Diagnosis Of Human Hepatocellular Carcinoma Research Articles

LOXL2 serves as a prognostic biomarker for hepatocellular carcinoma by mediating immune infiltration and vasculogenic mimicry.

The development of human hepatocellular carcinoma (HCC) is a multistep process that is accompanied by progressive changes in the liver microenvironment, including immune evasion and angiogenesis. Lysyl oxidase-like 2 (LOXL2) has been suggested to contribute to tumour progression and metastasis; however, the underlying mechanism remains unclear. The purpose of the present study was to explore the relationship between LOXL2 and immune infiltration and vasculogenic mimicry (VM) and to identify the role of LOXL2 in HCC diagnosis prognosis evaluation. The Cancer Genome Atlas (TCGA), UALCAN, GEPIA and Kaplan-Meier plotter databases were used to analyse LOXL2 expression and perform survival analysis. The Tumour Immune Estimation Resource (TIMER) was used to analyse immune cell infiltration, immune cell biomarkers and immune checkpoints. Immunohistochemistry (IHC) of 201 HCC samples was used to confirm the expression of LOXL2 and its relationship with VM. Coimmunoprecipitation (co-IP) and gain- and loss-of-function studies were performed to confirm the molecular mechanism of LOXL2 in VM. The expression of LOXL2 in HCC was higher than that in normal tissues at both the mRNA and protein levels. High expression of LOXL2 was associated with a poorer prognosis of HCC. The genetic alteration rate of LOXL2 was 5%. LOXL2 was positively related to immune cell infiltration and immune checkpoints (PD-1 and CTLA-4) in HCC. Co-IP showed that LOXL2 can interact directly with IQGAP1. Both gain- and loss-of-function studies showed that LOXL2 significantly induced cell migration, invasion and VM formation when IQGAP1 was upregulated. LOXL2 is involved in immune cell infiltration and promotes VM by upregulating IQGAP1. LOXL2 can be used as a novel biomarker for HCC diagnosis and prognosis prediction.

β-Catenin signaling in hepatocellular carcinoma.

Deregulated Wnt/β-catenin signaling is one of the main genetic alterations in human hepatocellular carcinoma (HCC). Comprehensive genomic analyses have revealed that gain-of-function mutation of CTNNB1, which encodes β-catenin, and loss-of-function mutation of AXIN1 occur in approximately 35% of human HCC samples. Human HCCs with activation of the Wnt/β-catenin pathway demonstrate unique gene expression patterns and pathological features. Activated Wnt/β-catenin synergizes with multiple signaling cascades to drive HCC formation, and it functions through its downstream effectors. Therefore, strategies targeting Wnt/β-catenin have been pursued as possible therapeutics against HCC. Here, we review the genetic alterations and oncogenic roles of aberrant Wnt/β-catenin signaling during hepatocarcinogenesis. In addition, we discuss the implication of this pathway in HCC diagnosis, classification, and personalized treatment.

Longitudinal Circulating Levels of miR-23b-3p, miR-126-3p and lncRNA GAS5 in HCC Patients Treated with Sorafenib.

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The multikinase inhibitor sorafenib is a systemic drug for unresectable HCC. The identification of molecular biomarkers for the early diagnosis of HCC and responsiveness to treatment are needed. In this work, we performed an exploratory study to investigate the longitudinal levels of cell-free long ncRNA GAS5 and microRNAs miR-126-3p and -23b-3p in a cohort of 7 patients during the period of treatment with sorafenib. We used qPCR to measure the amounts of GAS5 and miR-126-3p and droplet digital PCR (ddPCR) to measure the levels of miR-23b-3p. Patients treated with sorafenib displayed variable levels of GAS5, miR-126-3p and miR-23b-3p at different time-points of follow-up. miR-23b-3p was further measured by ddPCR in 37 healthy individuals and 25 untreated HCC patients. The amount of miR-23b-3p in the plasma of untreated HCC patients was significantly downregulated if compared to healthy individuals. The ROC curve analysis underlined its diagnostic relevance. In conclusion, our results highlight a potential clinical significance of circulating miR-23b-3p and an exploratory observation on the longitudinal plasmatic levels of GAS5, miR-126-3p and miR-23b-3p during sorafenib treatment.

Serum level of cytokeratin 19 as a diagnostic and prognostic marker in patients with HCV-related hepatocellular carcinoma

BackgroundThe heterogeneous nature of human hepatocellular carcinoma (HCC) impedes both treatment strategies and prognostic predictions. Several markers have been proposed for the diagnosis of HCC. Cytoskeleton-associated proteins have been known as cellular integrators in neoplasm formation. Hepatic progenitor cells are thought to express alpha-fetoprotein (AFP) and hematopoietic as well as biliary markers such as cytokeratin 19 (CK 19) and cytokeratin 7. The aim of this study was to verify the role of serum CK 19 alone or in combination with AFP as a diagnostic marker of HCC and to assess the changes in its levels after ablation of HCV-related HCC to evaluate its role as a predictor marker for recurrence of HCC after ablation. The study was conducted on 102 HCV-related cirrhotic patients categorized into three different groups according to the clinical, laboratory, and radiological evaluation: group I—62 patients with early or intermediate HCC who underwent locoregional intervention, group II—20 patients with advanced HCC not fit for any intervention apart from best supportive treatment, and group III—20 cirrhotic patients with no evidence of HCC as proved by two imaging techniques.ResultsThe mean serum levels of CK 19 were 6.5 ± 5.7, 10.5 ± 12.5, and 6.8 ± 2.8 ng/ml in groups I, II, and III, respectively, with no significant difference between groups. Sensitivity, specificity, positive, and negative predictive values of combined AFP and human CK 19 at cutoff levels of 25.5 ng/ml and 6.25 ng/ml were 93.9%, 45%, 87.5%, and 64.3%, respectively. In group I patients, CK 19 levels were comparable in patients with ablated focal lesion and those who did not at baseline; then, it was significantly higher in ablated patients than in patients with residual tumor 1 and 6 months after the intervention.ConclusionsCombination of both AFP and CK 19 levels could increase the diagnostic accuracy of suspected HCCs. CK 19 levels are good predictors of ablation/recurrence in patients who underwent interventional procedures minimizing the need for follow-up imaging modalities.

Anillin facilitates cell proliferation and induces tumor growth of hepatocellular carcinoma via miR-138/SOX4 axis regulation.

Actin-binding protein Anillin plays a pivotal role in regulating cytokinesis during the cell cycle, and involves in tumorigenesis and progress. However, the exact regulation mechanism of Anillin in human hepatocellular carcinoma (HCC) remains largely unknown. In this study, we examined and verified the anomalous high expression of Anillin in both HCC patients' specimens and HCC cell lines. High expression of Anillin is associated with dismal clinicopathologic features of HCC patients and poor prognosis. We conducted loss-of and gain-of function studies in HCC Hep3B cells. Anillin presented a significantly facilitating effect on cell proliferation in vitro and induced remarkable tumor growth in vivo. We found that the over-expression of Anillin was driven by a potential axis of miR-138/SOX4. Transcription factor SOX4 presented a high expression profile positive correlated with Anillin, and ChIP assay validated the interaction between SOX4 and the specific sequence of the promoter region of Anillin gene. While, we verified miR-138 as an upstream regulator of SOX4, which is abrogated in HCC cells and exerts degenerating effect on SOX4 mRNA. In our conclusion, Anillin facilitates the cell proliferation and enhances tumor growth of HCC, and is modulated by miR-138/SOX4 axis which regulates the transcriptional activity of Anillin. Findings above demonstrate us a probable axis for HCC diagnosis and treatment.Summary of the main pointAnillin facilitates the cell proliferation and enhances tumor growth in HCC. The transcriptional activity of Anillin is modulated by miR-138/SOX4 axis. Findings above demonstrate us a probable axis for HCC diagnosis and treatment.

Derlin-1 Promotes the Progression of Human Hepatocellular Carcinoma via the Activation of AKT Pathway.

IntroductionHepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. In the present research, we explored a new oncogene, derlin-1 (DERL1), and studied its role and mechanism in human HCC.MethodsWe assessed the expression and prognosis value of DERL1 in human HCC by using GEPIA dataset analysis and immunohistochemistry. To elucidate the specific function of DERL1, we suppressed its expression in two HCC cell lines, HuH7 and Hep3B, and overexpressed DERL1 in Hep3B cells. Cell proliferation and migration was detected by CCK8 and transwell assays. Cell flow cytometry was used to evaluate cell apoptosis.ResultsOur results demonstrated that DERL1 was highly expressed in HCC samples (n = 369) than in normal samples (n = 160). Similar results were obtained in 60 clinical samples that we collected from the local hospital. The high expression rate of DERL1 reached 78.3% (47/60). DERL1 overexpression samples were concentrated in patients with tumor diameters >5cm or lymph node metastases. Thus, we speculated that DERL1 operated as a tumor promotor in HCC, and its expression might be proposed as a predictor for tumor metastasis of human HCC. Interference of DERL1 markedly blocked cell proliferation and migration, and induced the apoptosis of HCC cells in vitro. Phosphorylation of Akt was significantly inhibited in cells transfected with DERL1 siRNA compared to their control cells in HuH7 and Hep3B cell lines. The opposite result was observed in the DERL1 overexpression cells.ConclusionOur findings prove that DERL1 promotes tumor progression via AKT pathway and provide a new potential target for the clinical treatment and diagnosis of human HCC.

Identification of hub genes in peripheral blood mononuclear cells for the diagnosis of hepatocellular carcinoma using a weighted gene co-expression network analysis.

Human hepatocellular carcinoma (HCC) is a common malignant tumor of the digestive tract that is prevalent worldwide. Improving diagnosis methods for HCC helps to improve patient survival rate. The present study aimed to identify novel HCC biomarkers for the diagnosis of HCC through analyzing gene changes on peripheral blood mononuclear cells (PBMCs) and verifying these in additional samples. The gene expression profiles GSE49515 (including 10 specimens from normal patients and 10 specimens from patients with HCC) and GSE58208 (including 5 specimens from normal patients and 10 specimens from patients with HCC) were downloaded from the online Gene Expression Omnibus database (GEO). Differentially expressed genes (DEGs) in PBMCs between healthy controls and patients with HCC were identified using R software. A total of 935 DEGs, including 686 upregulated DEGs and 249 downregulated DEGs, were identified in the present study. In order to identify any internal associations, these DEGs were used to construct weighted gene co-expression networks (WGCNA). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of genes in each module were conducted using the online database DAVID. Furthermore, hub genes with high module membership were identified in a co-expression network and receiver operating characteristic curves were used to verify the diagnostic values of these eight hub genes. Furthermore, the expression and diagnosis value of the eight hub genes were also verified in additional samples. The results of the present study suggested that secreted protein acidic and cysteine rich(SPARC), transmembrane protein 40 (TMEM40), solute carrier family 25 member 44, formyl peptide receptor 2 (FPR2), complement C8 β chain, N-myristoyltransferase 1, protein kinase C δ(PRKCD) and protein phosphatase, Mg2+/Mn2+ dependent 1M(PPM1M) were hub genes. SPARC, TMEM40, FPR2, PRKCD and PPM1M had prominent diagnostic value according to the results from the GEO data and the additional samples. The present study demonstrated that these hub genes may help to improve the diagnosis of HCC.

Chromobox homolog 8 (CBX8) Interacts with Y-Box binding protein 1 (YBX1) to promote cellular proliferation in hepatocellular carcinoma cells.

Polycomb group (PcG) proteins have recently been identified as critical regulators in tumor initiation and development. However, the function of CBX8 in human hepatocellular carcinoma (HCC) remains largely unknown. Our study was designed to explore the biological function and clinical implication of CBX8 in HCC. We investigated the interplay between CBX8 and cell cycle through Gene Set Enrichment Analysis and western blotting. Bioinformatics tools and co-immunoprecipitation were used to explore cell cycle regulation. Finally, we studied the expression and clinical significance of CBX8 in HCC through 3 independent datasets. CBX8 was upregulated in HCC and its expression correlated with cell cycle progression. CyclinD1 was downregulated by CBX8 knockdown but upregulated by CBX8 overexpression. YBX1 interacted with CBX8 and regulated the cell cycle. Moreover, targeting YBX1 with specific siRNA impaired CBX8-mediated regulation of CyclinD1. CBX8 overexpression boosted HCC cell growth, while CBX8 knockdown suppressed cell proliferation. Further, YBX1 interacted with CBX8. YBX1 knockdown compromised the proliferation of CBX8 overexpressing cells. CBX8 promotes HCC cell proliferation through YBX1 mediated cell cycle progression and is related to poor HCC prognoses. Therefore, CBX8 may serve as a potential target for the diagnosis and treatment of HCC.

An innovative peptide with high affinity to GPC3 for hepatocellular carcinoma diagnosis.

Glypican-3 (GPC3) is a key biomarker for early diagnosis of human hepatocellular carcinoma (HCC) due to its overexpression in most HCC tumor tissues. Recently, peptides with high affinity to GPC3 have attracted more attention because of their high biocompatibility, non-immunogenicity, fast clearing and easy modification. Herein, we have designed an innovative GPC3 targeting peptide (sequence: DYEMHLWWGTEL, denoted as IPA) by using structure-based virtual simulation. The higher binding abilities of IPA over the reported peptide (YP) were displayed on different cell lines, showing a positive correlation with GPC3 expressions, which were further verified by the GPC3 protein binding assay. The GPC3 targeting specificity of IPA was proved by peptide blocking and siRNA experiment. The localized anchor of peptide IPA on the cell membranes of HepG2 and Huh-7 with GPC3 overexpression confirmed the GPC3 binding capacity. By connecting a near-infrared dye MPA, the in vivo identification ability of IPA to GPC3 was also demonstrated on GPC3-positive (HepG2) and GPC3-negative (U87) xenograft-bearing mice. These results indicated that the designed IPA presented desirable GPC3 targeting ability, showing promising prospects in detecting the expression of GPC3 for HCC targeting imaging.

Extracellular mRNA detected by molecular beacons in tethered lipoplex nanoparticles for diagnosis of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains one of the major causes of cancer related deaths. Although ultrasonography (US), computed tomography (CT) and/or high-cost magnetic resonance imaging (MRI) have been shown to improve early detection of liver cancer and mortality rates in high-risk individuals, such imaging based methods are limited by high rates of false positivity leading to unnecessary patient anxiety and invasive procedures. Complementary blood biomarkers could increase the accuracy of early detection. Although Alpha-fetoprotein (AFP) in blood is widely used in HCC screening and diagnosis, the false-negative rate as high as 30% and 40% is found in advanced HCC and early stage HCC respectively. We detected AFP messenger RNA (mRNA) in extracellular vesicles (EVs) in patient plasma using designed molecular beacons and a novel tethered lipoplex nanoparticle (TLN) biochip. Together with glypican-3 (GPC-3) mRNA, another well-known HCC marker, we observed much improved performance of AFP protein-based HCC detection. Comparing normal donors (N = 38) and HCC patients (N = 40), our TLN biochip using EV AFP and GPC-3 mRNAs provided an AUC (area under the ROC curve) of 0.995, better than that of a single marker. This 2-mRNA combination also provided a perfect positive predictive value (PPV = 1) at a negative predictive value (NPV) of 0.95 and 20% prevalence, while the blood AFP protein or plasma EV GPC3 mRNA alone could only provide a PPV of 0.61 and 0.79 respectively at the same conditions. Thus, this facile new method may complement current models for risk stratification in liver cancer screening, therapeutic monitoring, and after-treatment surveillance. However, large scale validation will need to be conducted to confirm its clinical potential.

Identification and functional analysis of a core gene module associated with hepatitis C virus-induced human hepatocellular carcinoma progression.

Hepatitis C virus (HCV)-induced human hepatocellular carcinoma (HCC) progression may be due to a complex multi-step processes. The developmental mechanism of these processes is worth investigating for the prevention, diagnosis and therapy of HCC. The aim of the present study was to investigate the molecular mechanism underlying the progression of HCV-induced hepatocarcinogenesis. First, the dynamic gene module, consisting of key genes associated with progression between the normal stage and HCC, was identified using the Weighted Gene Co-expression Network Analysis tool from R language. By defining those genes in the module as seeds, the change of co-expression in differentially expressed gene sets in two consecutive stages of pathological progression was examined. Finally, interaction pairs of HCV viral proteins and their directly targeted proteins in the identified module were extracted from the literature and a comprehensive interaction dataset from yeast two-hybrid experiments. By combining the interactions between HCV and their targets, and protein-protein interactions in the Search Tool for the Retrieval of Interacting Genes database (STRING), the HCV-key genes interaction network was constructed and visualized using Cytoscape software 3.2. As a result, a module containing 44 key genes was identified to be associated with HCC progression, due to the dynamic features and functions of those genes in the module. Several important differentially co-expressed gene pairs were identified between non-HCC and HCC stages. In the key genes, cyclin dependent kinase 1 (CDK1), NDC80, cyclin A2 (CCNA2) and rac GTPase activating protein 1 (RACGAP1) were shown to be targeted by the HCV nonstructural proteins NS5A, NS3 and NS5B, respectively. The four genes perform an intermediary role between the HCV viral proteins and the dysfunctional module in the HCV key genes interaction network. These findings provided valuable information for understanding the mechanism of HCV-induced HCC progression and for seeking drug targets for the therapy and prevention of HCC.

The expression of annexin A10 in hepatocellular carcinoma and its correlation with matrix metalloproteinase 9 and vascular endothelial growth factor

Objective To explore the expression of annexin A10 (ANXA10) in human hepatocellular carcinoma (HCC), and analyze its correlation with matrix metalloproteinases-9 (MMP-9) and vascular endothelial growth factor (VEGF), thus to provide the basis for clinical diagnosis and assessment of HCC. Methods 88 HCC patients were selected, and they were all performed surgical treatment, HCC stage Ⅰin 11 cases, stageⅡ 25 cases, stage Ⅲ 31 cases, stage Ⅳ 21 cases.The expressions of ANXA10, MMP-9, VEGF of HCC cancer tissues, adjacent liver tissues and normal liver tissues were tested by immunohistochemical method.The correlation of ANXA10 with VEGF and MMP-9 was analyzed. Results The absorbance value of ANXA10 expression in the HCC cancer tissue was (0.074±0.012), which was lower than that in the adjacent liver tissues[(0.091±0.013)] and normal liver tissues[(0.131±0.025)], and ANXA10 expression of the adjacent liver tissue was lower than that of the normal liver tissues, the differences were statistically significant (t=8.96, 9.44, 8.71, all P<0.05). The absorbance values of MMP-9 and VEGF expression in the HCC cancer tissue were (0.147±0.017) and (0.127±0.028), respectively, which were higher than those in the adjacent liver tissues [(0.096±0.012), (0.091±0.015)] and normal liver tissues [(0.075±0.014), (0.077±0.019)]. The absorbance values of MMP-9 and VEGF of the adjacent liver tissues were higher than those of the normal liver tissues, the differences were statistically significant (t=8.05, 9.30, 8.11; 8.28, 9.51, 8.02, all P<0.05). The absorbance value of ANXA10 expression in the HCC stage Ⅲ+ Ⅳcancer tissue was (0.056±0.010), which was lower than that in the stage Ⅰ+ Ⅱcancer tissue [(0.082±0.016)], the difference was statistically significant (t=8.90, P<0.05). The absorbance values of MMP-9 and VEGF expression in the stage Ⅲ+ Ⅳcancer tissue were (0.157±0.022) and (0.169±0.033), respectively, which were higher than those in the stage Ⅰ+ Ⅱ cancer tissue [(0.114±0.015), (0.091±0.021)], the differences were statistically significant (t=9.13, 9.72, all P<0.05). ANXA10 was correlated with MMP-9 and VEGF (r=0.324, 0.295, all P<0.05). Conclusion ANXA10 presents lower expression in HCC cell, and the expression decreased with the increase of staging.It is negatively related with the MMP-9 and VEGF.ANXA10 expression missing or inactivation of malignant change may be one of the most important features in HCC. Key words: Carcinoma, hepatocellular; Annexins; Matrix metalloproteinase 9; Vascular endothelial growth factors

ZKSCAN1 gene and its related circular RNA (circZKSCAN1) both inhibit hepatocellular carcinoma cell growth, migration, and invasion but through different signaling pathways.

There is increasing evidence that circular RNA (circRNA) are involved in cancer development, but the regulation and function of human circRNA remain largely unknown. In this study, we demonstrated that ZKSCAN1, a zinc finger family gene, is expressed in both linear and circular (circZKSCAN1) forms of RNA in human hepatocellular carcinoma (HCC) tissues and cell lines. Here, we analyzed a cohort of 102 patients and found that expression of both ZKSCAN1 mRNA and circZKSCAN1 was significantly lower (P < 0.05) in the HCC samples compared with that in matched adjacent nontumorous tissues by reverse transcription PCR (RT‐PCR). The low expression level of ZKSCAN1 was only associated with tumor size (P = 0.032), while the cirZKSCAN1 levels varied in patients with different tumor numbers (P < 0.01), cirrhosis (P = 0.031), vascular invasion (P = 0.002), or microscopic vascular invasion (P = 0.002), as well as with the tumor grade (P < 0.001). Silencing both ZKSCAN1 mRNA and circZKSCAN1 promoted cell proliferation, migration, and invasion. In contrast, overexpression of both forms of RNA repressed HCC progression in vivo and in vitro. Silencing or overexpression of both forms of RNA did not interfere with each other. RNA‐seq revealed a very different molecular basis for the observed effects; ZKSCAN1 mRNA mainly regulated cellular metabolism, while circZKSCAN1 mediated several cancer‐related signaling pathways, suggesting a nonredundant role for ZKSCAN1 mRNA and circRNA. In conclusion, our results revealed two post‐translational products (ZKSCAN1 mRNA and circZKSCAN1) that cooperated closely with one another to inhibit growth, migration, and invasion of HCC. cirZKSCAN1 might be a useful marker for the diagnosis of HCC.

Aberrant expression of B7‑H4 may contribute to the development of hepatocellular carcinoma.

In order to determine the effect of B7-H4 on the development of human hepatocellular carcinoma (HCC), the expression levels of B7-H4 were evaluated using reverse transcription-polymerase chain reaction and flow cytometry in HL-7702 and Huh7 cells. B7-H4 protein expression levels were analyzed using western blotting and immunohistochemistry in HCC tissues collected from patients and from a mouse tumor model. Soluble B7-H4 (sB7-H4), interferon-γ (IFN-γ), and interleukin-4 (IL-4) in blood serum were assessed using ELISA in patients with HCC and mice injected with tumor cells. B7-H4 was expressed in HCC cell lines, mouse tumor tissues and HCC patient tissues. However, B7-H4 was not detected in HL-7702 cells or normal human liver tissues. The expression level of B7-H4 was positively correlated with tumor-node-metastasis (TNM) stage, lymph node metastasis, and differentiation degree in patients with HCC. sB7-H4 levels in blood serum samples collected from patients with HCC and tumorigenic mice were higher compared with healthy controls. Expression levels of IFN-γ were reduced, and IL-4 levels were increased in blood serum samples of patients with HCC and tumorigenic mice compared with healthy controls. sB7-H4 expression levels were negatively correlated with IFN-γ levels, and with the ratio of IFN-γ to IL-4. Additionally, sB7-H4 was positively correlated with IL-4 levels in mouse tumor tissues, serum samples obtained from tumorigenic mice and human HCC patients. Notably, the levels of sB7-H4 and IL-4 were positively correlated and IFN-γ was negatively correlated with the TNM stage of patients with HCC. In addition, sB7-H4 and IL-4 expression levels increased and levels of IFN-γ and the ratio of IFN-γ/IL-4 decreased as a function of time post tumor implantation in the mouse model. The present study determined that aberrant expression of B7-H4 contributed to HCC development. B7-H4 may be a potential target for therapy and diagnosis of HCC.

Histone acetyl transferase GCN5 promotes human hepatocellular carcinoma progression by enhancing AIB1 expression.

BackgroundGeneral control non-depressible 5 (GCN5) is a crucial catalytic component of a transcriptional regulatory complex that plays important roles in cellular functions from cell cycle regulation to DNA damage repair. Although GCN5 has recently been implicated in certain oncogenic roles, its role in liver cancer progression remains vague.ResultsIn this study, we report that GCN5 was overexpressed in 17 (54.8 %) of 31 human hepatocellular carcinoma (HCC) specimens. Down-regulation of GCN5 inhibited HCC cell proliferation and xenograft tumor formation. GCN5 knockdown decreased the protein levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and amplified in breast cancer 1 (AIB1), but increased the protein levels of cell cycle inhibitor p21Cip1/Waf1 in HepG2 cells. GCN5 regulated AIB1 expression, at least in part, by cooperating with E2F1 to enhance AIB1 transcription. Consistently, GCN5 expression was positively correlated with AIB1 expression in human HCC specimens in two GEO profile datasets.ConclusionSince AIB1 plays a promoting role in HCC progression, our results propose that GCN5 promotes HCC progression at least partially by regulating AIB1 expression. This study implicates that GCN5 might be a potential molecular target for HCC diagnosis and treatment.

Abstract 4936: The function role of Brf1 in alcohol-induced human and mouse hepatocellular carcinoma*

Abstract Brf1 (TFIIB-related factor 1) specifically regulates the transcription of Pol III genes (RNA polymerase III-dependent genes), including tRNAs and 5S rRNAs, which are elevated in both transformed and tumor cells, suggesting that they play a crucial role in tumorigenesis. Our recent studies have demonstrated that alcohol consumption increases Brf1 expression and Pol III gene transcription to promote tumor development. However, it remains to be investigated that Brf1 is expressed in human cancers. At the present studies, we have determined the significance of Brf1 overexpression in human hepatocellular carcinoma (HCC) and the impaction of repressing Brf1 expression in alcohol-promoted cell transformation. Biopsies of human HCC, liver tumor samples of mice and cell lines of normal and tumor liver were utilized to determine alteration of Brf1 expression using cytological and molecular biological approaches. The result indicates that Brf1 expression is increased in human cases of HCC. High expression of Brf1 displays shorter overall survival period. Levels of Brf1 and Pol III gene transcription in HCC patients with alcohol consumption show an additional increase, compared with the cases of non-HCC with or without alcohol intake. Induction of Brf1 and Pol III genes by ethanol in hepatoma cells is higher than in non-tumor cells. Ethanol increases the rate of cell transformation. Repression of Brf1 inhibits alcohol-promoted cell transformation. Together, these studies demonstrate that Brf1 is overexpressed in HCC cases and liver tumor cell lines. High level of Brf1 in human HCC is correlated with short survival time. Alcohol consumption enhances Brf1 expression to promote cell transformation. Brf1 is a new biomarker of diagnosis and prognosis of HCC. *: The project is supported by NIH grants: AA017288, AA021114 and AA02324 to Shuping Zhong Citation Format: Shuping Zhong. The function role of Brf1 in alcohol-induced human and mouse hepatocellular carcinoma*. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4936.

In-depth analysis of secretome and N-glycosecretome of human hepatocellular carcinoma metastatic cell lines shed light on metastasis correlated proteins

Cancer cell metastasis is a major cause of cancer fatality. But the underlying molecular mechanisms remain incompletely understood, which results in the lack of efficient diagnosis, therapy and prevention approaches. Here, we report a systematic study on the secretory proteins (secretome) and secretory N-glycoproteins (N-glycosecretome) of four human hepatocellular carcinoma (HCC) cell lines with different metastatic potential, to explore the molecular mechanism of metastasis and supply the clues for effective measurement of diagnosis and therapy. Totally, 6242 unique gene products (GPs) and 1637 unique N-glycosites from 635 GPs were confidently identified. About 4000 GPs on average were quantified in each of the cell lines, 1156 of which show differential expression (p<0.05). Ninety-nine percentage of the significantly altered proteins were secretory proteins and proteins correlated to cell movement were significantly activated with the increasing of metastatic potential of the cell lines. Twenty-three GPs increased both in the secretome and the N-glycosecretome were chosen as candidates and verified by western blot analysis, and 10 of them were chosen for immunohistochemistry (IHC) analysis. The cumulative survival rates of the patients with candidate (FAT1, DKK3) suggested that these proteins might be used as biomarkers for HCC diagnosis. In addition, a comparative analysis with the published core human plasma database (1754 GPs) revealed that there were 182 proteins not presented in the human plasma database but identified by our studies, some of which were selected and verified successfully by western blotting in human plasma.

Identification of a non-coding KLF4 transcript generated from intron retention and downregulated in human hepatocellular carcinoma.

The Krüppel-like factor 4 (KLF4) gene is related to various biological processes including stem cell reprogramming and tumorigenesis. In this study, we identified and characterized a non-coding transcript of KLF4, which was designated KLF4‑003, in human liver tissue samples. KLF4‑003 was identified in a number of cell lines by reverse transcription PCR and DNA sequencing. Its expression levels were determined in 54 pairs of human hepatocellular carcinoma (HCC) tissues and a number of HCC cell lines by real-time PCR (RT-PCR). Methylation status of KLF4‑003 CpG islands was determined by bisulfite sequencing. The regulatory effect of KLF4‑003 CpG islands hypermethylation in Hep3B cells was then validated by the 5-aza-dC demethylation treatment, followed by RT-PCR analysis. Receiver operating characteristic (ROC) curve was created to evaluate the diagnostic value for differentiating between HCC cancer and benign diseases. The association study between KLF4‑003 expression level and clinical traits of HCC patients was performed with SPSS. We found that KLF4‑003 was downregulated in 46 out of 54 HCC samples compared with their adjunct normal tissues. The reduced KLF4‑003 expression was significantly associated with HCC recurrence (P=0.045) in the follow-up of 31 HCC patients. Significant differences were detected between the methylation status of HCC specimens and their adjacent normal controls. Demethylation treatment significantly rescued the expression of KLF4‑003 in Hep3B cells. Such observation indicated that the CpG island hypermethylation was at least partially responsible for the downregulation of KLF4‑003 in HCC. The area under ROC curve for the prediction of HCC reached 0.803 (95% CI=0.719-0.886, P<0.001). Our results suggested that the expression of KLF4‑003 was epigenetically regulated by methylation status of a KLF4‑003 CpG island in HCC. The differential expression of KLF4‑003 might play an important role in HCC development and might serve as a potential biomarker for the diagnosis of HCC.

Detecting the golgi protein 73 of liver cancer with micro cantilever

Golgi protein 73 (GP73) is a potential serum biomarker used in diagnosing human hepatocellular carcinoma (HCC). Compared to alpha-fetoprotein, detection of GP73 is expected to give better sensitivity and specificity and thus offers a better method for diagnosis of HCC at an early stage. In this paper, silicon nitride microcantilever was used to detect GP73. The cantilever was modified through many steps to contain antibody of GP73. The result shows that the cantilever can be used as a label-free sensor to detect this kind of biomarker.

Expression and significance of microRNA-181b in hepatocellular carcinoma

Objective To explore the role of microRNA-181b （miR-181b） in the pathogenesis of human hepatocellular carcinoma （HCC）. Methods We detected the expressions of miR-181 b by using re- al-time quantitative polymerase chain reaction （Real-time PCR） in LO2 （normal human liver cell line）, HepG2 and SMMC7721 （human HCC cell lines） , normal liver tissues, para-cancerous tissue and cancer- ous tissues surgically resected from 35 cases of HCC. Cell Counting Kit-8 （ CCK-8 ） assay and growth curve were used to asses the effect of miR-181b inhibitor on proliferation of HCC cells. Results The expression of miR-181b in HepG2 and SMMC7721 cells was significantly higher than in L02 cells （0. 582 ± 0. 032 and 0. 716 ±0. 045 vs. 0. 255 ±0. 039,P 〈0. 05）, and that in cancerous tissue was significantly higher than in normal liver tissues and para-cancerous tissues （0. 636 ± 0. 038 vs. 0. 156 ± 0. 005 and 0. 167 ± 0. 023, P 〈0. 05）. miR-181b inhibitor could reduce the proliferative ability of HCC cells. Conclusion These re- suits indicate that the high expression of miR-181b is probably involved in HCC pathogenesis. Downregula- tion of miR-181b expression can inhibit the proliferation of HCC cells, miR-181b may act as a molecular tar- get for HCC diagnosis and targeted therapy. Key words: MicroRNA-181 b ; Carcinoma, hepatocellular; Real-time quantitative polymerase chain reaction