 in the non-cancerous liver tissue of 20 HCC patients was significantly higher than that in normal liver tissue of 13 colorectal cancer patients. In contrast, plasma MALAT1 levels were significantly low in HCC patients with hepatitis B infection, and significantly high in patients with liver damage B or liver cirrhosis. In a receiver-operator curve analysis of HCC and hepatic disease patients, the cut-off value of plasma MALAT1 was 1.60 and the area under the curve was 0.66. Plasma MALAT1 levels were not correlated with α-fetoprotein or protein induced by vitamin K absence II, whereas sensitivity and specificity for the detection of HCC with the combination of MALAT1, α-fetoprotein, and protein induced by vitamin K absence II were 88.6% and 75%, respectively. In conclusion, the plasma MALAT1 level is associated with liver damage, and has clinical utility for predicting development of HCC.

RRAD inhibits aerobic glycolysis, invasion, and migration and is associated with poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer worldwide. However, the mechanism underlying the HCC development remains unclear. Ras-related associated with diabetes (RRAD) is a small Ras-related GTPase which has been implicated in metabolic disease and several types of cancer, yet its functions in HCC remain unknown. A tissue microarray constructed by 90 paired HCC tissues and adjacent non-cancerous liver tissues was used to examine the protein levels of RRAD, and the messenger RNA (mRNA) expression of RRAD was also detected in a subset of this cohort. The prognostic significance of RRAD was estimated by the Kaplan-Meier analysis and Cox regression. The glucose utilization assay and lactate production assay were performed to measure the role of RRAD in HCC glycolysis. The effect of RRAD in HCC invasion and metastasis was analyzed by transwell assays. Our results suggested that the expression of RRAD was downregulated in HCC tissues compared to the adjacent non-tumorous liver tissues both in mRNA and protein levels and lower RRAD expression served as an independent prognostic indicator for the survival of HCC patients. Moreover, RRAD inhibited hepatoma cell aerobic glycolysis by negatively regulating the expression of glucose transporter 1 (GLUT1) and hexokinase II (HK-II). In addition, RRAD inhibition dramatically increased hepatoma cell invasion and metastasis. In conclusion, our study revealed that RRAD expression was decreased in HCC tumor tissues and predicted poor clinical outcome for HCC patients and played an important role in regulating aerobic glycolysis and cell invasion and metastasis and may represent potential targets for improving the treatment of HCC.

Hepatocellular carcinoma associated microRNA expression signature: integrated bioinformatics analysis, experimental validation and clinical significance.

microRNA (miRNA) expression profiles varied greatly among current studies due to different technological platforms and small sample size. Systematic and integrative analysis of published datesets that compared the miRNA expression profiles between hepatocellular carcinoma (HCC) tissue and paired adjacent noncancerous liver tissue was performed to determine candidate HCC associated miRNAs. Moreover, we further validated the confirmed miRNAs in a clinical setting using qRT-PCR and Tumor Cancer Genome Atlas (TCGA) dataset. A miRNA integrated-signature of 5 upregulated and 8 downregulated miRNAs was identified from 26 published datesets in HCC using robust rank aggregation method. qRT-PCR demonstrated that miR-93-5p, miR-224-5p, miR-221-3p and miR-21-5p was increased, whereas the expression of miR-214-3p, miR-199a-3p, miR-195-5p, miR-150-5p and miR-145-5p was decreased in the HCC tissues, which was also validated on TCGA dataset. A miRNA based score using LASSO regression model provided a high accuracy for identifying HCC tissue (AUC = 0.982): HCC risk score = 0.180E_miR-221 + 0.0262E_miR-21 - 0.007E_miR-223 - 0.185E_miR-130a. E_miR-n = Log 2 (expression of microRNA n). Furthermore, expression of 5 miRNAs (miR-222, miR-221, miR-21 miR-214 and miR-130a) correlated with pathological tumor grade. Cox regression analysis showed that miR-21 was related with 3-year survival (hazard ratio [HR]: 1.509, 95%CI: 1.079-2.112, P = 0.016) and 5-year survival (HR: 1.416, 95%CI: 1.057-1.897, P = 0.020). However, none of the deregulated miRNAs was related with microscopic vascular invasion. This study provides a basis for further clinical application of miRNAs in HCC.

Upregulation of Leucine Zipper Protein mRNA in Hepatocellular Carcinoma Associated With Poor Prognosis.

Leucine zipper protein (LUZP) plays key roles in development. Overexpression of LUZP was documented in several types of solid tumors. In this study, expression of LUZP messenger RNA (LUZP mRNA) in human hepatocellular carcinoma (HCC) was examined, and the correlations of LUZP mRNA level with patients' characteristics and prognosis were also investigated. Total RNA was extracted from HCC and paired noncancerous liver tissues of 77 patients. Expression of LUZP mRNA in the tissues was determined by real-time quantitative reverse transcriptase polymerase chain reaction. Using average LUZP mRNA level in noncancerous liver tissues as the cutoff, patients with HCC were categorized into high-expression group and low-expression group. Correlations of LUZP mRNA with clinical parameters were analyzed. Overall survival of the patients in the 2 groups was analyzed by Kaplan-Meier method. The LUZP mRNA level was significantly higher in HCC samples than in the noncancerous liver tissues (1.87 ± 0.11 vs 0.58 ± 0.05, P < .01). Significant differences were found between the 2 groups in terms of portal vein invasion, Tumor Lymph Node Metastasis (TNM) stage, and recurrence of HCC. The current study failed to find significant differences between the 2 groups in clinical characteristics such as age, gender, lymph node metastasis, hepatitis B virus infection, family HCC history, and alcohol intake. Overall survival in high-expression group was 12 months while that in the low-expression group was 34 months (P = .03). The LUZP mRNA is a prognostic indicator in HCC, and overexpression is associated with poor prognosis in patients with HCC.

SPHK1 inhibitor suppresses cell proliferation and invasion associated with the inhibition of NF-κB pathway in hepatocellular carcinoma

Sphingosine kinase 1 (SphK1) is an oncogenic enzyme promoting transformation, proliferation, and angiogenesis of a number of human tumors. However, its effect on hepatocellular carcinoma (HCC) behavior has not been fully clarified. The purpose of this study was to determine the correlation between HCC and SphK1, and to evaluate the effect of SphK1 inhibitor N,N-dimethylsphingosine (DMS) in HCC. The expression of SphK1 was measured in tissue samples from 76 HCC and paired adjacent noncancerous liver tissues (NT) by immunohistochemistry, quantitative real-time PCR, and Western blotting analysis. The effect of DMS was tested on HCC cells by evaluating cell viability in vitro. Transwell cell migration and invasion assay were carried out for functional analysis. Furthermore, Western blotting analysis was performed to examine the impact of DMS on the PI3K/Akt/NF-kB signaling. High expression of Sphk1 was observed in 84.21% (64/76) of the HCC versus 15.79% (12/76) of the adjacent non-tumorous liver tissues; the difference of Sphk1 expression between HCC and the adjacent non-tumorous liver tissues was statistically significant (P < 0.001). The results were confirmed by Western blot analyses and quantitative real-time PCR. DMS inhibited the proliferation of SK-Hep1 and MHCCLM3 cells which have a relatively high level of SphK1 in a time- and concentration-dependent manner, and the invasion and migration of SK-Hep1 cells were distinctly suppressed after undergoing treatment with DMS. Furthermore, DMS markedly suppressed the expression of phosphorylations of Akt and NF-κB in HCC cells. Our data suggest that the pathogenesis of human HCC maybe mediated by Sphk1, and the specific Sphk1 inhibitor DMS can play a therapeutic role in the treatment of HCC and thus, Sphk1 could represent selective targets for the molecularly targeted treatments of HCC.

736P - Identification of Intragenic Methylation in the Tusc1 Gene As a Novel Prognostic Marker of Hepatocellular Carcinoma

ABSTRACT Aim: Patients with hepatocellular carcinoma (HCC) have a poor prognosis, and novel molecular targets for treating recurrence and progression of the disease along with associated biomarkers are urgently required. In the present study, expression and the regulatory mechanism of TUSC1 (tumor suppressor candidate 1) were investigated to determine if it is a candidate tumor suppressor gene for HCC, which shows repressed transcription that involves aberrant DNA methylation. Methods: TUSC1 mRNA expression levels in HCC cell lines and 94 pairs of surgical specimens were determined using quantitative real-time reverse transcription polymerase chain reaction assay. Methylation status of HCC cell lines and clinical samples were analyzed to investigate the regulatory mechanism of TUSC1 transcription and the relationship between the methylation status of the TUSC1 gene and clinicopathological factors. The expression and distribution of the TUSC1 protein in liver tissues were determined using immunohistochemistry. Results: A majority of HCC cell lines (89%) and surgical specimens (84%) demonstrated reduced expression levels of TUSC1 mRNA compared with paired non-cancerous liver tissues. The mean mRNA expression level in HCC was significantly lower than in corresponding non-cancerous liver. In contrast, no significant difference was found in TUSC1 mRNA expression level between adjacent normal and cirrhotic liver tissue from HCC patients. The TUSC1 protein expression pattern in HCC and liver tissues was consistent with TUSC1 mRNA expression. Twenty-nine (31%) of 94 patients showed intragenic hypermethylation of the TUSC1 gene in HCC, and hypermethylation was significantly associated with advanced pathological stage. Subsequently, patients with hypermethylation of TUSC1 gene had a significantly poorer prognosis than patients without hypermethylation. Conclusions: Our results suggest that TUSC1 is a candidate tumor suppressor gene and intragenic hypermethylation is one of the suppressive mechanisms that regulate TUSC1 transcription in HCC. Intragenic methylation of the TUSC1 gene may serve as a novel prognostic marker of HCC. Disclosure: All authors have declared no conflicts of interest.