MicroRNA profile of hepatic epithelioid hemangioendothelioma: A case report.

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<0.01). Additionally, unsupervised hierarchical clustering analysis using Pearson's correlation revealed that the tumor tissues clustered separately from the normal tissues. The miRNA expression profile was analyzed in HEH and compared with angiosarcoma, which exhibits histology similar to HEH. Out of a total of 107 miRNAs, only miR-122-5p and miR-1290 demonstrated a differential expression pattern in angiosarcoma. Therefore, these miRNAs may be novel biological markers for the determination of a diagnosis of HEH in primary mesenchymal tumors of the liver. To the best of our knowledge, this study is the first report of a miRNA microarray analysis in HEH.

MicroRNA profiles in various hepatocellular carcinoma cell lines.

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.

Metformin-suppressed differentiation of human visceral preadipocytes: Involvement of microRNAs.

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 CCAAT­enhancer-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.

Identification of microRNA profiles associated with refractory primary biliary cirrhosis.

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 treatment­effective, and 5 were classed as being treatment­resistant. 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 treatment­resistant group compared with the treatment­effective 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 miRNA­122 upregulation. AST, ALT, and γ guanosine triphosphate were additionally associated with miRNA­378f upregulation. However, the reduction of miRNA­4311 was associated with reduced levels of AST and ALT. miRNA­4714­3p 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.

Diabetes mellitus and metformin in hepatocellular carcinoma.

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.

Primary hepatic neuroendocrine tumor: A case report.

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.

Diagnostic Dilemma in the Detection of Antibodies to Filamentous Actin.

BACKGROUND: Antibodies to filamentous-actin (anti-F-actin) were identified as the serological hallmark of type 1 autoimmune hepatitis (AIH). However, the standardized assay for the autoantibody has not yet been established. The purposes of this study were to verify the sensitivity and specificity for anti-F-actin by different assays and to investigate the benefits of anti-F-actin. METHODS: Twenty-two patients with type 1 AIH, 8 patients with autoimmune hepatitis/primary biliary cirrhosis overlap syndrome (AIH/PBC), and 18 patients with HCV related chronic liver disease (CLD-C) were enrolled in this study. Smooth muscle antibodies (SMAs) were assessed by an indirect immunofluorescent (HF) assay. Anti-F-actin was determined by an IIF method and an enzyme-linked immunosorbent assay (ELISA) method. Seropositivity for anti-F-actin was defined as positive in both methods. RESULTS: SMAs were present in the sera of 11 of 22 patients with AIH, 2 of 8 patients with AIH/PBC, and 3 of 18 patients with CLD-C. Ten AIH patients, 2 AIH/PBC patients, and no CLD-C patients were seropositive for anti-F-actin by the IIF method, while 13 AIH patients, 3 AIH/PBC patients, and no CLD-C patients were positive by the ELISA method. The prevalence of anti-F-actin in AIH, AIH/PBC, and CLD-C were estimated as 10 of 22 (48%), 1 of 8 (13%), and 0 of 26 (0%), respectively. Three AIH patients who showed a discrepancy in the anti-F-actin status were seropositive in ELISA, but seronegative in IIF. However, none of the AIH patients were seronegative in ELISA, but seropositive in IIF. The sera of 10 of 11 AIH patients with SMAs reacted with F-actin, while no sera of AIH patients without SMAs were directed against F-actin at all. AIH patients with anti-F-actin tended to have higher relapse rates during tapering of corticosteroid treatment. However, the declines in titers of anti-F-actin did not reflect the attenuation of the disease activity by the treatment with corticosteroids. CONCLUSIONS: SMAs in the sera of AIH patients predominantly recognized filamentous actin, while SMAs in the sera of CLD-C patients did not. Anti-F-actin by IIF was superior in the specificity to those by ELISA. AIH patients seropositive for anti-F-actin may predict the relapse.

Roles of Copper in Hepatocarcinogenesis via the Activation of Hypoxia-Inducible Factor-1α.

Hypoxia-inducible factor-1α (HIF-1α) is involved in the pathogenesis of hepatocellular carcinoma (HCC). However, the roles of trace elements in the activation of HIF-1α during hepatocarcinogenesis have been unclear. We investigated whether copper (Cu) and zinc (Zn) participated in the activation of HIF-1α in the process of hepatocarcinogenesis or not. Nine patients with chronic hepatitis (CH), five with liver cirrhosis (LC), 12 with HCC, and nine normal healthy controls were enrolled in this study. Their serum HIF-1α, Cu, and Zn levels were determined in the enrolled patients. Hepatic HIF-1α expression was evaluated, using an immunohistochemical procedure. The HCC patients had significantly higher serum HIF-1α levels than the CH patients (6.47 ± 1.57 vs. 5.09 ± 1.22 ng/ml, p = 0.0344). The serum Cu level in the HCC patients was also significantly higher than those in the CH and LC patients (137 ± 24 vs. 107 ± 15 µg/dl, 114 ± 24 µg/dl). Interestingly, a positive correlation was observed between serum HIF-1α and Cu levels in the enrolled patients (r = 0.425, p = 0.0137). In contrast, no significant differences in serum Zn levels were present between the HCC patients and the CH or LC patients. The serum HIF-1α was not positively correlated with the serum Zn level in the enrolled patients, either. Immunohistochemical analysis revealed that two of the five HCC patients had HIF-1α expression in the tumor tissues, whereas none of CH and LC had hepatic HIF-1α expression in the liver tissues. These data suggest that the activation of HIF-1α derived from a Cu accumulation in the liver may cause hepatocarcinogenesis.

Evaluation of in vivo efficacy of radiofrequency ablation with D-sorbitol in animal liver.

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.

Successful mucosal incision-assisted biopsy for the histological diagnosis of duodenal lymphoma: A case report.

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 suppresses the proliferation of gastric cancer cells in vitro.

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.

Galectin-9: An anticancer molecule for gallbladder carcinoma.

Gallbladder cancer (GBC) is the most common and aggressive type of biliary tract cancer. There are various histological types of GBC, and the vast majority of GBC cases are adenocarcinomas. Squamous and adenosquamous carcinomas are rare GBC subtypes that are traditionally considered to be more aggressive and to be associated with a poorer prognosis than adenocarcinoma. Galectin-9 (Gal-9), a tandem-repeat-type galectin, has been reported to induce apoptosis-mediated elimination of various cancers, including hepatocellular carcinoma, cholangiocarcinoma, and hematologic malignancies. Therefore, we investigated the antitumor effects of Gal-9 on GBC in vitro and in vivo. In our in vitro experiments, Gal-9 suppressed cell proliferation in various GBC cell lines but not in the OCUG-1 cell line, which represents a poorly differentiated type of adenosquamous carcinoma. Gal-9 induced the apoptosis of Gal-9-sensitive GBC cells by increasing the levels of caspase-cleaved keratin 18 and phosphorylated p53. However, Gal-9 did not affect the expression of various cell cycle-related proteins. In addition, Gal-9 suppressed tumor growth by implanted human GBC cells in a xenograft model. Furthermore, Gal-9 induced the phosphorylation of the Ephrin type-B receptor, and the microRNA (miRNA) expression profile was markedly altered by Gal-9. Based on these results, various miRNAs might contribute to the suppression of tumor growth. Our data reveal that Gal-9 suppresses the growth of GBC, possibly by inducing apoptosis and altering miRNA expression. Thus, Gal-9 might serve as a therapeutic agent for the treatment of GBC.

Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth.

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.

Laparoscopic splenectomy for patients with liver cirrhosis: Improvement of liver function in patients with Child-Pugh class B.

BACKGROUND: We aimed to assess the short-term outcomes of laparoscopic splenectomy (LS) and liver function at 1 year after splenectomy in the patients with liver cirrhosis. METHODS: Forty-five patients with liver cirrhosis and hypersplenism underwent LS. We reviewed electronic medical records regarding the liver functional reserve, the etiology of liver cirrhosis, and the presence of hepatocellular carcinoma and esophago-gastric varices. Prospectively collected data of perioperative variables, postoperative complications, and long-term liver function were analyzed. RESULTS: Forty-five patients had a chronic liver disease classified into Child-Pugh classes (A/B/C: 23/20/2). The etiologies of disease were hepatitis C virus infection in 34 patients, hepatitis B virus infection in 4, and others in 7. Fourteen patients underwent procedures in addition to LS, including hepatectomy (n = 7) and devascularization for esophagogastric varices (n = 8). Postoperative complications occurred in 11 patients (24%). Neither postoperative liver failure nor in-hospital mortality occurred. White blood cell and platelet counts determined 7 days, 1 month, and 1 year after LS doubled or increased more than twice compared with the preoperative values (P < .001). One year after LS, patients who had been classified preoperatively into Child-Pugh class B had decreased total serum bilirubin levels (P = .03), and increased prothrombin activity (P = 003) and decreased Child-Pugh scores (P = .001). The Child-Pugh classifications improved in 14 of 18 patients (78%) who had Child-Pugh class B preoperatively. CONCLUSION: LS is a safe and feasible procedure for hypersplenism in patients with liver cirrhosis. In addition, LS most likely ameliorates liver function at 1 year after LS in patients with Child-Pugh class B liver cirrhosis.

MicroRNA profiles in cisplatin-induced apoptosis of hepatocellular carcinoma cells.

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.

Galectin-9 suppresses the growth of hepatocellular carcinoma via apoptosis in vitro and in vivo.

Galectin-9, a soluble ß-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.

MicroRNA profiles following metformin treatment in a mouse model of non-alcoholic steatohepatitis.

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, miRNA­127, 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, miRNA­127, miRNA-34a, miRNA-300 and miRNA-342-3p were downregulated, but miRNA-122, miRNA-194, miRNA­101b 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.

Antidiabetic drug metformin inhibits esophageal adenocarcinoma cell proliferation in vitro and in vivo.

Esophageal carcinoma is the eighth most common cancer worldwide and the sixth leading cause of cancer-related deaths, with one of the worst prognoses of any form of cancer. Treatment with the anti-diabetic drug metformin has been associated with reduced cancer incidence in patients with type 2 diabetes. This study therefore evaluated the effects of metformin on the proliferation, in vitro and in vivo, of human esophageal adenocarcinoma cells, as well as the microRNAs associated with the antitumor effects of metformin. Metformin inhibited the proliferation of the esophageal adenocarcinoma cell lines OE19, OE33, SK-GT4 and OACM 5.1C, blocking the G0 to G1 transition in the cell cycle. This was accompanied by strong reductions in G1 cyclins, especially cyclin D1, cyclin-dependent kinase (Cdk)4, and Cdk6, and decreases in retinoblastoma protein phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor and insulin-like growth factor-1 receptor, as well as angiogenesis-related proteins, such as vascular endothelial growth factor, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2. Metformin also markedly altered microRNA expression. Treatment with metformin of athymic nude mice bearing xenograft tumors reduced tumor proliferation. These findings suggest that metformin may have clinical use in the treatment of esophageal adenocarcinoma.

Diversity of humoral responses to the centromere proteins among HCV-related chronic liver disease, PBC and AIH patients.

BACKGROUND: Anticentromere antibodies (ACAs) have been observed in patients with autoimmune hepatitis (AIH) and hepatitis C virus (HCV)-related chronic liver disease (CLD-C) as well as those with primary biliary cirrhosis (PBC). However, little is known about the differences in immune responses to the centromere proteins among these liver diseases. OBJECTIVE: By synthesizing recombinant proteins consisting of the N- and C-termini of major centromere proteins, we investigated the humoral responses against them in each disease. RESULTS: Eight of the 754 (1%) patients with CLD-C, 14 of the 57 (25%) patients with PBC and six of the 38 (16%) patients with AIH were seropositive for ACAs. There were no significant differences in ACA titers determined by an indirect immunofluorescent method among the groups of patients with CLD-C, PBC and AIH. However, the analysis of immunoreactivities against each recombinant protein revealed that the titers of IgG-subclass autoantibodies against the C-terminus of centromere protein (CENP)-B were significantly higher in the CLD-C patients than in the AIH patients. Likewise, the titers of IgM-subclass autoantibodies against the N-terminus of CENP-A were significantly higher in the PBC group than in the CLD-C group. The ACA-positive patients who developed liver cirrhosis had significantly higher titers of the IgA-subclass autoantibodies against the C-terminus of CENP-C than those who did not. CONCLUSION: These findings suggest that immunoreactivities against the fragments of centromere proteins show distinct patterns among CLD-C, PBC and AIH and that the determination of immunoreactivities against the centromere proteins may be useful for the prediction of disease progression.

Exacerbation of insulin resistance and hepatic steatosis deriving from zinc deficiency in patients with HCV-related chronic liver disease.

The role of zinc (Zn) in hepatic steatosis of patients with HCV-related chronic liver disease (CLD-C) remains uncertain, although persistent HCV infection often evokes hepatic steatosis. The primary purpose of this study was to elucidate the contribution of Zn deficiency to hepatic steatosis in patients with CLD-C. Fifty nondiabetic patients with CLD-C were enrolled. Hepatic 4-hydroxy-2-nonenal (4-HNE) expression was examined using an immunohistochemical procedure as a marker for lipid peroxidation. Serum ferritin levels were assessed for iron overload. Insulin resistance was evaluated using the values of the homeostasis model for assessment of insulin resistance (HOMA-IR). The severity of hepatic steatosis was graded on the classification system proposed by Brunt and colleagues. Serum Zn levels were inversely correlated with serum ferritin levels in the patients with CLD-C (r = -0.382, p = 0.0062). Serum ferritin levels were strongly associated with the HOMA-IR values (r = 0.476, p = 0.0005). Therefore, Zn deficiency resulted in insulin resistance through iron overload. Moreover, serum Zn levels were significantly decreased in proportion to the level of hepatic 4-HNE expression, which was enhanced as hepatic steatosis developed. Then, Zn deficiency eventually seemed to exacerbate hepatic steatosis by way of an increase in lipid peroxidation. However, the serum Zn levels were not associated with either loads of HCV-RNA or HCV genotypes. These data suggest that, in patients with CLD-C, Zn deficiency promotes insulin resistance by exacerbating iron overload in the liver and induces hepatic steatosis by facilitating lipid peroxidation.