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PDF file - 1223K, S1 Aberrant regulation of HDAC2 in human gastric cancer S2 Effect of HDAC2 knockdown on cell cycle distribution in MKN-1 cells S3 Western blot analysis of cell cycle regulatory proteins in MKN-1. S4 HDAC2 knockdown induces apoptosis and autophagic cell death in human gastric cancer cell line

Aberrant regulation of histone deacetylase 2 (HDAC2) was reported for gastric cancers. However, responsive cancer genes in disease onset and progression are less understood. HDAC2 expression was studied by quantitative RT-PCR and Western blotting. The functional consequences of HDAC2 knockdown on cell-cycle regulation, programmed cell death, and gene target identification was investigated by flow cytometry, Western blotting, electron microscopy, anchorage-independent colony formation, and cell migration assay and by whole-genome microarray. Therapeutic efficacy of HDAC2 knockdown was determined in nude mice with small hairpin expressing human gastric cancer cells. Epigenetic regulation of p16INK4a was studied by methylation-specific PCR and chromatin-IP to evidence HDAC2 or acetylated-histone-H4 binding at gene specific promoter sequences. HDAC2 gene and protein expression was significantly upregulated in different histopathologic grades of human gastric cancers and cancer cell lines. HDAC2 inactivation significantly reduced cell motility, cell invasion, clonal expansion, and tumor growth. HDAC2 knockdown-induced G1–S cell cycle arrest and restored activity of p16INK4a and the proapoptotic factors. This treatment caused PARP cleavage and hypophosphorylation of the Rb-protein, repressed cyclinD1, CDK4, and Bcl-2 expression and induced autophagic phenotype, that is, LC3B-II conversion. Some gastric tumors and cancer cells displayed p16INK4a promoter hypermethylation but treatment with 5-aza-deoxycitidine restored activity. With others the methylation status was unchanged. Here, chromatin-IP evidenced HDAC2 binding. Nonetheless, expression of p16INK4a was restored by HDAC2 knockdown with notable histone-H4-acetylation, as determined by chromatin-IP. Thus, p16INK4a is regulated by HDAC2. HDAC2 is a bona fide target for novel molecular therapies in gastric cancers. Mol Cancer Res; 11(1); 62–73. ©2012 AACR.

Supplementary Figures and Tables

The roles of chromatin remodelers and their underlying mechanisms of action in cancer remain unclear. In this study, SMARCB1, known initially as a bona fide tumor suppressor gene, was investigated in liver cancer. SMARCB1 was highly upregulated in patients with liver cancer and was associated with poor prognosis. Loss- and gain-of-function studies in liver cells revealed that SMARCB1 loss led to reduced cell proliferation, wound healing capacity, and tumor growth in vivo. Although upregulated SMARCB1 appeared to contribute to switch/sucrose nonfermentable (SWI/SNF) complex stability and integrity, it did not act using its known pathways antagonism with EZH2 or association between TP53 or AMPK. SMARCB1 knockdown induced a mild reduction in global H3K27 acetylation, and chromatin immunoprecipitation sequencing of SMARCB1 and acetylated histone H3K27 antibodies before and after SMARCB1 loss identified Nucleoporin210 (NUP210) as a critical target of SMARCB1, which bound its enhancer and changed H3K27Ac enrichment and downstream gene expression, particularly cholesterol homeostasis and xenobiotic metabolism. Notably, NUP210 was not only a putative tumor supporter involved in liver cancer but also acted as a key scaffold for SMARCB1 and P300 to chromatin. Furthermore, SMARCB1 deficiency conferred sensitivity to doxorubicin and P300 inhibitor in liver cancer cells. These findings provide insights into mechanisms underlying dysregulation of chromatin remodelers and show novel associations between nucleoporins and chromatin remodelers in cancer.Significance:This study reveals a novel protumorigenic role for SMARCB1 and describes valuable links between nucleoporins and chromatin remodelers in cancer by identifying NUP210 as a critical coregulator of SMARCB1 chromatin remodeling activity.

Aberrant regulation of histone deacetylase 2 (HDAC2) contributes to malignant progression in various cancers, but the underlying mechanism leading to the activation of oncogenic HDAC2 remains unknown. In this study, we show that HDAC2 expression is upregulated in a large cohort of patients with human hepatocellular carcinoma, and that high expression of HDAC2 was significantly associated with poor prognosis of patients with hepatocellular carcinoma. We found that mTORC1/NF-κBp50 signaling is necessary for the growth factor–induced HDAC2 and is sustained in hepatocellular carcinoma, but not in normal hepatic cells. Growth factor–induced mTORC1 activates the nuclear translocation of NF-κBp50, where it binds to the intragenic sequences of the HDAC2 gene and promotes its transcription. Hepatocellular carcinoma tissues derived from chemical-induced mouse and rat liver cancer models validated that mTORC1 activation and NF-κBp50 nuclear translocation are essential for the transcriptional activation of oncogenic HDAC2 in hepatocellular carcinoma. In addition, we demonstrate that HDAC2 is required to maintain mTORC1 activity by stabilizing the mTOR/RAPTOR complex. Elevated expression of HDAC2 triggers a positive feedback loop that activates AKT phosphorylation via the transcriptional modulation of phosphoinositide signaling molecules. Bioinformatics analysis of HDAC2 signature and immunoblot analysis of mesenchymal genes also evidenced that HDAC2 plays a role in the malignant behavior of tumor cells by Snail induction and simultaneously E-cadherin suppression in hepatocellular carcinoma cells. These findings establish a molecular mechanism responsible for the activation of oncogenic HDAC2, which explains how growth factor–induced HDAC2 maintains mitogenic signaling and function during hepatocellular malignant progression and provide a novel strategy for therapeutic intervention in liver cancer. Cancer Res; 74(6); 1728–38. ©2014 AACR.

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PDF file - 3559KB, Supplementary Figure S1. HDAC2 gene expression and GSEA results for genes up-regulated in the HDAC2-associated signature in HCC large cohort studies. Supplementary Figure S2. Representative immunohistochemical staining of phospho-mTOR, mTOR, and HDAC2 expression in non-cancerous hepatic cells and HCC cells. Supplementary Figure S3. The ectopic overexpression of HDAC2 induces AKT activation. Supplementary Figure S4. mRNA expression data of genes encoding kinase or phosphatase in the phosphoinositide signaling pathway. Supplementary Figure S5. A schematic illustration of the phosphoinositide signaling pathway regulated by HDAC2 in HCC cells. Supplementary Figure S6. HDAC2 induced the migration of liver cancer cells. Supplementary Figure S7. Synergistic anti-growth effect of Sorafenib and HDAC2 knock-down on HCC cells. Supplementary Figure S8. Growth factor-induced HDAC2 mediates a positive feedback loop to mTORC1 stabilization and AKT activation.

PDF file - 220KB, Supplementary Table S1. A List of 3,039 Genes Correlated with HDAC2 Gene Expression from a HCC Large Cohort Study. Supplementary Table S2. Overlap of the HDAC2-Correlated Gene Set with Other Existing Gene Sets. Supplementary Table S3. Molecular Concept Map Analysis of HDAC2-Specific Genes in Liver Cancer Cells.

Currently, a reliable serum biomarker for hepatocellular carcinoma (HCC) has not been established, particularly for early‐stage HCC (single tumor 0.8. In our validation study, serum exo‐miR‐4661‐5p could diagnose HCC in all stages (AUROC = 0.917), even in early stage (AUROC = 0.923), with a greater accuracy than other candidate serum exo‐miRs and serum AFP. The panel composed of exo‐miR‐4661‐5p and exo‐miR‐4746‐5p was identified as the most accurate biomarker for early‐stage HCC (AUROC = 0.947, 95% confidence interval = 0.889‐0.980, sensitivity = 81.8%, and specificity = 91.7%). In conclusion, exo‐miR‐4661‐5p–based serum panel is a promising diagnostic marker for early‐stage HCC., The present study derived potential serum exosomal microRNA panels for hepatocellular carcinoma (HCC) using a systematic, genome‐wide biomarker discovery approach. Serum exosomal microRNA‐4661‐5p–based panel is a potent diagnostic biomarker for early stage HCC and could also be used as a prognostic indicator in patients with HCC.

Pericardial effusion (PE) can develop in patients with virtually any condition that affects the pericardium, including acute pericarditis and a variety of systemic disorders. Thus, definite differentiation of malignant pericardial effusion and rapid diagnosis are known to have therapeutic and prognostic importance. The aim of this study was to identify novel molecular markers for the detection of cancer in patients with pericardial disorder. We performed one-way ANOVA analysis of whole transcriptome scans of 18 PEs from the patients with pericardial disorder including cancer. It resulted in 4385 outlier genes. Hierarchical clustering analysis showed two distinct clusters [cancer patient’s PEs (G1 + G2) vs. non-cancer PEs (G3)] within the dendrogram. To identify cancer-specific molecular signature, Welch’s t test of G1 and G3 was performed and 1639 gene elements were suggested as stringent classifiers between cancer PE and non-cancer PE. Gene set enrichment analysis of PE signature suggested that CD24, SDC1, and ST14 were strong molecular markers for identifying cancer patients among patients with pericardial disorder. Our results suggest that etiology-specific molecular signatures can discriminate cancer patients within pericardial disorder patients. CD24, SDC1, and ST14 are strong molecular markers for such discrimination.

Magnetic resonance elastography (MRE) is a non-invasive tool for measuring liver stiffness (LS) with high diagnostic accuracy. This study investigated whether quantified LS by MRE could predict early recurrence of patients with hepatocellular carcinoma (HCC) within the Milan criteria. A prospectively collected cohort, which included the HCC patients who underwent MRE before treatment (an HCC-MRE cohort), was analyzed. In the HCC-MRE cohort, only patients under the Milan criteria, who underwent hepatic resection, radiofrequency ablation (RFA), or transarterial chemoembolization (TACE), were reviewed. We investigated whether LS assessed by MRE was an independent predictor of early recurrence using Cox regressions and Kaplan-Meier analyses. A total of 192 HCC patients under the Milan criteria who underwent hepatic resection (n = 96), RFA (n = 23), or TACE (n = 73) were included. Higher LS ratings (kPa; hazard ratio [HR] = 1.12; 95% confidence interval [CI] = 1.01–1.25; p = 0.040) emerged as an independent risk factor for early tumor recurrence. In the subgroup analysis, higher LS ratings were associated with higher risks of early HCC recurrence in both the resection/RFA group (> 4.5 kPa; HR = 2.95; 95% CI = 1.26–6.94; p = 0.013) and the TACE group (> 6 kPa; HR = 2.94; 95% CI = 1.27–6.83; p = 0.012). LS assessed by MRE was an independent predictor of early recurrence among HCC patients under the Milan criteria after achieving a complete response. • Liver parenchymal stiffness measured by MRE predicts early recurrence of treated HCC under Milan criteria. • A liver stiffness > 5.5 kPa was associated with worse recurrence-free survival. • Patients with high pre-treatment LS may benefit from stringent follow-up.

Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. Wiskott-Aldrich syndrome protein family member 2 (WASF2) is an integral member of the actin cytoskeleton pathway that plays a crucial role in cell motility. In this study, we aimed to explore the role of WASF2 in HCC carcinogenesis and its regulatory mechanism. Methods: WASF2 expression in HCC was analyzed using six public RNA-seq datasets and 66 paired tissues from patients with HCC. Role of WASF2 in HCC cell phenotypes was evaluated using small interfering RNA (siRNA) in vitro and in vivo. Epigenetic regulatory mechanism of WASF2 was assessed in the Cancer Genome Atlas liver hepatocellular carcinoma project (TCGA_LIHC) dataset and also validated in 38 paired HCC tissues. Results: WASF2 is overexpressed in HCC and is clinically correlated with prognosis. WASF2 inactivation decreased the viability, growth, proliferation, migration, and invasion of Huh-7 and SNU475 HCC cells by restoring G2/M checkpoint function, inducing cell death, and inhibiting epithelial-mesenchymal transition, and hindering actin polymerization. In addition, WASF2 knockdown using siWASF2 in a xenograft mouse model exerted tumor suppressive effect. Furthermore, we observed a negative correlation between WASF2 methylation status and mRNA expression. The cg24162579 CpG island in the WASF2 5′ promoter region was hypomethylated in HCC compared to matched non-tumor samples. Patients with high WASF2 methylation and low WASF2 expression displayed the highest overall survival.Conclusions: WASF2 is overexpressed and hypomethylated in HCC and correlates with patient prognosis. Moreover, WASF2 inactivation exerts anti-tumorigenic effects on HCC cells in vitro and in vivo, suggesting that WASF2 could be a potential therapeutic target for HCC.

Reverse transcription-quantitative (RT-q) PCR is the most feasible and useful technique for identifying and evaluating cancer biomarkers; however, the method requires suitable reference genes for gene expression analysis. The aim of the present study was to identify the most suitable reference gene for the normalization of relative gene expression in human hepatocellular carcinoma (HCC) tissue and blood samples. First, 14 candidate reference genes were selected through a systematic literature search. The expression levels of these genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPLP0, RPL13A, SDHA, TBP, TFRC and YWHAZ) were evaluated using human multistage HCC transcriptome data (dataset GSE114564), which included normal liver (n=15), chronic hepatitis (n=20), liver cirrhosis (n=10), and early (n=18) and advanced HCC (n=45). From the 14 selected genes, five genes, whose expression levels were stable in all liver disease statuses (ACTB, GAPDH, HMBS, PPIA and RPLP0), were further assessed using RT-qPCR in 40 tissues (20 paired healthy tissues and 20 tissues from patients with HCC) and 40 blood samples (20 healthy controls and 20 samples from patients with HCC). BestKeeper statistical algorithms were used to identify the most stable reference genes, of which HMBS was found to be the most stable in both HCC tissues and blood samples. Therefore, the results of the present study suggest HMBS as a promising reference gene for the normalization of relative RT-qPCR techniques in HCC-related studies.

Protein markers of hepatocellular carcinoma (HCC)-derived exosomes (HEX) have not yet been fully evaluated. Here, we identified novel protein contents of HEX and their clinical significance as biomarkers. Exosomes were isolated from human HCC cell lines and an immortalized normal hepatocyte cell line. Proteomic analyses revealed 15 markedly overexpressed proteins in HEX. The clinical relevance of the 15 proteins was analyzed in public RNA-sequencing datasets, and 6 proteins were selected as candidate of potential biomarkers. Serum CCT8 and CFL1 were markedly overexpressed in test cohort (n = 8). In the validation cohort (n = 224), the area under the curve (AUC) of serum CCT8 and CFL1 for HCC diagnosis was calculated as 0.698 and 0.677, respectively, whereas that of serum alpha-fetoprotein (AFP) was 0.628. The combination of three serum markers (CCT8, CFL1, and AFP) demonstrated the highest AUC for HCC diagnosis. (AUC = 0.838, 95% confidence interval = 0.773–0.876) Furthermore, higher serum CCT8 and CFL1 concentrations were significantly associated with the presence of vascular invasion, advanced tumor stage, poor disease-free survival, and poor overall survival. Cofilin-1 and CCT8, enriched proteins in HEX, were identified as potential diagnostic and prognostic serum biomarkers for HCC patients.

Background/Aims: Phosphatase and tensin homolog (PTEN) is a known tumor suppressor gene that is downregulated in hepatocellular carcinoma (HCC). Here, we investigated the association between single nucleotide polymorphisms (SNPs) of PTEN and HCC development in patients with hepatitis B virus (HBV) infection. Methods: Six SNPs of PTEN at positions rs1234221, rs1903860, rs1234220, rs1903858, rs2299941, and rs17431184 were analyzed in a development population (417 chronic HBV carriers without HCC and 281 chronic HBV carriers with HCC). PTEN rs1903858, rs1903860, and rs2299941 SNPs were further assessed for the development of HCC in a validation population of 200 patients with HBV-related liver cirrhosis. Results: In the development population, PTEN rs1903860 C allele, rs1903858 G allele, and rs2299941 G allele were associated with a low risk of HCC. The haplotype A-T-A-A-A was associated with an increased risk of HCC (recessive model; odds ratio=2.277, 95% confidence interval [CI] =1.144-4.532, P=0.019). In the validation population, PTEN rs2299941 G allele was the only significant protective genetic polymorphism related to HCC development after adjustment for age and sex (hazard ratio=0.582, 95% CI =0.353-0.962, P=0.035). Conclusions: These findings suggest that genetic polymorphisms in PTEN may affect HCC development in patients with chronic HBV infection. (J Liver Cancer 2019;19:46-54)

Background/aims Posthepatectomy liver failure (PHLF) is a major complication that increases mortality in patients with hepatocellular carcinoma after surgical resection. The aim of this retrospective study was to evaluate the utility of magnetic resonance elastography-assessed liver stiffness (MRE-LS) for the prediction of PHLF and to develop an MRE-LS-based risk prediction model. Methods A total of 160 hepatocellular carcinoma patients who underwent surgical resection with available preoperative MRE-LS data were enrolled. Clinical and laboratory parameters were collected from medical records. Logistic regression analyses were conducted to identify the risk factors for PHLF and develop a risk prediction model. Results PHLF was present in 24 patients (15%). In the multivariate logistic analysis, high MRE-LS (kPa; odds ratio [OR] 1.49, 95% confidence interval [CI] 1.12 to 1.98, p=0.006), low serum albumin (≤3.8 g/dL; OR 15.89, 95% CI 2.41 to 104.82, p=0.004), major hepatic resection (OR 4.16, 95% CI 1.40 to 12.38, p=0.014), higher albumin-bilirubin score (>-0.55; OR 3.72, 95% CI 1.15 to 12.04, p=0.028), and higher serum α-fetoprotein (>100 ng/mL; OR 3.53, 95% CI 1.20 to 10.40, p=0.022) were identified as independent risk factors for PHLF. A risk prediction model for PHLF was established using the multivariate logistic regression equation. The area under the receiver operating characteristic curve (AUC) of the risk prediction model was 0.877 for predicting PHLF and 0.923 for predicting grade B and C PHLF. In leave-one-out cross-validation, the risk model showed good performance, with AUCs of 0.807 for all-grade PHLF and 0. 871 for grade B and C PHLF. Conclusions Our novel MRE-LS-based risk model had excellent performance in predicting PHLF, especially grade B and C PHLF.

Exosomal microRNAs (exo-miRs) contribute to cancer metastasis. To identify pro-metastatic circulating exo-miRs in hepatocellular carcinoma (HCC), next-generation sequencing-based plasma exo-miR profiles of 14 patients with HCC (eight non-metastatic and six with metastasis within 1 year of follow-up) were analyzed. Sixty-one miRs were significantly overexpressed among patients with metastatic HCC. Candidate miRs were selected through integrative analyses of two different public expression datasets, GSE67140 and The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC). Integrative analyses revealed 3 of 61 miRs (miR-106b-5p, miR-1307-5p, and miR-340-5p) commonly overexpressed both in metastasis and vascular invasion groups, with prognostic implications. Validation was performed using stored blood samples of 150 patients with HCC. Validation analysis showed that circulating exo-miR-1307-5p was significantly overexpressed in the metastasis group (p = 0.04), as well as in the vascular invasion and tumor recurrence groups. Circulating exo-miR-1307-5p expression was significantly correlated with tumor stage progression (p &lt, 0.0001). Downstream signaling pathways of miR-1307 were predicted using TargetScan and Ingenuity Pathway Analysis. On comprehensive bioinformatics analysis, the downstream pathway of miR-1307-5p, promoting epithelial&ndash, mesenchymal transition (EMT), showed SEC14L2 and ENG downregulation. Our results show that circulating exo-miR-1307-5p promotes metastasis and helps predict metastasis in HCC, and SEC14L2 and ENG are target tumor suppressor genes of miR-1307 that promote EMT.

Liquid biopsy can provide a strong basis for precision medicine. We aimed to identify novel single-nucleotide variants (SNVs) in circulating tumor DNA (ctDNA) in patients with hepatocellular carcinoma (HCC). Deep sequencing of plasma-derived ctDNA from 59 patients with HCC was performed using a panel of 2924 SNVs in 69 genes. In 55.9% of the patients, at least one somatic mutation was detected. Among 25 SNVs in 12 genes, four frequently observed SNVs, MLH1 (13%), STK11 (13%), PTEN (9%), and CTNNB1 (4%), were validated using droplet digital polymerase chain reaction with ctDNA from 62 patients with HCC. Three candidate SNVs were detected in 35.5% of the patients, with a frequency of 19% for MLH1 chr3:37025749T>A, 11% for STK11 chr19:1223126C>G, and 8% for PTEN chr10:87864461C>G. The MLH1 and STK11 SNVs were also confirmed in HCC tissues. The presence of the MLH1 SNV, in combination with an increased ctDNA level, predicted poor overall survival among 107 patients. MLH1 chr3:37025749T>A SNV detection in ctDNA is feasible, and thus, ctDNA can be used to detect somatic mutations in HCC. Furthermore, the presence or absence of the MLH1 SNV in ctDNA, combined with the ctDNA level, can predict the prognosis of patients with HCC.

Severe alcoholic hepatitis (AH) is the most aggressive form of alcohol-related liver disease with high mortality. The microbiome is an emerging therapeutic target in alcohol-related liver disease.To investigate the microbiome composition in patients with severe AH, and to determine microbiome recovery after rifaximin treatment in gut bacteria and bacteria derived-extracellular vesicles.We enrolled 24 patients with severe AH and 24 healthy controls. Additional faecal samples were collected after 4 weeks in 8 patients with severe AH who completed rifaximin treatment. Treatment response was defined based on Lille score model after 7 days of treatment. Metagenomic profiling was performed using 16S ribosomal RNA amplicon sequencing.Faecal microbiomes of patients with severe AH had lower alpha diversity and higher beta diversity than those of healthy controls in both gut bacteria and extracellular vesicles. Bacilli, Lactobacillales and Veillonella were significantly increased in the gut bacteria of patients with severe AH, and Veillonella, Veillonella parvula group and Lactobacillales were significantly increased in the extracellular vesicles of patients with severe AH. Eubacterium_g23, Oscillibacter and Clostridiales decreased in the gut bacteria of patients with severe AH, and Eubacterium_g23, Oscillibacter and Christensenellaceae decreased in the extracellular vesicles of patients with severe AH. After rifaximin treatment, 17 taxa in the gut bacteria and 23 taxa in extracellular vesicles were significantly restored in patients with severe AH. In common, Veillonella and Veillonella parvula group increased in patients with severe AH and decreased after rifaximin treatment, and Prevotella and Prevotellaceae decreased in patients with severe AH and increased after rifaximin treatment. Treatment non-responders showed a significantly lower abundance of Prevotella at baseline than did treatment responders.Dysbiosis was confirmed in severe AH but was alleviated by rifaximin treatment. Taxa associated with severe AH can be candidate biomarkers or therapeutic targets.

The roles of chromatin remodelers and their underlying mechanisms of action in cancer remain unclear. In this study, SMARCB1, known initially as a bona fide tumor suppressor gene, was investigated in liver cancer. SMARCB1 was highly upregulated in patients with liver cancer and was associated with poor prognosis. Loss- and gain-of-function studies in liver cells revealed that SMARCB1 loss led to reduced cell proliferation, wound healing capacity, and tumor growth in vivo. Although upregulated SMARCB1 appeared to contribute to switch/sucrose nonfermentable (SWI/SNF) complex stability and integrity, it did not act using its known pathways antagonism with EZH2 or association between TP53 or AMPK. SMARCB1 knockdown induced a mild reduction in global H3K27 acetylation, and chromatin immunoprecipitation sequencing of SMARCB1 and acetylated histone H3K27 antibodies before and after SMARCB1 loss identified Nucleoporin210 (NUP210) as a critical target of SMARCB1, which bound its enhancer and changed H3K27Ac enrichment and downstream gene expression, particularly cholesterol homeostasis and xenobiotic metabolism. Notably, NUP210 was not only a putative tumor supporter involved in liver cancer but also acted as a key scaffold for SMARCB1 and P300 to chromatin. Furthermore, SMARCB1 deficiency conferred sensitivity to doxorubicin and P300 inhibitor in liver cancer cells. These findings provide insights into mechanisms underlying dysregulation of chromatin remodelers and show novel associations between nucleoporins and chromatin remodelers in cancer. Significance: This study reveals a novel protumorigenic role for SMARCB1 and describes valuable links between nucleoporins and chromatin remodelers in cancer by identifying NUP210 as a critical coregulator of SMARCB1 chromatin remodeling activity.

Exosomal microRNAs (exo-miRs) have been promising cancer biomarkers. MiRs in hepatocellular carcinoma (HCC) cell-derived exosomes (HEX) were analyzed to identify reliable serum biomarkers for HCC. To detect overexpressed miRs in HEX, extracted exosomal small RNAs from human HCC cell lines and normal hepatocytes were sequenced and analyzed. Clinical significance of the overexpressed miRs in HEX was evaluated using quantitative real-time PCR (qRT-PCR) on serum samples of a validation cohort consisting of 28 healthy individuals, 60 with chronic liver disease, and 90 with HCC. We found 49 significantly overexpressed miRs in HEX compared to a normal hepatocyte. Among them, miR-10b-5p, miR-18a-5p, miR-215-5p, and miR-940 were overexpressed in HCC tissues and also associated with prognosis of HCC in the analysis of a public omics database. qRT-PCR analysis of the four serum exo-miRs in the validation cohort revealed serum exo-miR-10b-5p as a promising biomarker for early-stage HCC with 0.934 area under the curve (AUC) (sensitivity, 90.7%, specificity, 75.0%, cutoff value, 1.8-fold). Overexpression of serum exo-miR-215-5p was found to be significantly associated with poor disease-free survival in patients with HCC. Serum exo-miR-10b-5p is a potential biomarker for early-stage HCC, while serum exo-miR-215-5p can be used as prognostic biomarker for HCC.

Magnetic resonance elastography (MRE) is a non-invasive tool for measuring liver stiffness (LS) with high diagnostic accuracy. This study investigated whether quantified LS by MRE could predict early recurrence of patients with hepatocellular carcinoma (HCC) within the Milan criteria.A prospectively collected cohort, which included the HCC patients who underwent MRE before treatment (an HCC-MRE cohort), was analyzed. In the HCC-MRE cohort, only patients under the Milan criteria, who underwent hepatic resection, radiofrequency ablation (RFA), or transarterial chemoembolization (TACE), were reviewed. We investigated whether LS assessed by MRE was an independent predictor of early recurrence using Cox regressions and Kaplan-Meier analyses.A total of 192 HCC patients under the Milan criteria who underwent hepatic resection (n = 96), RFA (n = 23), or TACE (n = 73) were included. Higher LS ratings (kPa; hazard ratio [HR] = 1.12; 95% confidence interval [CI] = 1.01-1.25; p = 0.040) emerged as an independent risk factor for early tumor recurrence. In the subgroup analysis, higher LS ratings were associated with higher risks of early HCC recurrence in both the resection/RFA group ( 4.5 kPa; HR = 2.95; 95% CI = 1.26-6.94; p = 0.013) and the TACE group ( 6 kPa; HR = 2.94; 95% CI = 1.27-6.83; p = 0.012).LS assessed by MRE was an independent predictor of early recurrence among HCC patients under the Milan criteria after achieving a complete response.• Liver parenchymal stiffness measured by MRE predicts early recurrence of treated HCC under Milan criteria. • A liver stiffness 5.5 kPa was associated with worse recurrence-free survival. • Patients with high pre-treatment LS may benefit from stringent follow-up.

MALAT1 is deregulated in various cancers. The underlying mechanisms of MALA T1-mediated tumorigenesis are unclear. We found that MALAT1 was highly overexpressed and its overexpression was significantly associated with poor prognosis of liver cancer patients analyzed from the TCGA Liver Hepatocellular Carcinoma and Gene Expression Omnibus databases of the National Center for Biotechnology Information. Microarray analysis to identify the miRNAs deregulated by silencing MALAT1 in two liver cancer cell lines, HepG2 and Hep3B, revealed the common deregulation of 16 miRNAs including miR-574 and miR-20b in both cell lines. The predicted targets of miR-574 and miR-20b were cancer-related pathways including the RAS, MAPK and Wnt/β-catenin pathways. Aberrant expression of MALAT1 might contribute to liver cancer tumorigenesis by deregulation of cancer-associated miRNAs.

MicroRNAs (miRNAs) engage in complex interactions with the machinery that controls the transcriptome and concurrently target multiple mRNAs. Here, we demonstrate that microRNA-495-3p (miR-495-3p) functions as a potent tumor suppressor by governing ten oncogenic epigenetic modifiers (EMs) in gastric carcinogenesis. From the large cohort transcriptome datasets of gastric cancer (GC) patients available from The Cancer Genome Atlas (TCGA) and the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), we were able to recapitulate 15 EMs as significantly overexpressed in GC among the 51 EMs that were previously reported to be involved in cancer progression. Computational target prediction yielded miR-495-3p, which targets as many as ten of the 15 candidate oncogenic EMs. Ectopic expression of miRNA mimics in GC cells caused miR-495-3p to suppress ten EMs, and inhibited tumor cell growth and proliferation via caspase-dependent and caspase-independent cell death processing. In addition, in vitro metastasis assays showed that miR-495-3p plays a role in the metastatic behavior of GC cells by regulating SLUG, vimentin, and N-cadherin. Furthermore, treatment of GC cells with 5-aza-2'-deoxcytidine restored miR-495-3p expression; sequence analysis revealed hypermethylation of the miR-495-3p promoter region in GC cells. A negative regulatory loop is proposed, whereby DNMT1, among ten oncogenic EMs, regulates miR-495-3p expression via hypermethylation of the miR-495-3p promoter. Our findings suggest that the functional loss or suppression of miR-495-3p triggers overexpression of multiple oncogenic EMs, and thereby contributes to malignant transformation and growth of gastric epithelial cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

// Sung Kyung Choi 1, * , Kurinji Pandiyan 2, 3, * , Jung Woo Eun 4 , Xiaojing Yang 2 , Seong Hwi Hong 1 , Suk Woo Nam 4 , Peter A. Jones 5 , Gangning Liang 2 and Jueng Soo You 1, 6 1 Department of Biochemistry, School of Medicine, Konkuk University, Seoul, Korea 2 Departments of Urology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 3 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA 4 Department of Pathology, College of Medicine, The Catholic University, Seoul, Korea 5 Van Andel Research Institute, Grand Rapids, MI, USA 6 Research Institute of Medical Science, KonKuk University School of Medicine, Seoul, Korea * These authors contributed equally to this work Correspondence to: Jueng Soo You, email: jsyou@kku.ac.kr Keywords: epigenetic landscape, EMT, DNA methylation, chromatin accessibility, TRIM29 Received: August 01, 2017 Accepted: September 22, 2017 Published: October 09, 2017 ABSTRACT Epithelial to mesenchymal transition (EMT) is a key trans-differentiation process, which plays a critical role in physiology and pathology. Although gene expression changes in EMT have been scrutinized, study of epigenome is in its infancy. To understand epigenetic changes during TWIST-driven EMT, we used the AcceSssIble assay to study DNA methylation and chromatin accessibility in human mammary epithelial cells (HMECs). The DNA methylation changes were found to have functional significance in EMT – i.e. methylated genes were enriched for E-box motifs that can be recognized by TWIST, at the promoters suggesting a potential targeting phenomenon, whereas the demethylated regions were enriched for pro-metastatic genes, supporting the role of EMT in metastasis. TWIST-induced EMT triggers alterations in chromatin accessibility both independent of and dependent on DNA methylation changes, primarily resulting in closed chromatin conformation. By overlapping the genes, whose chromatin structure is changed during early EMT and a known “core EMT signature”, we identified 18 driver candidate genes during EMT, 14 upregulated and 4 downregulated genes with corresponding chromatin structure changes. Among 18 genes, we focused on TRIM29 as a novel marker of EMT. Although loss of TRIM29 is insufficient to suppress CDH, it is enough to induce CDH2 and VIM. Gene functional annotation analysis shows the involvement of TRIM29 in epidermal development, cell differentiation and cell migration. Taken together, our results provide a robust snapshot of chromatin state during human EMT and identify TRIM29 as a core mediator of EMT.

// Jung Woo Eun 1 , Hee Doo Yang 1 , Soo Hyun Kim 2 , Sungyoup Hong 3 , Kyu Nam Park 2 , Suk Woo Nam 1 and Sikyoung Jeong 3 1 Department of Pathology, Functional RNomics Research Center, Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea 2 Department of Emergency Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea 3 Department of Emergency Medicine, College of Medicine, The Catholic University of Korea, Daejeon, Republic of Korea Correspondence to: Sikyoung Jeong, email: // Keywords : peripheral blood transcriptome, molecular signature, neurological prognosis, cardiac arrest, cerebral performance category, Pathology Section Received : October 27, 2016 Accepted : January 19, 2017 Published : January 28, 2017 Abstract Background: Early prognostication of neurological outcome in comatose patients after cardiac arrest (CA) is important for devising patient treatment strategies. However, there is still a lack of sensitive and specific biomarkers for easy identification of these patients. We evaluated whether molecular signatures from blood of CA patients might help to improve the prediction of neurological outcome. Methods: We examined 22 comatose patients resuscitated after CA and obtained peripheral blood samples 48 hours after CA. To identify novel blood biomarkers, we aimed to measure neurological outcomes according to the Cerebral Performance Category (CPC) score at 6 months after CA and to determine blood transcriptome-based molecular signature of poor neurological outcome group. Results: According to the CPC score, 10 patients exhibited a CPC score of one and 12 patients, a CPC score four to five. Blood transcriptomics revealed differently expressed profiles between the good outcome group and poor outcome group. A total of 150 genes were down-regulated and 237 genes were up-regulated in the poor neurological outcome group compared with good outcome group. From the blood transcriptome-based signatures, we identified that MAPK3, BCL2 and AKT1 were more specific and sensitive diagnostic biomarkers in poor neurological outcome with an area under the curve of 0.867 ( p

BACKGROUND/AIM Fibroblast growth factor (FGF), vascular endothelial growth factor, and hepatocyte growth factor play a critical role in the pathogenesis of hepatocellular carcinoma (HCC). MATERIALS AND METHODS We assessed nine single nucleotide polymorphisms (SNPs) in the FGF1, FGF2, FGF receptor (FGFR)-2, Flt-1, and c-MET genes in 245 HCC patients and 483 chronic hepatitis B virus (HBV) carriers without HCC. RESULTS Kaplan-Meier analysis showed that patients with the FGF2 rs308447 TT genotype had shorter overall survival than patients with the CC or CT genotype (p=0.016) and that FGF2 rs308379 A allele carriers had shorter overall survival than patients with the TT genotype (p=0.020). CONCLUSION Multivariate Cox proportional analysis revealed that the FGF2 rs308379 A allele (hazard ratio(HR)=1.663, p=0.004) and advanced tumor stage (HR=3.430, p