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Aims/hypothesis Non-alcoholic fatty liver disease (NAFLD) associated with type 2 diabetes may more easily progress towards severe forms of non-alcoholic steatohepatitis (NASH) and cirrhosis. Although the Wnt effector transcription factor 7-like 2 (TCF7L2) is closely associated with type 2 diabetes risk, the role of TCF7L2 in NAFLD development remains unclear. Here, we investigated how changes in TCF7L2 expression in the liver affects hepatic lipid metabolism based on the major risk factors of NAFLD development. Methods Tcf7l2 was selectively ablated in the liver of C57BL/6N mice by inducing the albumin (Alb) promoter to recombine Tcf7l2 alleles floxed at exon 5 (liver-specific Tcf7l2-knockout [KO] mice: Alb-Cre;Tcf7l2f/f). Alb-Cre;Tcf7l2f/f and their wild-type (Tcf7l2f/f) littermates were fed a high-fat diet (HFD) or a high-carbohydrate diet (HCD) for 22 weeks to reproduce NAFLD/NASH. Mice were refed a standard chow diet or an HCD to stimulate de novo lipogenesis (DNL) or fed an HFD to provide exogenous fatty acids. We analysed glucose and insulin sensitivity, metabolic respiration, mRNA expression profiles, hepatic triglyceride (TG), hepatic DNL, selected hepatic metabolites, selected plasma metabolites and liver histology. Results Alb-Cre;Tcf7l2f/f essentially exhibited increased lipogenic genes, but there were no changes in hepatic lipid content in mice fed a normal chow diet. However, following 22 weeks of diet-induced NAFLD/NASH conditions, liver steatosis was exacerbated owing to preferential metabolism of carbohydrate over fat. Indeed, hepatic Tcf7l2 deficiency enhanced liver lipid content in a manner that was dependent on the duration and amount of exposure to carbohydrates, owing to cell-autonomous increases in hepatic DNL. Mechanistically, TCF7L2 regulated the transcriptional activity of Mlxipl (also known as ChREBP) by modulating O-GlcNAcylation and protein content of carbohydrate response element binding protein (ChREBP), and targeted Srebf1 (also called SREBP1) via miRNA (miR)-33-5p in hepatocytes. Eventually, restoring TCF7L2 expression at the physiological level in the liver of Alb-Cre;Tcf7l2f/f mice alleviated liver steatosis without altering body composition under both acute and chronic HCD conditions. Conclusions/interpretation In mice, loss of hepatic Tcf7l2 contributes to liver steatosis by inducing preferential metabolism of carbohydrates via DNL activation. Therefore, TCF7L2 could be a promising regulator of the NAFLD associated with high-carbohydrate diets and diabetes since TCF7L2 deficiency may lead to development of NAFLD by promoting utilisation of excess glucose pools through activating DNL. Data availability RNA-sequencing data have been deposited into the NCBI GEO under the accession number GSE162449 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162449). Graphical abstract

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

Reprogramming M2-type, pro-tumoral tumor-associated macrophages (TAMs) into M1-type, anti-tumoral macrophages is a key strategy in cancer therapy. In this study, we exploited epigenetic therapy using the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) and the histone deacetylation inhibitor trichostatin A (TSA), to reprogram M2-type macrophages into an M1-like phenotype. Treatment of M2-type macrophages with the combination of 5-aza-dC and TSA decreased the levels of M2 macrophage cytokines while increasing those of M1 macrophage cytokines, as compared to the use of either therapy alone. Conditioned medium of M2 macrophages treated with the combination of 5-aza-dC and TSA sensitized the tumor cells to paclitaxel. Moreover, treatment with the combination inhibited tumor growth and improved anti-tumor immunity in the tumor microenvironment. Depletion of macrophages reduced the anti-tumor growth activity of the combination therapy. Profiling of miRNAs revealed that the expression of miR-7083-5p was remarkably upregulated in M2 macrophages, following treatment with 5-aza-dC and TSA. Transfection of miR-7083-5p reprogrammed the M2-type macrophages towards an M1-like phenotype, and adoptive transfer of M2 macrophages pre-treated with miR-7083-5p into mice inhibited tumor growth. miR-7083-5p inhibited the expression of colony-stimulating factor 2 receptor alpha and CD43 as candidate targets. These results show that epigenetic therapy upon treatment with the combination of 5-aza-dC and TSA skews M2-type TAMs towards the M1-like phenotype by upregulating miR-7083-5p, which contributes to the inhibition of tumor growth.

Ubiquitin-specific protease 1 (USP1) is a deubiquitinase involved in DNA damage repair by modulating the ubiquitination of major regulators, such as PCNA and FANCD2. Because USP1 is highly expressed in many cancers, dysregulation of USP1 contributes to cancer therapy. However, the role of USP1 and the mechanisms underlying chemotherapy remain unclear. In this study, we found high USP1 expression in tumor tissues and that it correlated with poor prognosis in RCC. Mechanistically, USP1 enhanced survivin stabilization by removing ubiquitin. Pharmacological inhibitors (ML23 and pimozide) and siRNA targeting USP1 induced downregulation of survivin expression. In addition, ML323 upregulated DR5 expression by decreasing miR-216a-5p expression at the post-transcriptional level, and miR-216a-5p mimics suppressed the upregulation of DR5 by ML323. Inhibition of USP1 sensitized cancer cells. Overexpression of survivin or knockdown of DR5 markedly prevented the co-treatment with ML323 and TRAIL-induced apoptosis. These results of in vitro were proved in a mouse xenograft model, in which combined treatment significantly reduced tumor size and induced survivin downregulation and DR5 upregulation. Furthermore, USP1 and survivin protein expression showed a positive correlation, whereas miR-216a-5p and DR5 were inversely correlated in RCC tumor tissues. Taken together, our results suggest two target substrates of USP1 and demonstrate the involvement of survivin and DR5 in USP1-targeted chemotherapy.

Background Mac-2 binding protein glycosylation isomer (M2BPGi) has been established as a non-invasive biomarker for liver fibrosis. We evaluated the diagnostic efficacy of M2BPGi compared with those of other liver fibrosis markers in liver fibrosis in non-alcoholic fatty liver disease (NAFLD). Methods We analyzed serum M2BPGi levels in 113 NAFLD patients. A pathologist graded liver fibrosis histopathologically. The diagnostic efficacies of serum M2BPGi and other liver fibrosis markers (aspartate aminotransferase to platelet ratio index, fibrosis index based on four factors, and NAFLD fibrosis score [NFS]) were evaluated using correlation, area under the ROC curve (AUC), logistic regression, and C-statistics. Results Serum M2BPGi level and other liver fibrosis markers showed a moderate correlation with fibrosis grade. The AUC values of M2BPGi were 0.761, 0.819, 0.866, and 0.900 for diagnosing fibrosis (F)>0, F>1, F>2, and F>3, respectively. Logistic regression analysis showed M2BPGi as the only independent factor associated with F>2 and F>3. Although C-statistics showed that NFS was the best diagnostic factor for F>2 and F>3, M2BPGi with NFS had an increased C-statistics value, indicating that it is a better diagnostic model. Conclusions The serum M2BPGi level increased with liver fibrosis severity and could be a good biomarker for diagnosing advanced fibrosis and cirrhosis in NAFLD patients. A well-controlled, prospective study with a larger sample size is needed to validate the diagnostic power of M2BPGi and other fibrosis markers in NAFLD.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. It lacks sufficient diagnostic and prognostic markers, which leads to a poor prognosis. The survival rate of HCC patients is still low due to its highly metastatic nature. According to recent studies, non-coding RNAs play a vital role in cancer pathogenesis and may be a new diagnostic target for HCC. Circular RNA (circRNA) is a type of non-coding RNA with a closed loop structure formed by mRNA back-splicing. Its functions are miRNA sponging, translating to proteins and gene regulation. Recently, it has been found that circRNAs play a potential role at angiogenesis, metastasis, and drug resistance in many types of cancers. Thus, we aim to investigate the clinical relevance of hsa_circ_0116796 in HCC.Total RNAs from 100 pairs of HCC and corresponding normal liver (NL) tissues with different stages were extracted using QIAzol. CircRNA primers were coded including the gap junction for specificity. The expression levels were determined by quantitative real-time PCR (qRT-PCR), and its expression was normalized with GAPDH. In addition, we analyzed the correlation between hsa_circ_0116796 expression and various clinicopathological features of HCC patients. The target miRNAs were predicted in silico analyses. We found that hsa_circ_0116796 was significantly decreased in HCC tumor tissues compared to corresponding NL tissues (P Citation Format: Huiwon Do, Gyeonghwa Kim, Yu Rim Lee, Won Young Tak, Soo Young Park, Keun Hur. Hsa_circ_0116796 as a novel biomarker for predicting prognosis of human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1011.

Circular RNAs (CircRNAs) are type of noncoding RNAs without 5’ caps and 3’ tails that has been created by back splicing of mRNA. Recently, circRNAs are identified in many types of cancers, and its function has been elucidated as multiple miRNAs sponge. Hepatocellular carcinoma (HCC) is the most common type of malignancies. The incidence and mortality of HCC have been on the rise in recent years. Thus, it is necessary to identify further molecular mechanisms of HCC initiation and progression. In this study, we aim to investigate the clinical relevance of circRNAs encoded by the ZKSCAN1 (Zinc finger protein with KRAB and SCAN domains 1) gene in HCC.We analyzed clinical specimens from 100 pairs of HCC and corresponding normal liver (NL) tissues. Total RNAs were isolated from clinical tissues using qiagen kit. CircRNAs (circZKSCAN1) derived from ZKSCAN1 gene. The expression levels were determined by quantitative real-time PCR (qRT-PCR), and its expression was normalized with GAPDH. In addition, we analyzed the correlation between circZKSCAN1 expression and various clinicopathological features of HCC patients.CircZKSCAN1 expression was significantly down-regulated in HCC tissues compared to corresponding NL tissues (P Citation Format: Minji Bang, Gyeonghwa Kim, Eun Hwangbo, Mi Yeon Jeon, Keun Hur. Clinical significance of circZKSCAN1 RNA in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3305.

Aims: Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we aimed to investigate the differences between NDTRs and NDMVs. Methods: The generation of neutrophil-derived EVs were visualized by live-cell fluorescence images and the physical characteristics were further analyzed using nanotracking analysis assay, scanning electron microscopic analysis, and marker expressions. Functional characteristics of neutrophil-derived EVs were analyzed using assays for bactericidal activity, monocyte chemotaxis, phenotype polarization of macrophages, and miRNA sequencing. Finally, the effects of neutrophil-derived EVs on the acute and chronic inflammation were examined in vivo. Results: Both EVs share similar characteristics including stimulators, surface marker expression, bactericidal activity, and chemoattractive effect on monocytes via MCP-1. However, the integrin-mediated physical interaction was required for generation of NDTRs whereas NDMV generation was dependent on PI3K pathway. Interestingly, NDTRs contained proinflammatory miRNAs such as miR-1260, miR-1285, miR-4454, and miR-7975, while NDMVs contained anti-inflammatory miRNAs such as miR-126, miR-150, and miR-451a. Although both EVs were easily uptaken by monocytes, NDTRs enhanced proinflammatory macrophage polarization whereas NDMVs induced anti-inflammatory macrophage polarization. Moreover, NDTRs showed protective effects against lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. Conclusion: These results suggest that NDTR is a proinflammatory subtype of neutrophil-derived EVs distinguished from NDMV.

Background Sarcopenia frequently occurs in metastatic cancer patients. Emerging evidence has revealed that various secretory products from metastatic tumours can influence host organs and promote sarcopenia in patients with malignancies. Furthermore, the biological functions of microRNAs in cell‐to‐cell communication by incorporating into neighbouring or distal cells, which have been gradually elucidated in various diseases, including sarcopenia, have been elucidated. Methods We evaluated psoas muscle mass index (PMI) and intramuscular adipose tissue content (IMAC) using pre‐operative computed tomography imaging in 183 colorectal cancer (CRC) patients. miR‐203 expression levels in CRC tissues and pre‐operative serum were evaluated using quantitative polymerase chain reaction. Functional analysis of miR‐203 overexpression was investigated in human skeletal muscle cells (SkMCs), and cells were analysed for proliferation and apoptosis. Expressions of several putative miR‐203 target genes (CASP3, CASP10, BIRC5, BMI1, BIRC2, and BIRC3) in SKMCs were validated. Results A total of 183 patients (108 men and 75 women) were included. The median age of enrolled patients at diagnosis was 68.0 years (range 35–89 years). High IMAC status significantly correlated with female gender (P = 0.004) and older age (P = 0.0003); however, no other clinicopathological factors correlated with IMAC status in CRC patients. In contrast, decreased PMI significantly correlated with female gender (P = 0.006) and all well‐established disease development factors, including advanced T stage (P = 0.035), presence of venous invasion (P = 0.034), lymphovascular invasion (P = 0.012), lymph node (P = 0.001), distant metastasis (P = 0.002), and advanced Union for International Cancer Control tumour–node–metastasis stage classification (P = 0.0004). Although both high IMAC status and low PMI status significantly correlated with poor overall survival (IMAC: P = 0.0002; PMI: P

MicroRNAs (miRNAs) are important diagnostic and prognostic biomarkers for various tumors. Currently, many diagnostic systems have been developed to detect miRNAs, but simple techniques for detecting miRNAs are still required. Recently, we reported that the expression of miRNA-135b is upregulated in gastric epithelial cells during gastric inflammation and carcinogenesis. Our aim was to develop an in vitro diagnostic platform to analyze the expression of gastric cancer-related biomarkers in the blood. The diagnostic platform comprised an isothermal amplification-based lateral flow biosensor (IA-LFB) that enables easy diagnosis of gastric cancer through visual observation. In this platform, trace amounts of biomarkers are isothermally amplified through rolling circle amplification (RCA), and the amplified product is grafted to the LFB. The performance of the IA-LFB was confirmed using RNAs extracted from in vitro and in vivo models. The platform could detect target miRNAs within 3 h with excellent sensitivity and selectivity. In particular, the IA-LFB could detect the overexpression of gastric cancer-related markers (miRNA-135b and miRNA-21) in RNAs extracted from the blood of patients with various stages (stages 1-4) of gastric cancer compared to that in healthy volunteers. Therefore, IA-LFB is a simple and sensitive in vitro diagnostic system for detecting gastric cancer-related biomarkers and can contribute to the early diagnosis and prognosis monitoring of gastric cancer. Furthermore, this technology can be applied to systems that can detect multiple biomarkers related to various diseases (such as infectious and genetic diseases).

Exosomal noncoding RNAs (ncRNAs) have unique expression profiles reflecting the characteristics of a tumor, and their role in tumor progression and metastasis is emerging. However, the significance of circulating exosomal ncRNAs in the prognosis of hepatocellular carcinoma (HCC) remains to be elucidated. We therefore determined the prognostic significance of circulating exosomal ncRNAs (miRNA-21 and lncRNA-ATB) for human HCC. This prospective study enrolled 79 HCC patients between October 2014 and September 2015. Exosomes were extracted from serum samples using the ExoQuick Exosome Precipitation Solution. To validate the isolation of the exosomes from serum, immunoblotting for exosome markers and characterization of nanoparticle using NanoSight were performed. NcRNAs were isolated from exosomes using the miRNeasy serum/plasma micro kit. Both circulating exosomal miRNA-21 and lncRNA-ATB were related to TNM stage and other prognostic factors, including the T stage and portal vein thrombosis. Multivariate analysis using the Cox regression test identified that both higher miRNA-21 and higher lncRNA-ATB were independent predictors of mortality and disease progression, along with larger tumor size and higher C-reactive protein (all p < 0.05). The overall survival and progression-free survival were significantly lower in patients with higher circulating levels of exosomal miRNA-21 (≥0.09) and lncRNA-ATB (≥0.0016) (log-rank test: p < 0.05). In conclusion, our study has provided strong evidence that circulating exosomal ncRNAs (miRNA-21 and lncRNA-ATB) are novel prognostic markers and therapeutic targets for HCC.

Circular RNAs (circRNAs) represent potential biomarkers because of their highly stable structure and robust expression pattern in clinical samples. The aim of this study was to evaluate the expression of a recently identified circRNA, hsa_circ_0005986; determine its clinical significance; and evaluate its potential as a biomarker of hepatocellular carcinoma (HCC). We evaluated hsa_circ_0005986 expression in 123 HCC tissue samples, its clinical significance, and its association with patients’ clinicopathological characteristics and survival. Hsa_circ_0005986 expression was downregulated in HCC tissues. Low hsa_circ_0005986 expression was more common in tumors larger than 5 cm [odds ratio (OR), 3.19; 95% confidence interval (CI), 1.51–6.76; p = 0.002], advanced TNM stage (III/IV; OR, 2.39; 95% CI, 1.16–4.95; p = 0.018), and higher BCLC stage (B/C; OR, 2.71; 95% CI, 1.30–5.65; p = 0.007). High hsa_circ_0005986 expression was associated with improved survival and was an independent prognostic factor for overall [hazard ratio (HR), 0.572; 95% CI, 0.339–0.966; p = 0.037] and progression-free (HR, 0.573; 95% CI, 0.362–0.906; p = 0.017) survival. Moreover, the circRNA–miRNA–mRNA network was constructed using RNA-seq/miRNA-seq data and clinical information from TCGA-LIHC dataset. Our findings indicate a promising role for hsa_circ_0005986 as a prognostic biomarker in patients with HCC.

Among the three isoforms encoded by Rtn4, Nogo-A has been intensely investigated as a central nervous system inhibitor. Although Nogo-A expression is increased in muscles of patients with amyotrophic lateral sclerosis, its role in muscle homeostasis and regeneration is not well elucidated. In this study, we discovered a significant increase in Nogo-A expression in various muscle-related pathological conditions. Nogo−/− mice displayed dystrophic muscle structure, dysregulated muscle regeneration following injury, and altered gene expression involving lipid storage and muscle cell differentiation. We hypothesized that increased Nogo-A levels might regulate muscle regeneration. Differentiating myoblasts exhibited Nogo-A upregulation and silencing Nogo-A abrogated myoblast differentiation. Nogo-A interacted with filamin-C, suggesting a role for Nogo-A in cytoskeletal arrangement during myogenesis. In conclusion, Nogo-A maintains muscle homeostasis and integrity, and pathologically altered Nogo-A expression mediates muscle regeneration, suggesting Nogo-A as a novel target for the treatment of myopathies in clinical settings.

The level of long interspersed nuclear element-1 (LINE-1) methylation, representing the global deoxyribonucleic acid methylation level, could contribute to the prognosis of cancer via the activation of oncogenes. This study was performed to evaluate the prognostic implications of LINE-1 hypomethylation in patients with hepatocellular carcinoma (HCC) and the possible mechanisms related to oncogene activation. Seventy-seven HCC patients between October 2014 and September 2015 were enrolled in this prospective study. Quantitative pyrosequencing was performed to assess the LINE-1 methylation level of HCC and matched non-HCC tissue samples. The expression of suppression of tumorigenicity 18 was measured by immunohistochemistry and its correlation with LINE-1 methylation levels was examined. LINE-1 was significantly hypomethylated in the HCC tissue compared with the matched nontumor tissue (64.0 ± 11.6% vs 75.6 ± 4.0%, P

To compare the clinical value of acoustic radiation force impulse (ARFI) elastography and transient elastography (TE) for hepatocellular carcinoma (HCC) recurrence prediction after radiofrequency ablation (RFA) and to investigate other predictors of HCC recurrence.Between 2011 and 2016, 130 patients with HCC who underwent ARFI elastography and TE within 6 months before curative RFA were prospectively enrolled. Independent predictors of HCC recurrence were analyzed separately using ARFI elastography and TE. ARFI elastography and TE accuracy to predict HCC recurrence was determined by receiver operating characteristic curve analysis.Of all included patients (91 men; mean age, 63.5 years; range: 43-84 years), 51 (42.5%) experienced HCC recurrence during the follow-up period (median, 21.9 months). In multivariable analysis using ARFI velocity, serum albumin and ARFI velocity [hazard ratios: 2.873; 95% confidence interval (CI): 1.806-4.571; P0.001] were independent predictors of recurrence, and in multivariable analysis using TE value, serum albumin and TE value (hazard ratios: 1.028; 95% CI: 1.013-1.043; P0.001) were independent predictors of recurrence. The area under the receiver operating characteristic curve of ARFI elastography (0.821; 95% CI: 0.747-0.895) was not statistically different from that of TE (0.793; 95% CI: 0.712-0.874) for predicting HCC recurrence (P=0.827). The optimal ARFI velocity and TE cutoff values were 1.6 m/s and 14 kPa, respectively.ARFI elastography and TE yield comparable predictors of HCC recurrence after RFA.

Aim: Aberrant microRNA (miR) expression is associated with hepatocellular carcinoma (HCC). Materials & methods: Here, we investigated the clinical significance of miR-21 and miR-31 for HCC-specific prognostic effect. HCC patients (n = 93) who underwent liver biopsy or surgical resection were enrolled, and HCC tissues and paired adjacent nontumor liver tissues were collected and analyzed for miRs expression. Results: MiR-21 expression was significantly upregulated in HCC tissues relative to nontumor tissues. Both miR-21 and miR-31 expression in HCC tissues did not predict overall survival; however, miR-21 was considered an independent predictor of disease progression together with portal vein thrombosis and higher Barcelona Clinical Liver Cancer stage. Conclusion: Elevated miR-21 expression might represent a biomarker for HCC prognosis.

Adipose tissue and skeletal muscle is associated with non-alcoholic fatty liver disease (NAFLD). This study evaluates the association between body composition and histologic severity in patients with NAFLD. Using the cross-sectional CT images at the level of L3 vertebra and the histologic findings of 178 patients with biopsy-proven NAFLD, we analyzed the correlation of the histologic findings to the skeletal muscle index (SMI), subcutaneous adipose tissue index (SATI), and visceral adipose tissue index (VATI), which is defined as the body composition area (cm2) by height squared (m2). The clinical and laboratory features with body composition were analyzed to determine the risk factors for advanced fibrosis. The VATI significantly increased in severe non-alcoholic steatohepatitis (NASH) or advanced fibrosis. In addition, the VATI was correlated with the NAFLD activity score (NAS) and the fibrosis stage. In multivariate analyses, age (odds ratio (OR), 1.09, 95% confidence interval (CI), 1.02–1.19, p = 0.025), severe NASH (OR, 8.66, 95% CI, 2.13–46.40, p = 0.005), and visceral adiposity (OR, 6.77, 95% CI, 1.81–29.90, p = 0.007) were independently associated with advanced fibrosis in patients with NAFLD. Visceral adiposity is correlated with the histologic severity of NAFLD, which is independently associated with advanced fibrosis.

Peritoneal carcinomatosis (PC) of gastric origin has a poor prognosis with short survival due to lack of effective therapeutic modalities. Here, we evaluated the therapeutic efficacy of an injectable thermosensitive poly (organophosphazene) (PPZ) hydrogel with docetaxel (DTX-gel) to develop an effective therapeutic agent for patient with PC. Three days after inoculation of highly metastatic 44As3Luc cells into peritoneal cavity, the mice were intravenously or intraperitoneally administered with docetaxel alone (DTX-sol IV or IP), and intraperitoneally injected with DTX-gel. The anti-tumor activity was monitored by bioluminescence live imaging system. Compared to DTX-sol IV or IP, the tumor growth was significantly reduced in the DTX-gel treated mice (p

// Se Young Jang 1, * , Gyeonghwa Kim 2, * , Soo Young Park 1 , Yu Rim Lee 1 , Sang Hoon Kwon 1 , Hyeong Seok Kim 1 , Jun Sik Yoon 1 , Jun Seob Lee 1 , Young-Oh Kweon 1 , Heon Tak Ha 3 , Jae Min Chun 3 , Young Seok Han 3 , Won Kee Lee 4 , Jun Young Chang 5 , Jung Gil Park 6 , Byungheon Lee 2 , Won Young Tak 1 and Keun Hur 2 1 Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, Republic of Korea 2 Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, Republic of Korea 3 Department of Surgery, Kyungpook National University School of Medicine, Daegu, Republic of Korea 4 Biostatistics, Medical Research Collaboration Center in Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, Republic of Korea 5 Department of Neurology, Gyeongsang National University Changwon Hospital, Changwon, Republic of Korea 6 Department of Internal Medicine, College of Medicine, Yeungnam University, Daegu, Republic of Korea * These authors contributed equally to this work Correspondence to: Keun Hur, email: KeunHur@knu.ac.kr Won Young Tak, email: wytak@knu.ac.kr Keywords: hepatocellular carcinoma, long noncoding RNA ATB, survival, prognosis, epithelial mesenchymal transition Received: June 28, 2017 Accepted: September 04, 2017 Published: September 20, 2017 ABSTRACT Hepatocellular carcinoma (HCC) is a worldwide health problem and it is important to understand the mechanistic roles of the biomolecules involved in its pathogenesis. Long non-coding RNAs (lncRNAs) are frequently and aberrantly expressed in various human cancers and are known to play a role in cancer pathogenesis. The aim of this study was to analyze the expression of lncRNA-ATB in HCC and investigate the implications for prognoses. In total, 100 samples of HCC tissues and their corresponding, adjacent, non-cancerous liver tissues were collected. Total RNAs were extracted and the expression levels of lncRNA-ATB were measured by qRT-PCR. The association of lncRNA expression with clinicopathological features and patient survival were then analyzed. LncRNA-ATB was significantly upregulated in HCC tissues compared with the levels in corresponding non-cancerous tissues. Expression of lncRNA-ATB was significantly associated with portal vein thrombosis, intrahepatic or extrahepatic metastases, mUICC stage, and the BCLC stage. Large tumors (> 5 cm, HR = 3.851, 95% CI = 1.431–10.364, p = 0.008) and higher lncRNA-ATB expression (HR = 4.158, 95% CI = 1.226–14.107, p = 0.022) were the significant prognostic factors for overall survival. With this novel evidence of the involvement of lncRNA-ATB in HCC pathogenesis and clinical features, lncRNA-ATB can be concluded to have potential as a biomarker for the prognosis of HCC and as a targeted therapy for afflicted patients.

Background and Aim Liver stiffness (LS) value determined using transient elastography (TE) can be used to assess the degree of liver fibrosis. The study investigated whether TE can predict the recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA). Methods This study retrospectively enrolled 228 patients with HCC who received TE and RFA as the first-line treatment for HCC between 2008 and 2015. Cox regression analysis was used to identify independent predictors of HCC recurrence. Results The median age of the study population (170 men and 58 women) was 61 years. During the study period, HCC recurrence and mortality developed in 125 (54.8%) and 37 (16.2%) patients after RFA, respectively. Liver cirrhosis, platelet count, multiple tumors, and LS value were the independent predictors of HCC recurrence. When the study population was stratified into early (

// Byung Woog Kang 1, * , YongHun Choi 2, 3, * , Oh Kyoung Kwon 4 , Seung Soo Lee 4 , Ho Young Chung 4 , Wansik Yu 4 , Han Ik Bae 5 , An Na Seo 5 , Hyojeung Kang 6 , Suk Kyeong Lee 7 , Seong Woo Jeon 8 , Keun Hur 2, 3 , Jong Gwang Kim 1 1 Department of Oncology/Hematology, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Kyungpook National University Cancer Research Institute, Kyungpook National University, Daegu, South Korea 2 Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, South Korea 3 BK21 Plus KNU Biomedical Convergence Program, Department of Biomedical Science, Kyungpook National University, Daegu, South Korea 4 Department of Surgery, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, South Korea 5 Department of Pathology, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, South Korea 6 College of Pharmacy, Institute of Microorganisms and Research Institute of Pharmaceutical Sciences, Kyunpook National University, Daegu, South Korea 7 Department of Medical Lifescience, College of Medicine, Catholic University of Korea, Seoul, South Korea 8 Department of Gastroeneterology, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, South Korea * These authors contributed equally to this work Correspondence to: Jong Gwang Kim, email: jkk21c@knu.ac.kr Keun Hur, email: KeunHur@knu.ac.kr Keywords: gastric cancer, epstein-barr virus, microRNA, prognosis, BART Received: August 02, 2016 Accepted: January 10, 2017 Published: January 19, 2017 ABSTRACT This study analyzed the relationship between several Epstein-Barr virus (EBV) microRNA (miRNA) expression profiles and the clinicopathologic features of patients with EBV-associated gastric cancer. The miRNA expression was examined in 59 tumor and 39 paired normal mucosal tissues from available formalin-fixed paraffin embedded tissue samples. The expression levels of miR-BamHI fragment A rightward transcript (BART)1-5p, miR-BART4-5p, and miR-BART20-5p were determined using a quantitative real-time polymerase chain reaction. The expression of all three analyzed EBV microRNAs was significantly higher in the tumor tissue than in the paired normal tissue ( P < 0.001 for each). When the median value of the EBV microRNA expression levels was used as the cutoff point, a high BART20-5p expression was associated with worse recurrence-free survival ( P = 0.034) in a multivariate analysis including age and pathologic stage. In conclusion, the expression level of BART20-5p may predict recurrence-free survival for patients with EBV-associated gastric cancer. Further studies are warranted to clarify the roles of EBV BART microRNAs in the carcinogenesis, and their potential as a biomarker and therapeutic target for EBV-associated gastric cancer.

Purpose MicroRNAs (miRNAs) regulate various cellular functions, including development, cell proliferation, apoptosis, and tumorigenesis. Different signatures associated with various tissue types, diagnosis, progression, prognosis, staging, and treatment response have been identified by miRNA expression profiling of human tumors. miRNAs function as oncogenes or as tumor suppressors. The relationship between gastric cancer and miRNA garnered attention due to the high incidence of gastric cancer in Asian countries. miR-222/221 expression increases in gastric tumor tissues. The oncogenic effect of miR-222/221 was previously determined in functional studies and xenograft models. In this study, transgenic mice over-expressing miR-222/221 were generated to confirm the effect of miR-222/221 on gastric carcinogenesis. Materials and methods At 6 weeks of age, 65 transgenic mice and 53 wild-type mice were given drinking water containing N-nitroso-N-methylurea (MNU) for 5 alternating weeks to induce gastric cancer. The mice were euthanized at 36 weeks of age and histologic analysis was performed. Results Hyperplasia was observed in 3.77% of the wild-type mice and in 18.46% of the transgenic mice (p=0.020). Adenoma was observed in 20.75% of the wild-type mice and 26.15% of the transgenic mice (p=0.522). Carcinoma was observed in 32.08% of the wild-type mice and 41.54% of the transgenic mice (p=0.341). The frequency of hyperplasia, adenoma, and carcinoma was higher in transgenic mice, but the difference was statistically significant only in hyperplasia. Conclusion These results suggest that hyperplasia, a gastric pre-cancerous lesion, is associated with miR-222/221 expression but miR-222/221 expression does not affect tumorigenesis itself.

SUMMARYExtracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we investigated the similarities and differences between neutrophil-derived EV subtypes. Neutrophil-derived EVs shared similar characteristics regarding stimulators, generation mechanisms, and surface marker expression. Both neutrophil-derived EV subtypes exhibited similar functions, such as direct bactericidal activity and induction of monocyte chemotaxis via MCP-1. However, NDTR generation was dependent on the integrin signaling, while NDMV generation was dependent on the PI3K pathway. The CD16 expression level differentiated the neutrophil-derived EV subtypes. Interestingly, both subtypes showed different patterns of miRNA expression and were easily phagocytosed by monocytes. NDTRs induced M1 macrophage polarization, whereas NDMVs induced M2 macrophage polarization. Moreover, NDTRs but not NDMVs exerted protective effects against sepsis-induced lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. These results suggest a new insight into neutrophil-derived EV subtypes: proinflammatory NDTRs and anti-inflammatory NDMVs.Key pointsNeutrophil-derived trails are proinflammatory extracellular vesicles that induce M1 macrophage polarization and protect against inflammationNeutrophil-derived microvesicles are anti-inflammatory extracellular vesicles that induce M2 macrophage polarization

Background: Chronic inflammation promotes aging and carcinogenesis in colorectal mucosa, particularly in ulcerative colitis (UC); providing a rationale for the development of these molecular alterations as useful biomarkers for diagnosis and risk prediction of UC-associated colorectal cancer (UC-CRC) and sporadic colorectal cancer (S-CRC). Methods: We performed array-based methylation analyses using tissues from 76 patients with S-CRC (vs. normal mucosa) and UC-CRC (vs. UC). In the evaluation phase, 945 colorectal specimens from 352 patients with S-CRC, 31 with UC-CRC, and 57 with UC, were analyzed for methylation levels of identified genes by quantitative pyrosequencing. In the validation phase, 199 non-cancerous rectal mucosa specimens from 20 patients with S-CRC, 61 with UC-CRC, 90 with UC, and 28 healthy volunteers were examined. Findings: Comprehensive analysis identified OPLAH as a hypermethylated CpG site in S-CRC and UC-CRC patients. In the evaluation phase, OPLAH methylation (met-OPLAH) differentiated S-CRC tissue from normal mucosa (area under the receiver operating characteristic curve (AUC):0.95, sensitivity:0.89, specificity:0.95), and met-OPLAH levels in normal mucosa were positively correlated with age. However, met-OPLAH was specifically hypermethylated in UC-CRC tissues and differentiated UC-CRC from UC mucosa (AUC:0.95, sensitivity:0.88, specificity:0.94). Levels of met-OPLAH were significantly higher in rectal tissues vs. proximal mucosa, and were associated with age and disease duration in rectal mucosa. In non-cancerous rectal mucosa, met-OPLAH could discriminate UC patients with or without UC-CRC (AUC:0.84, sensitivity:0.83, specificity:0.72). We further developed ∆met-OPLAH, which could distinguish S-CRC from UC-CRC (P=0.0002, AUC:0.81, sensitivity:0.92, specificity:0.65). Interpretation: Assessment of met-OPLAH can be used for screening of UC-CRC using non-cancerous rectal mucosa, diagnosis using cancerous tissues, and decision-making of surgical approaches using both tissue types in patients with UC and cancer. Funding: Grants in Aid for Scientific Research (17K10628, 18K08591) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Declaration of Interest: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. The authors have no conflict of interests to disclose. Ethical Approval Statement: Specimen collection and experiments were approved by the Institutional Review Boards of all participating institutions. All participants provided a written informed consent for participation in research.

Context: Gastric cancer (GC) is the fourth most common cause of cancer-related deaths worldwide.Objective: To determine the mRNA-expression of the MAL, TMEM220, MMP28, IL-19 and HOPX genes and analyse the methylation statuses of MAL and TMEM220.Materials and methods: Gene-expression levels were analysed in 10 GC cell lines and 30 matched pairs of GC and normal mucosa (NM) gastric tissue specimens in real-time reverse-transcriptase polymerase chain reactions. Gene methylation was evaluated by bisulphite sequencing. Detailed gene-methylation patterns were confirmed by pyrosequencing analysis.Results: MAL, TMEM220, MMP28 and IL-19 were significantly down-regulated in GC cell lines and GC tissues compared to NM tissues. MAL and TMEM220 were highly methylated in GC tissues, and methylation inversely correlated with expression. MAL and TMEM220 expression were restored by treatment with 5-aza-2′-deoxycytidine. MAL and TMEM220 were specifically methylated and were down-regulated in human GC.Discussion and...

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This study was designed to determine the prognostic impact and clinical significance of FGFR2 in residual disease after preoperative chemoradiotherapy (CRT) in patients with rectal cancer. The surgical specimens of 145 patients with residual rectal cancer after preoperative CRT were analyzed. To evaluate FGFR2 expression, immunohistochemistry was performed on whole section tissues. KRAS exon 2 (codon 12 and 13), BRAF V600E mutational status, and microsatellite instability (MSI) were determined using polymerase chain reactions. Of the eligible 141 patients, FGFR2 over-expression was observed in 75.9 % (n = 107) and was correlated with perineural invasion (P = 0.005) and inferior tumor regression grading (TRG) (P = 0.009). However, FGFR2 expression had no relationship with KRAS and BRAF mutation results or with MSI results. On univariate analysis, FGFR2 over-expression was significantly associated with worse rectal cancer-specific survival (RCSS) (P = 0.005) and disease-free survival (DFS) (P = 0.035). However, multivariate analysis revealed that FGFR2 over-expression was not independently associated with RCSS and DFS (all P > 0.05). Although FGFR2 over-expression did not independently influence patient outcome, FGFR2 over-expression was associated with worse prognosis and inferior TRG. Our data may aid in understanding the therapeutic approaches targeting FGFR2 in patients with residual rectal cancer after preoperative CRT.

Background and aims Distant metastasis is a major cause of deaths in patients with colorectal cancer (CRC), which is partly due to lack of robust metastasis-predictive biomarkers. In spite of the important function of microRNA (miR)-203 in cancer metastasis, its clinical significance in CRC metastasis remains unknown. Here, we evaluated the potential role of serum miR-203 as a non-invasive biomarker for CRC metastasis. Methods MiR-203 expression was quantified by quantitative reverse-transcription PCR in 58 pairs of primary CRC (pCRC) and corresponding matched liver metastasis (LM), as well as 186 serum and 154 matched tissue specimens from patients with CRC in cohort 1. Next, we performed validation of miR-203 levels in serum from 144 patients with CRC in an independent cohort (cohort 2). Mouse models of CRC-associated metastases were established to identify the source of circulating miR-203. Expression patterns of miR-203 in tissues were determined by in situ hybridisation. Results MiR-203 expression was significantly upregulated in LM compared with matched pCRC tissues. Serum miR-203 levels were significantly upregulated in a stage-dependent manner, and high miR-203 expression was associated with poor survival in patients with CRC in both patient cohorts. Increased miR-203 levels in serum indicated high risk for poor prognosis (HR=2.1), as well as metastasis to lymph nodes (OR=2.5), liver (OR=6.2), peritoneum (OR=7.2) and distant organs (OR=4.4). Serum miR-203 levels were significantly higher in animals with liver or systemic metastasis compared with controls. Conclusions High levels of serum miR-203 associate with poor survival and metastasis, suggesting it to be a promising non-invasive prognostic and metastasis-predictive biomarker in patients with CRC.

Simple Summary Non-coding RNAs such as microRNAs, long non-coding RNAs, and circular RNAs contribute to the development and progression of hepatocellular carcinoma through epigenetic association. Long non-coding RNAs and circular RNAs act as competing endogenous RNAs that contain binding sites for miRNAs and thus compete with the miRNAs, which results in promotion of miRNA target gene expression, thereby leading to proliferation and metastasis of hepatocellular carcinoma. Competing endogenous RNAs have the potential to become diagnostic biomarkers and therapeutic targets for treatment of hepatocellular carcinoma. Abstract The three major members of non-coding RNAs (ncRNAs), named microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), play an important role in hepatocellular carcinoma (HCC) development. Recently, the competing endogenous RNA (ceRNA) regulation model described lncRNA/circRNA as a sponge for miRNAs to indirectly regulate miRNA downstream target genes. Accumulating evidence has indicated that ceRNA regulatory networks are associated with biological processes in HCC, including cancer cell growth, epithelial to mesenchymal transition (EMT), metastasis, and chemoresistance. In this review, we summarize recent discoveries, which are specific ceRNA regulatory networks (lncRNA/circRNA-miRNA-mRNA) in HCC and discuss their clinical significance.

Background:Long noncoding RNAs (lncRNAs) have recently emerged as important biological regulators and the aberrant expression of lncRNAs has been reported in various diseases including cancer, cardiovascular disease, and diabetes mellitus. However, the role of lncRNAs in the pathogenesis of rheumatoid arthritis (RA) remains unknown.Objectives:Thus, we studied lncRNAs influenced by IL-1, which is one of the key mediators in the pathogenesis of RA, and also investigated whether regulation of NF-κB activation, which is known to be induced by IL-1, could lead to the changes of expression of those lncRNAs.Methods:Fibroblast-like synoviocytes (FLS) were obtained from the knee joints of the patients with RA. The next-generation sequencing (NGS) data were analyzed to identify differentially expressed lncRNAs between unstimulated RA FLS and IL-1-stimulated RA FLS. The expression levels of the top 5 candidates in NGS data were validated by RT-qPCR using extended number of unstimulated RA FLS and IL-1-stimulated RA FLS. IMD-0560, an inhibitor of IκB kinase (IKK) was used for the regulation of NF-κB activation. Activation and inhibition of NF-κB were confirmed by Western blotting. Changed expressions of the lncRNAs were identified by RT-qPCR.Results:NGS analysis revealed up-regulated 30 lncRNAs and down-regulated 15 lncRNAs in IL-1-treated RA FLS compared with unstimulated RA FLS. Top 5 lncRNAs were selected among 30 lncRNAs up-regulated by IL-1 in RA FLS based on fold-change with P-value cutoff. The up-regulated lncRNAs including NR_046035, NR_027783, NR_033422, NR_003133, and NR_049759 were validated by RT-qPCR. IMD-0560 inhibited phosphorylation of IκBα induced by IL-1 in RA FLS. Overexpression of lncRNAs induced by IL-1 was also inhibited by IMD-0560 in RA FLS.Conclusion:Our study revealed that IL-1 increased the expression of NR_046035, NR_027783, NR_033422, NR_003133, and NR_049759 in RA FLS. In addition, the expression of these lncRNAs was regulated by inhibition of NF-κB activation. Thus, our data suggest that the lncRNAs might be involved in the pathogenesis of RA through NF-κB signaling pathway.References:[1]Long noncoding RNAs and human disease. Trends Cell Biol. 2011 Jun;21(6):354-61.[2]A long noncoding RNA mediates both activation and repression of immune response genes. Science. 2013 Aug 16;341(6147):789-92.[3]Long noncoding RNA expression profile in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Arthritis Res Ther. 2016 Oct 6;18(1):227.Disclosure of Interests:None declared

Background: Advanced liver fibrosis is the most important prognostic factor in nonalcoholic fatty liver disease (NAFLD). The long noncoding RNA (lncRNA), growth arrest-specific transcript 5 (GAS5), is associated with the inhibition of liver fibrogenesis, and its levels are decreased in cirrhotic liver. Methods: We analyzed 51 patients with NAFLD, the diagnosis of which was confirmed by liver biopsy. Expression of GAS5 in both the liver and plasma of the patients was analyzed using a quantitative real-time polymerase chain reaction according to the fibrosis stage. Results: Plasma GAS5 expression was significantly higher in patients with advanced fibrosis than in those without. As the fibrosis progressed, GAS5 expression in plasma increased, with the exception of that in cirrhotic livers. Plasma levels of GAS5 were lower in patients with cirrhosis than in those with advanced fibrosis. Conclusion: Elevated circulating levels of the lncRNA GAS5 are associated with the progression of liver fibrosis prior to the development of cirrhosis.

181 Background: Sarcopenia is commonly observed in advanced cancer patients with distant metastases. In addition, biological functions of microRNAs (miRNAs) in cell-to-cell communication by incorporating into neighboring or distal cells has been gradually elucidated in various diseases, including sarcopenia, have been established. Methods: First, we quantified miR-203 expression, a key miRNA involved in cell-to-cell communication, by qRT-PCR and in situ hybridization in 58 pairs of primary CRC (pCRC) and corresponding matched liver metastasis (LM) tissues. We further evaluated miR-203 levels using pCRC tissues and matched preoperative serum to clarify its clinical significance in independent 183 CRC patients. Second, we assessed psoas muscle mass index (PMI) and intramuscular adipose tissue content (IMAC) using preoperative computed tomography imaging to clarify its clinical burden and correlations with miR-203 levels in CRC patients. Functional analysis of miR-203 overexpression was investigated in human skeletal muscle cells (SkMC), and cells were analyzed for proliferation and apoptosis. Expression of several, putative, miR-203-target genes was also validated in SkMC cells. Results: MiR-203 expression was significantly upregulated in LM compared with matched pCRC tissues. Serum miR-203 levels were significantly upregulated in a stage-dependent manner, and high miR-203 expression was associated with poor survival in patients with CRC in both patient cohorts. In contrast to IMAC, decreased PMI significantly correlated with well-established disease development factors, and decreased PMI was an independent prognostic factor for both overall survival, and disease-free survival in CRC patients. Although tissue miR-203 expression did not significantly correlate with the body composition status, serum miR-203 expression negatively correlated with preoperative PMI level. Overexpression of miR-203 inhibited cell proliferation and induced apoptosis via downregulation of BIRC5 (Survivin) expression in SkMC cell. Conclusions: Assessment of serum miR-203 could be used for risk assessment of metastasis-related myopenia in CRC.

Hypoxia-inducible factors (HIFs) are transcription factors that play central roles in cellular responses against hypoxia. In most cancers, HIFs are closely associated with tumorigenesis by regulating cell survival, angiogenesis, metastasis, and adaptation to the hypoxic tumor microenvironment. Recently, non-coding RNAs (ncRNAs) have been reported to play critical roles in the hypoxic response in various cancers. Here, we review the roles of hypoxia-response ncRNAs in gastrointestinal cancer, with a particular focus on microRNAs and long ncRNAs, and discuss the functional relationships and regulatory mechanisms between HIFs and ncRNAs.

Exosomal noncoding RNAs (ncRNAs) have unique expression profiles reflecting the characteristics of a tumor, and their role in tumor progression and metastasis is emerging. However, the significance of circulating exosomal ncRNAs in the prognosis of hepatocellular carcinoma (HCC) remains to be elucidated. We therefore determined the prognostic significance of circulating exosomal ncRNAs (miRNA-21 and lncRNA-ATB) for human HCC. This prospective study enrolled 79 HCC patients between October 2014 and September 2015. Exosomes were extracted from serum samples using the ExoQuick Exosome Precipitation Solution. To validate the isolation of the exosomes from serum, immunoblotting for exosome markers and characterization of nanoparticle using NanoSight were performed. NcRNAs were isolated from exosomes using the miRNeasy serum/plasma micro kit. Both circulating exosomal miRNA-21 and lncRNA-ATB were related to TNM stage and other prognostic factors, including the T stage and portal vein thrombosis. Multivariate analysis using the Cox regression test identified that both higher miRNA-21 and higher lncRNA-ATB were independent predictors of mortality and disease progression, along with larger tumor size and higher C-reactive protein (all p0.05). The overall survival and progression-free survival were significantly lower in patients with higher circulating levels of exosomal miRNA-21 (≥0.09) and lncRNA-ATB (≥0.0016) (log-rank test: p0.05). In conclusion, our study has provided strong evidence that circulating exosomal ncRNAs (miRNA-21 and lncRNA-ATB) are novel prognostic markers and therapeutic targets for HCC.

Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death worldwide. Distant metastasis is a major cause of mortality in CRC. MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the post-transcriptional and translational regulation of gene expression. Many miRNAs are aberrantly expressed in cancer and influence tumor progression. Accumulating studies suggest that multiple miRNAs are actively involved in the CRC metastasis process. Thus, we aim to introduce the role of miRNAs in multi-steps of CRC metastasis, including cancer cell invasion, intravasation, circulation, extravasation, colonization, angiogenesis, and epithelial-mesenchymal transition (EMT). Moreover, we suggest the potential application of miRNAs as biomarkers for CRC patients with metastasis. [BMB Reports 2015; 48(4): 217-222]

To compare the efficacy of and mortality after lamivudine (LAM), tenofovir (TDF), and entecavir (ETV) treatment in patients with severe acute chronic hepatitis B (CHB) exacerbation.We analyzed 91 patients with severe acute CHB exacerbation treated with LAM (n=28), TDF (n=26), or ETV (n=37) for 10 years. The primary endpoint was overall mortality or liver transplantation (LT) by 48 weeks. The determined predictors of mortality, virologic and biochemical responses, and drug resistance were also evaluated.The overall mortality or LT rate was not significantly different among the LAM (14.3%), ETV (10.8%), and TDF (3.8%) groups (P=0.435). In the multivariate analysis, the occurrence of ascites (hazard ratio [HR] 10.467, 95% confidence interval [CI] 1.596-68.645, P=0.014) and model for end-stage liver disease (MELD) scores above 25 (HR 28.920, CI 4.719-177.251, P=0.000) increased the risk of mortality or LT. All groups showed similar biochemical responses (P=0.134), virologic responses (HBV DNA116copies/mL, P=0.151), and HBeAg seroconversion (P=0.560). Antiviral resistance emerged in five patients treated with LAM by 48 weeks (17.9%, P=0.003).LAM, ETV, and TDF selection is not related with mortality and LT in patients with severe acute CHB exacerbation and hepatic decompensation. To reduce mortality, patients with ascites and MELD scores above 25 should be considered for LT.

Long interspersed nuclear element-1 (LINE-1) is a retrotransposon that contains a CpG island in its 5'-untranslated region. The CpG island of LINE-1 is often heavily methylated in normal somatic cells, which is associated with poor prognosis in various cancers. DNA methylation can differ between formalin-fixed paraffin-embedded (FFPE) and frozen tissues. Therefore, this study aimed to compare the LINE-1 methylation status between the two tissue-storage conditions in gastric cancer (GC) clinical samples and to evaluate whether LINE-1 can be used as an independent prognostic marker for each tissue-storage type. We analyzed four CpG sites of LINE-1 and examined the methylation levels at these sites in 25 FFPE and 41 frozen GC tissues by quantitative bisulfite pyrosequencing. The LINE-1 methylation status was significantly different between the FFPE and frozen GC tissues (p < 0.001). We further analyzed the clinicopathological features in the two groups separately. In the frozen GC tissues, LINE-1 was significantly hypomethylated in GC tissues compared to their corresponding normal gastric mucosa tissues (p < 0.001), and its methylation status was associated with gender, differentiation state, and lymphatic and venous invasion of GC. In the FFPE GC tissues, the methylation levels of LINE-1 differed according to tumor location and venous invasion of GC. In conclusion, LINE-1 can be used as a useful methylation marker for venous invasion in both FFPE and frozen tumor tissues of GC.

To evaluate the ability of epithelial-to-mesenchymal transition-related microRNAs (miRNAs) as serum biomarkers for prognosis and prediction of metastasis in patients with colorectal cancer (CRC).Epithelial-to-mesenchymal transition-related miRNAs drive CRC progression and metastasis. However, their potential as serum biomarkers in CRC has not been studied.This was a 3-phase study using 446 colorectal specimens. In the first phase, we selected candidate miRNAs associated with metastasis by analyzing the expression of 4 miR-200 family members (miR-200b, -200c, -141, and -429) in serum samples from 12 patients with stage I and IV CRC. The second phase involved independent validation of candidate miRNAs in serum from 182 patients with CRC and 24 controls. Finally, we analyzed expression in matched 156 tumor tissues from 182 patients with CRC and an independent set of 20 matched primary CRC and corresponding liver metastases to identify the source of circulating miRNAs.After initial screening, miR-200c was selected as the candidate serum miRNA best associated with metastasis. Validation analysis revealed that serum miR-200c levels were significantly higher in stage IV than in stage I-III CRCs. High serum miR-200c demonstrated a significant positive correlation with lymph node metastasis, distant metastasis, and prognosis (P = 0.0026, P = 0.0023, and P = 0.0064, respectively). More importantly, serum miR-200c was an independent predictor for lymph node metastasis (odds ratio: 4.81, 95% confidence interval: 1.98-11.7, P = 0.0005) and tumor recurrence (hazard ratio: 4.51, 95% confidence interval: 1.56-13.01, P = 0.005) and emerged as an independent prognostic marker for CRC (hazard ratio: 2.67, 95% confidence interval: 1.28-5.67, P = 0.01).Serum miR-200c has strong potential to serve as a noninvasive biomarker for CRC prognosis and predicting metastasis.

e15161 Background: Preoperative chemoradiotherapy (CRT) followed by total mesorectal excision has become the standard treatment for locally advanced rectal cancer (LARC). However, the effect of CRT varies from complete response to complete resistance, and predicting response to CRT have not been well characterized yet. Previous studies have shown the potential of micro-RNA (miRNA) based approaches to enhance tumor radiation response. Accordingly, the present study attempted to identify biomarkers to predict response for preoperative CRT using comprehensive miRNA analysis in patients with LARC. Methods: This study included 65 rectal cancer tissues and 90 serum samples from patients who diagnosed with LACR and received preoperative CRT at Kyungpook National University Chilgok Hospital. Tissue specimens and serum samples were collected before CRT to evaluate the biologic differences between the good CRT response group and the poor CRT response group. For discovery of specific miRNAs, 800 miRNAs were analyzed using NanoString in 30 rectal cancer tissues. Thereafter, a total of 65 tissues, and 90 serum samples were investigated using real-time PCR for validation. Results: The median age was 59 years (range, 30-82), and the ratio of male to female was 3.05 to 1. The pathologic stages after preoperative CRT were as follows: pathologic complete response (n=13, 14.4%), pathologic stage I (n=13, 14.4%), pathologic stage II (n=27, 30.0%), pathologic stage III (n=28, 31.1%), and pathologic stage IV (n=9, 10.0%). In the discovery set, 16 target miRNAs were detected. In the validation set with tissue specimens, expression of 3 miRNAs (miR-199a/b-3p ( p=0.032), miR-199a-5p ( p=0.023), miR-199b-5p ( p=0.005)) was significantly upregulated which was associated with better response of CRT. Moreover, among the 3 candidate miRNAs, miR-199b-5p level was significantly upregulated in serum, and it was also found to be related with better response of CRT in LARC (pathologic stage 0/I versus II/III/IV, p=0.027). Conclusions: In the present study, high level of exosomal miR-199b-5p was associated with better response, suggesting it to be a promising non-invasive biomarker to predict response of CRT in patients with LARC. Accordingly, specific miRNAs can be predictive biomarker or therapeutic target to overcome radiotherapy resistance in LARC with a functional study.

Epigenetic alterations, such as histone modification, DNA methylation, and miRNA-mediated processes, are critically associated with various mechanisms of proliferation and metastasis in several types of cancer. To overcome the side effects and limited effectiveness of drugs for cancer treatment, there is a continuous need for the identification of more effective drug targets and the execution of mechanism of action (MOA) studies. Recently, epigenetic modifiers have been recognized as important therapeutic targets for hepatocellular carcinoma (HCC) based on their reported abilities to suppress HCC metastasis and proliferation in both in vivo and in vitro studies. Therefore, here, we introduce epigenetic modifiers and alterations related to HCC metastasis and proliferation, and their molecular mechanisms in HCC metastasis. The existing data suggest that the study of epigenetic modifiers is important for the development of specific inhibitors and diagnostic targets for HCC treatment.

Objective Hypomethylation of LINE-1 elements has emerged as a distinguishing feature in human cancers. Limited evidence indicates that some LINE-1 elements encode an additional internal antisense promoter, and increased hypomethylation of this region may lead to inadvertent activation of evolutionarily methylation-silenced downstream genes. However, the significance of this fundamental epigenetic mechanism in colorectal cancer (CRC) has not been investigated previously. Design We analysed tissue specimens from 77 CRC patients with matched sets of normal colonic mucosa, primary CRC tissues (PC), and liver metastasis tissues (LM). LINE-1 methylation levels were determined by quantitative bisulfite pyrosequencing. MET, RAB3IP and CHRM3 protein expression was determined by western blotting and IHC. MET proto-oncogene transcription and 5-hydroxymethylcytosine (5-hmc) were evaluated by quantitative real-time-PCR. Results Global LINE-1 methylation levels in LM were significantly lower compared with the matched PC (PC=66.2% vs LM=63.8%; p

Liver stiffness (LS) value determined using transient elastography (TE) can be used to assess the degree of liver fibrosis. The study investigated whether TE can predict the recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA).This study retrospectively enrolled 228 patients with HCC who received TE and RFA as the first-line treatment for HCC between 2008 and 2015. Cox regression analysis was used to identify independent predictors of HCC recurrence.The median age of the study population (170 men and 58 women) was 61 years. During the study period, HCC recurrence and mortality developed in 125 (54.8%) and 37 (16.2%) patients after RFA, respectively. Liver cirrhosis, platelet count, multiple tumors, and LS value were the independent predictors of HCC recurrence. When the study population was stratified into early ( 12 months) and late (≥ 12 months) recurrence groups, LS value was an independent predictor of late recurrence, along with liver cirrhosis and spleen diameter. The risk of late recurrence was higher in patients with LS values of ≥ 13 kPa than in those with LS values of 13 kPa (adjusted hazard ratio [HR] = 4.507, 95% confidence interval [CI] 2.131-7.724, P 0.001). Recurrence was the only predictor of overall survival (HR = 18.583, 95% CI 2.424-142.486, P = 0.005).Findings of this study suggest that LS measurement using TE can be a useful predictor of HCC recurrence after RFA.