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Background and aim Direct-acting antiviral (DAA) therapies have been proven to be highly effective for the eradication of hepatitis C virus (HCV) without resistance-associated substitutions (RASs). However, even in cases with no detected RASs, treatment sometimes fails, suggestive of the existence of some host-related factors involved in HCV eradication by DAAs. To explore such factors, we analyzed the serum microRNAs (miRNAs) of patients who received DAA treatment. Methods The serum miRNA expression levels of 39 patients with chronic HCV infection without any detectable RASs, who achieved sustained virological response with asunaprevir/daclatasvir or grazoprevir/elbasvir therapy, were investigated cyclopedically, using oligonucleotide microarrays. The effects of specific miRNAs on the replication of HCV were measured in the HCV genomic replicon containing Huh-7 hepatoma cells. Results Along with the disappearance of HCV, the expression quantiles of 16 miRNAs in the asunaprevir/daclatasvir group and 18 miRNAs in the grazoprevir/elbasvir group showed a tendency to increase or decrease. Among these molecules, adjustments for multiple testing yielded a significant differential expression at a false discovery rate of less than 5% for only one molecule, hsa-miR-762. Its expression quantile increased after HCV exclusion in all patients who had achieved sustained virological response. Quantitative polymerase chain reaction analysis validated a significant increase in the serum hsa-miR-762 after disappearance of HCV. On the contrary, hsa-miR-762 was decreased in the relapse and breakthrough of HCV in DAA failures. Transfection of hsa-miR-762 into cultured HCV-infected hepatocytes significantly decreased HCV-RNA replication. Conclusion These data suggest that hsa-miR-762 is one of the host factors participating in HCV exclusion by DAA therapy.

AIM The aim of this study was to determine whether oral L-carnitine administration reduces the blood ammonia concentration and number of hospital admissions for hepatic encephalopathy in patients with advanced cirrhosis. METHODS Of 68 patients with hepatic encephalopathy treated with oral L-carnitine supplementation from April 2013 to March 2016, we enrolled 19 patients who had received full standard treatment. We analyzed blood ammonia concentration, number of hospital admissions, and prognosis to determine how effective L-carnitine was in achieving mid-term to long-term suppression of recurrent hepatic encephalopathy. RESULTS Median blood ammonia concentrations at the start, 1 week, 12 weeks, 24 weeks, and 48 weeks were 159, 79, 75, and 82 μg/dL, respectively. Blood ammonia concentrations 12 week, 24 weeks, and 48 weeks after L-carnitine administration were significantly lower than those at the start (P

Simple Summary In this study, the rate of hepatocellular carcinoma (HCC) recurrence (HCC-R) after hepatitis C virus eradication by direct-acting antiviral (DAA) treatment was relatively high, corresponding to 63.8% (83/130) of the studied cases. The patients who electively received palliative treatment before DAA treatment and showed a shorter interval period between the last treatment for HCC and DAA initiation, also displayed a significant increase in alpha-fetoprotein during follow-up after therapy, relative to cases without HCC-R. Overall, the survival was comparable in both groups because of the improvement in liver function tests after the DAA therapy, and thus no differences in survival rates were observed between patients with or without HCC-R. The results of this study indicate that interferon (IFN)-free DAA treatment after treatment for HCC could be recommended to improve prognosis in this subset of cases. However, it remains imperative to observe the timing, i.e., at least 9–12 months after the last treatment for HCC, before proposing DAA therapy. Abstract There are limited studies that have evaluated the long-term outcomes in patients with hepatocellular carcinoma (HCC) recurrence after direct-acting antiviral (DAA) treatment. In this retrospective study, we aimed to investigate the recurrence rates, recurrence factors, and prognosis of 130 patients who were treated with IFN-free DAA treatment after treatment for HCC. The median observation time was 41 ± 13.9 months after DAA treatment. The recurrence rates of HCC were 23.2%, 32.5%, 46.3%, and 59.4% at 6, 12, 24, and 36 months, respectively. A multivariate analysis showed that palliative treatment prior to DAA treatment (HR = 3.974, 95% CI 1.924–8.207, p = 0.0006) and alpha-fetoprotein at sustained virological response 12 (HR = 1.048, 95% CI 1.016–1.077, p = 0.0046) were associated with independent factors for HCC recurrence (HCC-R). The 12-, 24-, and 36-month overall survival rates were 97.6%, 94.0%, and 89.8%, respectively. The 12-, 24-, and 36-month survival rates of the non-recurrence and recurrence groups were 97.7%, 97.7%, and 94.1% and 97.6%, 92.3%, and 87.9%, respectively (p = 0.3404). The size of the main tumor lesion and the serological data were significantly improved at the time of HCC-R after DAA treatment. This study showed an improved prognosis regardless of recurrence rate, which suggests that DAA treatment in HCV patients should be considered.

We treated a 51-year-old Japanese man with chronic hepatitis B (viral load 7.6 LC/mL, genotype C). Hepatitis B virus DNA and HBe antigen were undetectable during the administration of the nucleic acid analogs (NUCs) lamivudine and adefovir, although the concentration of HBs antigen (HBsAg) was 851.2 IU/mL. The HBsAg levels were reduced 150-fold when pegylated-interferon (Peg-IFN) α-2a was administered weekly for 48 weeks and did not increase during the rest period. Therefore, Peg-IFNα-2a was administered twice each week. During this time, HBsAg reached undetectable concentrations, and HBs antibody was detected and continued to be detectable during the three-year follow-up. These unprecedented findings suggest that IFN may contribute to the seroclearance of HBsAg in patients treated with NUCs.

The granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates cell proliferation and differentiation of precursor cells in the bone marrow. Several cases of G-CSF-producing malignant tumors in various organs have been reported, but there are only nine cases of G-CSF-producing hepatocellular carcinoma (HCC) reported in the English literature. G-CSF-producing tumors grow rapidly and have a high probability of distant metastases; thus, they generally have a poor prognosis. Given that the mechanism of the carcinogenesis of G-CSF-producing HCC remains unclear, an efficient treatment strategy also remains to be elucidated. We report herein a case of G-CSF-producing HCC accompanied by leukocytosis and high serum G-CSF concentrations in the disease progression stage after long-term complete response. We also reviewed previous reports to investigate the clinical behaviors of G-CSF-producing HCC, including our case. Clinicians should consider G-CSF-producing HCC in patients with a hepatic mass and drastic leukocytosis, without any evidence of infection and blood disorders. Early diagnosis and prompt therapy, including radical resection, may provide a more favorable prognosis.

Hepatitis C virus (HCV) infection causes sustained inflammation and fibrosis. Several oral direct-acting antivirals (DAAs) including ombitasvir/paritaprevir/ritonavir (OBV/PTV/r) were recently developed for HCV elimination. The combination of DAAs brought a higher sustained viral response (SVR) rate to anti-HCV therapy compared to interferon (IFN)-based regimens. However, 5% of hepatitis C patients who undergo DAA therapy still suffer from a sustained HCV infection. MicroRNA (miRNA) is essentially interfering, endogenous noncoding RNA that has been investigated as a new biomarker for the response to DAA in hepatitis C patients. Here we used a miRNA array and real-time polymerase chain reaction (PCR) to determine the targetable miRNA before and 12 weeks after OBV/PTV/r treatment for refractory hepatitis C. We used replicon cells, in which genotype 1b type HCV is stably transfected in Huh7 cells, to determine whether miRNA can inhibit HCV replication. Among 2,555 miRNAs, three were significantly up-regulated and eight miRNAs were down-regulated in serum 12 weeks after OBV/PTV/r treatment. An unsupervised hierarchical clustering analysis, using Pearson's correlation, showed that the miRNA profiles between before and 12 weeks after OBV/PTV/r treatment were clustered separately. At 12 weeks after OBV/PTV, miR-636 was targeted among the eight down-regulated miRNAs, and the expression level of circulating miR-636 was significantly diminished. The amount of HCV-RNA was significantly diminished 48 hours after miR-636 inhibitor transfection in HCV replicon cells. In conclusion, miR-636 might be one of the essential targetable molecules in HCV patients who undergo DAA therapy and still suffer from a sustained HCV infection.

Background and Aim Chronic liver diseases (CLDs) progress from chronic inflammation to fibrosis to tumorigenesis. Galectin-9, a β-galactoside-specific animal lectin, is indicated to contribute to all three steps of progression. The aim of this study was to determine which of the three steps was most dominant in elevating the serum galectin-9 concentration and to test the possibility of galectin-9 as a serum biomarker. Methods Japanese patients with chronic hepatitis (CH), liver cirrhosis (LC), hepatocellular carcinoma (HCC), nonalcoholic fatty liver disease or alcoholic liver disease who provided informed consent were enrolled in this study. Serum galectin-9 levels were measured using a sandwich ELISA. Multiple regression analyses were performed using EZR to identify factors which determined serum galectin-9 concentration. Results One hundred one patients with 50 of CH and 51 of LC were enrolled; the cohort included 45 cases of HCV infection, 13 cases of HBV infection, 46 cases with HCC-related complications. The median serum galectin-9 concentration was 77.54pg/ml (inter quartile range: 18.89-241.9pg/ml). Multiple linear regression analyses proved Fib-4 index and APRI, indexes of liver fibrosis, were able to predict the serum galectin-9 levels with statistical significance. A multiple logistic regression analysis determined 10pg/ml increase of the serum galectin-9 concentration presented an odds ratio of 3.90 for liver fibrosis progression. Conclusions The serum galectin-9 concentration represents a potential biomarker of liver fibrosis in patients with CLDs, regardless of chronic inflammation or the presence of HCC complications. Furthermore, higher serum galectin-9 levels are a predictor for liver fibrosis progression.

The role of free testosterone, that not bound to sex hormone-binding globulin, in male patients with HCV infection remains uncertain. We investigated whether free testosterone is involved in the progression to hepatic fibrosis/steatosis or insulin resistance in male patients with HCV-related chronic liver disease or not. Free androgen indices, which reflect circulating free testosterone levels, were calculated as 100 × total testosterone levels/sex hormone-binding globulin levels in 30 male patients with HCV-related chronic liver disease. Degrees of hepatic fibrosis and steatosis were evaluated by the New Inuyama Classification and the classification proposed by Brunt and colleagues, respectively. Insulin resistance was estimated by HOMA-IR values. Serum total testosterone levels were independent of hepatic fibrosis staging in the enrolled patients. However, circulating sex hormone-binding globulin levels were significantly increased in proportion to the severity of hepatic fibrosis. Therefore, free androgen indices were inversely correlated with the severity of hepatic fibrosis. Moreover, free androgen indices were inversely correlated with the grades of hepatic steatosis and HOMA-IR values in those patients. Our data suggest that lower circulating free testosterone levels may be recognized as the risk factor for more advanced hepatic fibrosis, steatosis and/or higher insulin resistance in male patients with HCV-related chronic liver disease.

Liver metastasis from gastrointestinal cancer defines a patient's prognosis. Despite medical developments, pancreatic cancer with liver metastasis confers a very poor prognosis. Galectin-9 (Gal‑9) is a tandem-repeat-type galectin that has recently been demonstrated to exert antitumor effects on various types of cancer cells by inducing apoptosis. However, the apoptotic pathway of Gal‑9 in solid tumors is unclear. The aim of the present study was to evaluate the effects of Gal‑9 on human liver metastasis from pancreatic cancer. Gal‑9 suppressed cell proliferation in metastatic liver cancer cell lines derived from pancreatic cancer (KMP2, KMP7, and KMP8) and increased the levels of caspase-cleaved keratin 18 and fluorescein isothiocyanate (FITC)-conjugated Annexin V. Furthermore, expression of apoptosis-related molecules such as caspase-7, cleaved caspase-3, cleaved PARP, cytochrome c, Smac/Diablo and HtrA2/Omi was enhanced. However, Gal‑9 did not affect expression of various cell cycle-related proteins. The microRNA (miRNA) expression profile was markedly altered by Gal‑9, and various miRNAs might contribute to tumor growth suppression. Our data reveal that Gal‑9 suppresses the growth of liver metastasis, possibly by inducing apoptosis through a mechanism involving mitochondria and changes in miRNA expression. Thus, Gal‑9 might serve as a therapeutic agent for the treatment of liver metastasis from pancreatic cancer.

A 72-year-old female was referred for further evaluation of epigastralgia. Abdominal contrast computed tomography revealed numerous tumors in the two lobes of the liver. Liver biopsy and immunohistochemical staining revealed that the tumor cells were positive for factor VIII-associated antigen, platelet endothelial cell adhesion molecule 1 and human hematopoietic progenitor cell antigen, concordant with a diagnosis of hepatic epithelioid hemangioendothelioma (HEH). To elucidate the etiology of HEH, particularly the microRNA (miRNA) profiles, tissue samples obtained from normal and tumor tissues were analyzed using a miRNA array system. A total of 14 miRNAs were significantly upregulated and 93 miRNAs were downregulated in the tumor tissues (P

The prognosis of hepatocellular carcinoma (HCC) patients exhibiting macroscopic vascular invasion (MVI) is poor, and the most appropriate treatment approach remains unclear. The current study aimed to investigate the efficacy and safety of sorafenib treatment following chemoradiotherapy for advanced HCC exhibiting MVI. A newly reported regimen, including 5-fluorouracil and cisplatin therapy (NewFP), plus three-dimensional conformal radiotherapy (3D-CRT) for MVI was used as the initial treatment. Additionally, sorafenib, as a secondary treatment, was administered after NewFP plus 3D-CRT for MVI. The present retrospective study enrolled patients with unresectable advanced HCC that was treated with NewFP plus 3D-CRT for MVI between January 2009 and December 2017. In total, 32 HCC patients with MVI were registered. Of these 32 patients, 18 were treated with NewFP plus 3D-CRT for MVI (NewFP + 3D-CRT group) and 14 were treated with sorafenib following NewFP plus 3D-CRT for MVI (sorafenib after NewFP + 3D-CRT group). The study endpoints were overall survival, overall response rate and disease control rate. Clinical factors influencing overall survival were identified using univariate and multivariate analyses. The median survival time in the NewFP + 3D-CRT group and sorafenib following NewFP + 3D-CRT group was 6.7 and 49.2 months, respectively (P=0.0003). For patients with advanced HCC exhibiting MVI, the initial treatment with NewFP plus 3D-CRT for MVI was well tolerated. The administration of sorafenib as the secondary treatment following NewFP plus 3D-CRT for MVI was associated with a significantly higher overall response rate, disease control rate and increased overall survival as compared with the NewFP plus 3D-CRT treatment.

Direct acting antivirals (DAA) have recently been developed to treat patients with hepatitis C virus (HCV) infection, and interferon-free DAA treatment has improved the cure rate of patients. However, the occurrence rate of hepatocellular carcinoma (HCC) following HCV eradication remains unknown. Therefore, the present study aimed to identify predictors of HCC occurrence following DAA treatment. Among 1,454 patients infected with HCV, 1,088 patients who achieved sustained virologic response and who had no history of HCC treatment were recruited between September 2014 and November 2018. The incidence of HCC in patients infected with HCV following DAA treatment, and the predictors contributing to HCC occurrence were identified using clinicopathological characteristics and blood test results. During the present study, 26 patients developed HCC. The incidence of HCC was 0.61, 1.88, 2.82 and 3.71% at 6, 12, 18 and 24 months after treatment with DAA, respectively. The results of multivariate analysis identified age [hazard ratio (HR), 1.0729; P=0.0044] and α-fetoprotein (AFP) level after DAA treatment (HR, 1.0486; P=0.0486) as independent factors that may contribute to HCC occurrence following DAA treatment. By using these two factors, a novel scoring system (0-2 points) was established to predict HCC occurrence following HCV eradication by DAA treatment. The incidence of HCC at 2 years was 0.3% in the 0 points group, 6.27% in the 1 point group and 18.37% in the 2 points group. In conclusion, AFP level after DAA treatment and age at DAA administration were identified as independent predictors of HCC occurrence in patients that were treated with DAA. The scoring system that was established in the present study is simple and easy, and using pre-treatment factors may be a convenient tool to predict the risk of HCC occurrence in HCV-free patients following DAA treatment.

During diagnosis of early stage hepatocellular carcinoma (HCC), single or small lesions are difficult to identify using screening ultrasonography, and conventional tumor markers are frequently negative. MicroRNAs (miRNAs) are small non-coding RNAs that suppress the translation of target mRNAs and exert significance as biomarkers. The aim of the present study was to use samples of patients with HCC and those with other liver diseases caused by hepatitis C virus (HCV) infection to investigate the expression profile of serum miRNAs, and identify a miRNA that can serve as a HCC biomarker. Initially, changes in 2,555 miRNAs between pre- and post-curative treatment serum from 12 patients with early stage HCC were examined using microarray analysis. The serum levels of miR-125a-5p in 40 individuals with HCV-associated chronic hepatitis (CH), liver cirrhosis (LC) or HCC were measured using reverse transcription-quantitative polymerase chain reaction, and 5 miRNAs, including miR-125a-5p, miR-423-5p, miR-1247, miR-1304 and miR-3648, were identified to be downregulated following curative treatment in patients with HCC. Among these, miR-125a-5p was identified to be similarly decreased following treatment in all patients. Additionally, the expression levels of miR-125a-5p were significantly upregulated in patients with HCC in the early and advanced stages of disease, compared with patients with CH or LC (P

We investigated the correlation between serum complement component 3 (C3) levels and metabolic and histological abnormalities in patients with chronic hepatitis C (CH-C).Obesity and insulin resistance were estimated by calculating body mass index (BMI) and the values of the homeostasis model for assessment of insulin resistance (HOMA-IR), respectively. Severity of hepatic steatosis and fibrosis were evaluated by New Inuyama Classification and the classification proposed by Brunt and colleagues, respectively. The degree of hepatic C3 expression was examined, using an immunohistochemical procedure.Serum C3 levels were significantly correlated with BMI, HOMA-IR value, and serum triglyceride levels in CH-C patients. Histological analysis revealed that serum C3 levels were significantly elevated in proportion to the severity of hepatic steatosis in such patients. The serum C3 level tended to increase as the severity of hepatic fibrosis progressed. However, the degree of C3 expression in hepatocytes was not associated with serum C3 level among those patients.These results suggest that the elevation of serum C3 levels may reflect obesity, insulin resistance, and/or hepatic steatosis in patients with CH-C, and that the increase in the synthesis of C3 may derive from the activation of cells other than hepatocytes in those patients.

Visceral adipose tissue contributes to the pathophysiology of metabolic syndrome. Metformin has been reported to suppress lipogenesis in a murine preadipocyte cell line. However, the effect of metformin on the differentiation of human visceral adipose tissue remains unknown. MicroRNAs (miRNAs or miRs) have been suggested as therapeutic targets because of their involvement in the differentiation and maturation of fatty cells. The aim of this study was to determine whether metformin suppresses the differentiation of human preadipocytes and to identify miRNAs associated with the regulation of lipid metabolism. Human visceral preadipocytes (HPrAD-vis) were preincubated in growth media and then cultured with differentiation media containing metformin for 1 or 2 weeks. Adipogenic differentiation of the cells was assessed by Oil Red O staining, and soluble adiponectin in the culture media was measured using an enzyme-linked immunosorbent assay. Cell proliferation was assessed using a WST-8 assay, and the gene and protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT‑enhancer-binding protein α (C/EBPα) was determined by RT-qPCR and western blot analysis, respectively. miRNAs were profiled using human miRNA Oligo chips after total RNA was extracted and labeled. Oil Red O staining showed that metformin suppressed the accumulation of lipid droplets in HPrAD-vis cells. The adiponectin concentration in the culture media was also decreased in metformin-treated cells. The WST-8 assay revealed no effect on proliferation or growth inhibition following metformin treatment, although metformin suppressed the expression of PPARγ and C/EBPα. miRNA profiling further revealed differences between the metformin-treated group and control HPrAD-vis cells. Thus, the findings of the present study demonstrated that metformin suppressed the differentiation of human preadipocytes in vitro and altered the miRNA profile of these cells. Thus, the miRNAs whose expression levels were altered by metformin may contribute to the observed suppression of HPrAD-vis cell differentiation.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortality worldwide. Although surgery is considered the most effective treatment for patients with HCC, its indication is restricted by limited criteria and a high relapse rate following surgery; therefore, systemic chemotherapy is required for patients with advanced-stage HCC to prolong their survival. MicroRNAs (miRNAs) are endogenous non-coding RNAs of 18-22 nucleotides in length. It has been reported that aberrant expression of miRNAs is a feature shared by various types of human cancer. Previous studies have indicated that the modulation of non-coding RNAs, particularly miRNAs, may be a valuable therapeutic target for HCC. The aim of the present study was to elucidate the miRNA profiles associated with differentiation and hepatitis B virus (HBV) infection observed in HCC cell lines. The human Alex, Hep3B, HepG2, HuH1, HuH7, JHH1, JHH2, JHH5, JHH6, HLE, HLF and Li-7 HCC cell lines were used for an miRNA array. Replicate data were analyzed following their classification into: i) Poorly- and well-differentiated human HCC cells and ii) HBV-positive and -negative human HCC cells. Out of the 1,719 miRNAs, 4 were found to be significantly upregulated and 52 significantly downregulated in the poorly-differentiated cells, as compared with the well-differentiated cells. Conversely, in the HBV-positive cells 125 miRNAs were found to be upregulated and 2 downregulated, as compared with the HBV-negative cells. Unsupervised hierarchical clustering analysis with Pearson's correlation revealed that the miRNA expression levels were clustered both together and separately in each group. In conclusion, miRNA profile characterization based on various parameters may be a novel approach to determine the etiology of HCC.

Gastric cancer is the second-leading cause of cancer-related mortality worldwide, and the prognosis of advanced gastric cancer remains poor. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been demonstrated to exert anti-proliferative effects on various types of cancer cells. The aim of our present study was to evaluate the effects of Gal-9 on human gastric cancer cells and the expression levels of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. In our initial experiments, Gal-9 suppressed the proliferation of gastric cancer cell lines in vitro. Our data further revealed that Gal-9 increased caspase-cleaved keratin 18 (CCK18) levels in gastric cancer cells. Additionally, Gal-9 reduced the phosphorylation of vascular endothelial growth factor receptor-3 (VEGFR-3) and insulin-like growth factor-1 receptor (IGF-1R). Furthermore, miRNA expression levels were markedly altered with Gal-9 treatment in vitro. In conclusion, Gal-9 suppressed the proliferation of human gastric cancer cells by inducing apoptosis. These findings suggest that Gal-9 could be a potential therapeutic target in the treatment of gastric cancer.

Tissue sampling of primary duodenal lymphoma is essential for its histological diagnosis. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), which is frequently used for submucosal tumor (SMT)-like duodenal tumors, is adequate for cytological diagnosis, but not for histological diagnosis. Therefore, in the present study, a mucosal incision-assisted biopsy (MIAB) was performed in an 81-year-old woman for the diagnosis of an SMT-like duodenal mass, as tissue sampling for histological analysis using a regular endoscopic biopsy had failed to establish a definite diagnosis of malignant lymphoma. EUS-FNA had also led to poor tissue sampling due to the difficult location of the duodenal tumor. The pathological examination of biopsy samples using MIAB revealed the presence of a diffuse proliferation of atypical lymphocytes, and the expression of cluster of differentiation (CD)20 and CD79a, but no expression of CD3 in the tumor specimens. The patient was diagnosed with diffuse large B-cell lymphoma. To the best of knowledge, this is first report of a case using MIAB as a sampling method for the histological diagnosis of SMT-like primary duodenal lymphoma. This case suggests that MIAB may be an essential method for obtaining tissue samples from SMT-like duodenal tumors.

Galectin-9, a soluble β-galactoside-binding animal lectin, evokes apoptosis in various human cancer cell lines. The galectin-9 antitumor effect against hepatocellular carcinoma (HCC) is, however, unknown. We investigated whether galectin-9 suppresses HCC growth in vitro and in vivo. We assessed the antitumor effect of galectin-9 on HCC cells by conducting WST-8 assay in vitro and xenograft model analysis in vivo. Galectin-9-induced apoptosis was evaluated by FACS and ELISA in vitro and by TUNEL stain in vivo. Cell cycle alteration was profiled by FACS. Caspases were profiled by colorimetry. MicroRNAs related to the galectin-9 antitumor effects were determined using microarrays, and their antitumor effect was confirmed in a transfection study in vitro. The expression levels of the target proteins of the miRNAs extracted above were analyzed by western blot analysis. To summarize the results, galectin-9 inhibited the growth of the HCC cell lines HLE and Li-7 in vitro and Li-7 in vivo inducing apoptosis. Cell cycle turnover was not arrested in HLE and Li-7 cells in vitro. miR-1246 was similarly extracted both in vitro and in vivo, which sensitized Li-7 cells to apoptosis when transfected into the cells. DYRK1A, a target protein of miR-1246 was downregulated in Li-7 cells. Caspase-9 was upregulated in Li-7 cells in vitro and in vivo. In conclusion, galectin-9 inhibited the growth of HCC cells by apoptosis, but not cell cycle arrest, in vitro and in vivo. miR-1246 mediated signals of galectin-9, possibly through miR-1246-DYRK1A-caspase-9 axis. Galectin-9 might be a candidate agent for HCC chemotherapy.

We herein present a case of an 87-year-old patient with multiple liver tumors identified on abdominal ultrasound. The assessment performed on admission included physical examination, computed tomography (CT) during hepatic angiography and CT during arterial portography. The examination revealed contrast enhancement of a proportion of the liver tumors (20 mm maximum diameter) during the arterial phase and mild contrast washout of those tumors during the delayed phase. On contrast-enhanced magnetic resonance imaging using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid, certain liver tumors exhibited contrast enhancement during the early phase and contrast washout during the hepatocyte phase in both lobes. By contrast, no lesions were identified during positron emission tomography imaging of the liver. A liver biopsy was performed and immunohistochemical staining revealed enhanced expression of cytokeratin AE1/AE3, synaptophysin, chromogranin A and CD56 and no expression of hepatocyte antigen or CΚ7. The mindbomb E3 ubiquitin protein ligase-1 index was ~2% in most of the tumor. The liver tumors were finally diagnosed as multiple intrahepatic metastases from a primary hepatic neuroendocrine tumor (PHNET). The patient underwent transarterial chemoembolisation with a combination of miriplatin (84 mg) mixed with gelatin sponge particles and lipiodol. To the best of our knowledge, this is the first report of PHNET in an patient aged >85 years.

Chronic liver diseases progress from chronic inflammation to fibrosis to tumorigenesis. Galectin-9, a β-galactoside-specific animal lectin, is indicated to contribute to all three steps of progression. The aim of this study was to determine which of the three steps was most dominant in elevating the serum galectin-9 concentration and to test the possibility of galectin-9 as a serum biomarker.Japanese patients with chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), non-alcoholic fatty liver disease, or alcoholic liver disease who provided informed consent were enrolled in this study. Serum galectin-9 levels were measured using a sandwich ELISA. Multiple regression analyses were performed using ezr to identify factors that determined serum galectin-9 concentration.One hundred one patients with 50 of chronic hepatitis and 51 of liver cirrhosis were enrolled; the cohort included 45 cases of hepatitis C virus infection, 13 cases of hepatitis B virus infection, and 46 cases with HCC-related complications. The median serum galectin-9 concentration was 77.54 pg/mL (interquartile range: 18.89-241.9 pg/mL). Multiple linear regression analyses proved Fibrosis-4 index and aspartate aminotransferase to platelet ratio index, indexes of liver fibrosis, were able to predict the serum galectin-9 levels with statistical significance. A multiple logistic regression analysis determined 10 pg/mL increase in the serum galectin-9 concentration presented an odds ratio of 3.90 for liver fibrosis progression.The serum galectin-9 concentration represents a potential biomarker of liver fibrosis in patients with chronic liver diseases, regardless of chronic inflammation or the presence of HCC complications. Furthermore, higher serum galectin-9 levels are a predictor for liver fibrosis progression.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third leading cause of cancer-related death. Telmisartan, a widely used antihypertensive drug, is an angiotensin II type 1 (AT1) receptor blocker (ARB) that might inhibit cancer cell proliferation, but the mechanisms through which telmisartan affects various cancers remain unknown. The aim of the present study was to evaluate the effects of telmisartan on human HCC and to assess the expression of microRNAs (miRNAs). We studied the effects of telmisartan on HCC cells using the HLF, HLE, HepG2, HuH-7 and PLC/PRF/5 cell lines. In our experiments, telmisartan inhibited the proliferation of HLF, HLE and HepG2 cells, which represent poorly differentiated types of HCC cells. However, HuH-7 and PLC/PRF/5 cells, which represent well-differentiated types of HCC cells, were not sensitive to telmisartan. Telmisartan induced G0/G1 cell cycle arrest of HLF cells by inhibiting the G0-to-G1 cell cycle transition. This blockade was accompanied by a marked decrease in the levels of cyclin D1, cyclin E and other cell cycle-related proteins. Notably, the activity of the AMP-activated protein kinase (AMPK) pathway was increased, and the mammalian target of rapamycin (mTOR) pathway was inhibited by telmisartan treatment. Additionally, telmisartan increased the level of caspase-cleaved cytokeratin 18 (cCK18), partially contributed to the induction of apoptosis in HLF cells and reduced the phosphorylation of ErbB3 in HLF cells. Furthermore, miRNA expression was markedly altered by telmisartan in vitro. In conclusion, telmisartan inhibits human HCC cell proliferation by inducing cell cycle arrest.

Recent studies suggest that small non‑coding microRNAs (miRNAs or miRs) play an important role in the regulation of genes involved in various cellular and developmental processes. However, the expression of miRNAs during the aging process remains largely unknown. The aim of the present study was to analyze miRNA expression profiles in rat livers during the aging process. The livers of male Wistar rats at different stages of development (fetal, aged 3 days, and 1, 2, 4, 8 and 36 weeks of age) were used. Total RNA was extracted from the livers. We analyzed the expression levels of 679 rat miRNA probes. In addition, immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was performed. Several up- and downregulated miRNAs were identified in the rat livers at 7 different fetal developmental stages and at 36 weeks of age. We observed the upregulation of miR‑29a, miR‑29c, miR‑195 and miR‑497, whereas miR‑301a, miR‑148b-3p, miR‑7a, miR‑93, miR‑106b, miR‑185, miR‑450a, miR‑539 and miR‑301b were downregulated in the aging rat livers. The number of PCNA-positive hepatocytes was decreased with age. In conclusion, our findings suggest that these up- and downregulated miRNAs play an important role in aging by regulating cell cycles that are involved in liver senescence. Further investigation is required to reveal additional target genes of the miRNAs expressed in the liver and the roles of miRNAs in the developmental process of aging in the liver.

Metformin is a commonly used oral anti-hyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle-related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.

Percutaneous ethanol injection therapy (PEIT) has been administered as a safe therapeutic modality for patients with small hepatocellular carcinoma (HCC). Due to the nature of the straight approaching line of a PEIT or radiofrequency ablation needle, penetrating the vessels that are interposed between the dermal insertion point and the nodule is unavoidable. A device with an overcoat needle and coaxial curved PEIT needle was created that facilitated a detour around interposing large vessels in order to avoid unnecessary harmful effects that result from the PEIT procedure. Two cases of HCC located adjacent to a neighboring large vessel were treated with a curved PEIT needle. The curved PEIT needle, which is connected to an outer needle, enabled deviation around the interposing vessels and successful connection with the HCC. Careful use of the curved line of the PEIT needle enabled the safe and successful performance of the PEIT without any requirement for specific training. This hand-assisted technique may be an applicable treatment for small HCC located beneath large vessels as a direct therapeutic method using ultrasound guidance.

Angiogenesis plays a pivotal role in the progression and metastasis of hepatocellular carcinoma (HCC). However, the expression of a wide range of angiogenic factors remains obscure in HCC. The purpose of the present study was to determine the expression of various angiogenic factors related to hepatocarcinogenesis. We examined the expression of 19 angiogenic factors using antibody arrays in human tissues of various liver diseases, including HCC. We also studied the expression of 19 angiogenic factors in the human HCC cell lines PLC/PRF/5, Hep 3B, HuH7, HLE, HLF and Li-7 and the normal hepatocyte cell line ACBRI3716. In human tissues, although the expression of acidic fibroblast growth factor (aFGF) was found to increase from normal liver to chronic hepatitis, its expression remained unchanged in the transition from chronic hepatitis to HCC. Vascular endothelial growth factor (VEGF) was elevated in liver cirrhosis, but the amounts remained unchanged in the transition from liver cirrhosis to HCC. In contrast, either interleukin-8 (IL-8) or basic fibroblast growth factor (bFGF) was upregulated in HCC. In the HCC cell lines PLC/PRF/5, Hep 3B and HuH-7, the expression of IL-8 was elevated. Although IL-8 was not elevated, bFGF was upregulated in the other HCC cell lines HLE, HLF and Li-7. Thus, either IL-8 or bFGF was upregulated in HCC cell lines and in HCC tissue samples. These data suggest that the upregulation of either IL-8 or bFGF is closely related to the transition from liver cirrhosis into HCC. Therefore, the analysis of the expression of these cytokines using protein arrays may identify novel therapies for individual patients with HCC.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide. Diabetes mellitus, a risk factor for cancer, is also globally endemic. The clinical link between these two diseases has been the subject of investigation for a century, and diabetes mellitus has been established as a risk factor for HCC. Accordingly, metformin, a first-line oral anti-diabetic, was first proposed as a candidate anti-cancer agent in 2005 in a cohort study in Scotland. Several subsequent large cohort studies and randomized controlled trials have not demonstrated significant efficacy for metformin in suppressing HCC incidence and mortality in diabetic patients; however, two recent randomized controlled trials have reported positive data for the tumor-preventive potential of metformin in non-diabetic subjects. The search for biological links between cancer and diabetes has revealed intracellular pathways that are shared by cancer and diabetes. The signal transduction mechanisms by which metformin suppresses carcinogenesis in cell lines or xenograft tissues and improves chemoresistance in cancer stem cells have also been elucidated. This review addresses the clinical and biological links between HCC and diabetes mellitus and the anti-cancer activity of metformin in clinical studies and basic experiments.

Percutaneous radiofrequency ablation (RFA) enables cauterization of liver cancer in a limited number of sessions without major complications. In contrast to the efficacy of this technique, the size of coagulation necrosis is limited due to increased impedance. D-sorbitol has been used as an irrigating fluid during transurethral resection of the prostate, since it is considered to be a dielectric fluid. In order to determine whether D-sorbitol enhances the effect of RFA, RFA was performed by slowly injecting 3% D-sorbitol near the tip of the RFA needle. The maximum of the total injected volume of D-sorbitol was 20 ml and RFA was terminated if the threshold of impedance was exceeded. RFA and D-sorbitol RFA were performed in 5 different parts of pig livers and dog livers in vivo. The total volumes of coagulation necrosis in the D-sorbitol RFA group were significantly higher compared with those in the RFA group. The total delivered energy in the D-sorbitol RFA group was also higher compared with that in the RFA group, due to the suppression of impedance elevation. No significant complications, such as bleeding or damage, were observed during the D-sorbitol RFA procedure in the in vivo model. In conclusion, RFA combined with D-sorbitol increases the total volume of coagulation necrosis through controlling impedance in the ablated liver and, therefore, D-sorbitol may be useful for the treatment of liver cancers.

MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that control the target gene translation by RNA interference; miRNAs are associated with cellular processes, including proliferation, differentiation, apoptosis, and cell survival. Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology. One third of patients with PBC demonstrate suboptimal responses, which result in worse outcomes. It has been previously reported that miRNAs are involved in drug resistance, however, the association between miRNA expression levels and refractory PBC remains to be fully elucidated. In the present study, among the 20 patients with PBC treated with ursodeoxycholic acid or bezafibrate, 15 patients were classed as treatment‑effective, and 5 were classed as being treatment‑resistant. Using the miRNA array technique, miRNA profiles were identified for each group. A total of 35 miRNAs were significantly upregulated, and 23 were significantly downregulated in the treatment‑resistant group compared with the treatment‑effective group. In order to examine the association between the highly altered miRNAs and clinical features of the two groups, numerous parameters were analyzed. Elevated levels of direct bilirubin, aspartate transaminase (AST), and alanine transaminase (ALT) were identified to be associated with miRNA‑122 upregulation. AST, ALT, and γ guanosine triphosphate were additionally associated with miRNA‑378f upregulation. However, the reduction of miRNA‑4311 was associated with reduced levels of AST and ALT. miRNA‑4714‑3p was also negatively correlated with total bilirubin and lactate dehydrogenase. Therefore, identifying the miRNA profile was demonstrated to be a useful approach in the characterization of PBC development. It is suggested that highly altered miRNAs may be potential biomarkers for use in the development of treatment of patients with refractory PBC.

Although gemcitabine (2',2'-difluorocytidine monohydrochloride) is a common anticancer agent of cholangiocellular carcinoma (CCC), its growth inhibitory effects and gemcitabine resistance in CCC cells are poorly understood. Our aims were to uncover the mechanism underlying the antitumor effect of gemcitabine and to analyze the mechanism regulating in vitro CCC cell gemcitabine resistance. In addition, we sought to identify miRNAs associated with the antitumor effects of gemcitabine in CCCs. Using a cell proliferation assay and flow cytometry, we examined the ability of gemcitabine to inhibit cell proliferation in three types of human CCC cell lines (HuCCT-1, Huh28, TKKK). We also employed western blotting to investigate the effects of gemcitabine on cell cycle-related molecules in CCC cells. In addition, we used array chips to assess gemcitabine-mediated changes in angiogenic molecules and activated tyrosine kinase receptors in CCC cells. We used miRNA array chips to comprehensively analyze gemcitabine-induced miRNAs and examined clusters of differentially expressed miRNAs in cells with and without gemcitabine treatment. Gemcitabine inhibited cell proliferation in a dose- and time-dependent manner in HuCCT-1 cells, whereas cell proliferation was unchanged in Huh28 and TKKK cells. Gemcitabine inhibited cell cycle progression in HuCCT-1 cells from G0/G1 to S phase, resulting in G1 cell cycle arrest due to the reduction of cyclin D1 expression. In addition, gemcitabine upregulated the angiogenic molecules IL-6, IL-8, ENA-78 and MCP-1. In TKKK cells, by contrast, gemcitabine did not arrest the cell cycle or modify angiogenic molecules. Furthermore, in gemcitabine-sensitive HuCCT-1 cells, gemcitabine markedly altered miRNA expression. The miRNAs and angiogenic molecules altered by gemcitabine contribute to the inhibition of tumor growth in vitro.

Cisplatin [cis-diamminedichloroplatinum (II)], is a platinum coordination compound that is commonly used to treat hepatocellular carcinoma (HCC). It is also one of the most compelling anticancer drugs. Recent studies suggest that cisplatin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of cisplatin in several types of cancers, including HCC, has not been elucidated. The goal of the present study was to evaluate the effects of cisplatin on the proliferation of HCC cells in vitro and to determine which microRNAs (miRNAs) are associated with the anticancer effects of cisplatin in vitro. We used various human HCC-derived cell lines to study the effects of cisplatin on human HCC cells. Cisplatin led to a strong dose- and time- dependent inhibition of cell proliferation in HLE, HLF, HuH7, Li-7, Hep3B and HepG2 cells in vitro. Cisplatin also blocked the progression of the cell cycle in the G0/G1 phase, which inhibited cyclin D1 and induced apoptosis. In addition, miRNA expression was markedly altered by treatment with cisplatin in vitro. Therefore, various miRNAs induced by cisplatin may also contribute to the suppression of cellular proliferation and apoptosis. Our results demonstrate that cisplatin inhibits the growth of HCC, possibly through the induction of G1 cell cycle arrest and apoptosis through the alteration of microRNA expression.

Since the links consisting of road network have high interdependency, a single decision maker should generally manage road network However, some organizational requirements often force to choose decentralized management scheme. In such a situation, a management scheme should be designed to realize efficient management of the network.In this paper, decentralized managers'maintenance decisions are modeled based on game theory.Maintenance strategies at an equilibrium of the game model are compared with optimum maintenance strategies taken by a single decision maker, and the relationship between network decomposition and efficiency is analyzed.

Non-alcoholic steatohepatitis (NASH) is one of the most common causes of chronic liver disease and is considered to be a causative factor of cryptogenic cirrhosis and hepatocellular carcinoma. microRNAs (miRNAs) are small non-coding RNAs that negatively regulate messenger RNA (mRNA). Recently, it was demonstrated that the aberrant expression of certain miRNAs plays a pivotal role in liver disease. The aim of the present study was to evaluate changes in miRNA profiles associated with metformin treatment in a NASH model. Eight-week-old male mice were fed a methionine- and choline-deficient (MCD) diet alone or with 0.08% metformin for 15 weeks. Metformin significantly downregulated the level of plasma transaminases and attenuated hepatic steatosis and liver fibrosis. The expression of miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p was enhanced among the 71 upregulated miRNAs, and the expression of miRNA-122, miRNA-194, miRNA-101b and miRNA-705 was decreased among 60 downregulated miRNAs in the liver of MCD-fed mice when compared with control mice. Of note, miRNA profiles were altered following treatment with metformin in MCD-fed mice. miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p were down-regulated, but miRNA-122, miRNA-194, miRNA-101b and miRNA-705 were significantly upregulated in MCD-fed mice treated with metformin. miRNA profiles were altered in MCD-fed mice and metformin attenuated this effect on miRNA expression. Therefore, miRNA profiles are a potential tool that may be utilized to clarify the mechanism behind the metformin-induced improvement of hepatic steatosis and liver fibrosis. Furthermore, identification of targetable miRNAs may be used as a novel therapy in human NASH.

Metformin is a commonly used oral anti-hyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle‑related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.

Molecular-targeted therapy is one of the most promising therapies for patients with advanced-stage colorectal cancer (CRC). However, a wide range of proteins have unknown expression levels in CRC. The purpose of the present study was to determine the expression levels of various proteins related to colorectal carcinogenesis and cancer development. We examined the expression levels of 507 target proteins using a biotin label-based antibody array in 6 human CRC tissues. We also analyzed the clinicopathological features of CRC patients. In CRC tissues, IL-1α, GRO, Glut5, MIG, ICAM-5, VE-cadherin, uPA and Leptin R were increased when compared to levels in normal colon tissues. MPIF-1/CCL23, FGF R5, MIP2, SAA and IL-18 Rβ were strongly upregulated in rectal cancer when compared to the levels in non-rectal cancer. These data suggest that differential protein expression profiles exist under different conditions, including carcinogenesis and CRC localization. Therefore, an exhaustive analysis of protein expression levels using a biotin label-based antibody protein array is a potentially useful tool for identifying novel individual therapies for CRC patients.