Kentaro Kojima

MicroRNA122 is a key regulator of α-fetoprotein expression and influences the aggressiveness of hepatocellular carcinoma

α-fetoprotein (AFP) is not only a widely used biomarker in hepatocellular carcinoma (HCC) surveillance, but is also clinically recognized as linked with aggressive tumour behaviour. Here we show that deregulation of microRNA122, a liver-specific microRNA, is a cause of both AFP elevation and a more biologically aggressive phenotype in HCC. We identify CUX1, a direct target of microRNA122, as a common central mediator of these two effects. Using liver tissues from transgenic mice in which microRNA122 is functionally silenced, an orthotopic xenograft tumour model, and human clinical samples, we further demonstrate that a microRNA122/CUX1/microRNA214/ZBTB20 pathway regulates AFP expression. We also show that the microRNA122/CUX1/RhoA pathway regulates the aggressive characteristics of tumours. We conclude that microRNA122 and associated signalling proteins may represent viable therapeutic targets, and that serum AFP levels in HCC patients may be a surrogate marker for deregulated intracellular microRNA122 signalling pathways in HCC tissues.

Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Although novel drugs against HCV are under development, the current standard therapy consists principally of interferon (IFN). To improve the response to IFN treatment by enhancing interferon-stimulated response element (ISRE)-mediated gene transcription, we screened 75 microRNAs highly expressed in hepatocytes for their ability to modulate ISRE activity. Overexpression of microRNA-122 (miR122) significantly suppressed ISRE activity. Conversely, silencing of miR122 function enhanced IFN-induced ISRE activity, by decreasing expression of suppressor of cytokine signaling 3 (SOCS3). This decrease in SOCS3 level was not mediated by microRNA target gene suppression, but rather by enhanced methylation at SOCS3 gene promoter. Taken together, our data, along with the fact that antisense oligonucleotides of miR122 also directly inhibit HCV replication, suggest that a combination therapy comprising IFN and silencing of miR122 function may be a promising therapeutic option in the near future.

MicroRNA-22 and microRNA-140 suppress NF-κB activity by regulating the expression of NF-κB coactivators.

Nuclear factor κB (NF-κB) is a transcription factor that regulates a set of genes that are critical to many biological phenomena, including liver tumorigenesis. To identify microRNAs (miRNAs) that regulate NF-κB activity in the liver, we screened 60 miRNAs expressed in hepatocytes for their ability to modulate NF-κB activity. We found that miRNA-22 and miRNA-140-3p significantly suppressed NF-κB activity by regulating the expression of nuclear receptor coactivator 1 (NCOA1) and nuclear receptor-interacting protein 1 (NRIP1), both of which are NF-κB coactivators. Our results provide new information about the roles of miRNAs in the regulation of NF-κB activity.

The flavonoid apigenin improves glucose tolerance through inhibition of microRNA maturation in miRNA103 transgenic mice

Polyphenols are representative bioactive substances with diverse biological effects. Here, we show that apigenin, a flavonoid, has suppressive effects on microRNA (miRNA) function. The effects were mediated by impaired maturation of a subset of miRNAs, probably through inhibition of the phosphorylation of TRBP, a component of miRNA-generating complexes via impaired mitogen-activated protein kinase (MAPK) Erk activation. While glucose intolerance was observed in miRNA103 (miR103)-overexpressing transgenic mice, administration of apigenin improved this pathogenic status likely through suppression of matured miR103 expression levels. These results suggest that apigenin may have favorable effects on the pathogenic status induced by overexpression of miRNA103, whose maturation is mediated by phosphorylated TRBP.

Receptor for Activated Protein Kinase C: Requirement for Efficient MicroRNA Function and Reduced Expression in Hepatocellular Carcinoma

MicroRNAs (miRNAs) are important regulators of gene expression that control physiological and pathological processes. A global reduction in miRNA abundance and function is a general trait of human cancers, playing a causal role in the transformed phenotype. Here, we sought to newly identify genes involved in the regulation of miRNA function by performing a genetic screen using reporter constructs that measure miRNA function and retrovirus-based random gene disruption. Of the six genes identified, RACK1, which encodes “receptor for activated protein kinase C” (RACK1), was confirmed to be necessary for full miRNA function. RACK1 binds to KH-type splicing regulatory protein (KSRP), a member of the Dicer complex, and is required for the recruitment of mature miRNAs to the RNA-induced silencing complex (RISC). In addition, RACK1 expression was frequently found to be reduced in hepatocellular carcinoma. These findings suggest the involvement of RACK1 in miRNA function and indicate that reduced miRNA function, due to decreased expression of RACK1, may have pathologically relevant roles in liver cancers.

Efficacy of tolvaptan in patients with refractory ascites in a clinical setting

AIM To elucidate the efficacies of tolvaptan (TLV) as a treatment for refractory ascites compared with conventional treatment. METHODS We retrospectively enrolled 120 refractory ascites patients between January 1, 2009 and September 31, 2014. Sixty patients were treated with oral TLV at a starting dose of 3.75 mg/d in addition to sodium restriction (> 7 g/d), albumin infusion (10-20 g/wk), and standard diuretic therapy (20-60 mg/d furosemide and 25-50 mg/d spironolactone) and 60 patients with large volume paracentesis in addition to sodium restriction (less than 7 g/d), albumin infusion (10-20 g/wk), and standard diuretic therapy (20-120 mg/d furosemide and 25-150 mg/d spironolactone). Patient demographics and laboratory data, including liver function, were not matched due to the small number of patients. Continuous variables were analyzed by unpaired t-test or paired t-test. Fishers exact test was applied in cases comparing two nominal variables. We analyzed factors affecting clinical outcomes using receiver operating characteristic curves and multivariate regression analysis. We also used multivariate Coxs proportional hazard regression analysis to elucidate the risk factors that contributed to the increased incidence of ascites. RESULTS TLV was effective in 38 (63.3%) patients. The best cut-off values for urine output and reduced urine osmolality as measures of refractory ascites improvement were > 1800 mL within the first 24 h and > 30%, respectively. Multivariate regression analysis indicated that > 25% reduced urine osmolality [odds ratio (OR) = 20.7; P < 0.01] and positive hepatitis C viral antibodies (OR = 5.93; P = 0.05) were positively correlated with an improvement of refractory ascites, while the total bilirubin level per 1.0 mg/dL (OR = 0.57; P = 0.02) was negatively correlated with improvement. In comparing the TLV group and controls, only the serum sodium level was significantly lower in the TLV group (133 mEq/L vs 136 mEq/L; P = 0.02). However, there were no significant differences in the other parameters between the two groups. The cumulative incidence rate was significantly higher in the control group with a median incidence time of 30 d in the TLV group and 20 d in the control group (P = 0.01). Cox hazard proportional multivariate analysis indicated that the use of TLV (OR = 0.58; P < 0.01), uncontrolled liver neoplasms (OR = 1.92; P < 0.01), total bilirubin level per 1.0 mg/dL (OR = 1.10; P < 0.01), and higher sodium level per 1.0 mEq/L (OR = 0.94; P < 0.01) were independent factors that contributed to incidence. CONCLUSION Administration of TLV results in better control of refractory ascites and reduced the incidence of additional invasive procedures or hospitalization compared with conventional ascites treatments.

Development of a new extracorporeal whole-liver perfusion system

We have developed a new extracorporeal whole-liver accommodation device in which a whole swine liver is placed in a physiological state by modeling the intraabdominal arrangement in the pig body, with the liver supported by a special inferior vena cava tube. Furthermore, we employed a diaphragm-type artificial heart in our system to produce pulsatile blood flow through the hepatic artery, which is considered to be indispensable to dilate peripheral vessels and supply oxygenated whole blood to the peripheral liver tissue. Beneficial effects were demonstrated in visual findings and bile juice secretion. The color of the liver surface in our system remained bright red, indicating that the liver vessels were well drained and free from congestion, and bile juice secretion was maintained at more than 10 ml/h throughout the perfusion period. Our system exhibited excellent ammonia removal and urea nitrogen synthesis, and serum aspartate aminotransferase levels showed no increase, indicating the absence of hepatocyte destruction. Histological findings showed that the liver could expand appropriately and was free from compression caused by its own weight. In conclusion, our original liver accommodation device enabled appropriate expansion of the whole liver and supplied adequate oxygenated blood to peripheral areas by means of a pulsatile pump.

Xenogeneic direct hemoperfusion using whole swine liver for liver failure in dogs

BACKGROUND We developed a new method of xenogeneic direct hemoperfusion of a bioartificial liver support system consisting of a leukocyte-adsorbent column, an immunoglobulin-adsorbent column, and the substitute unit for hepatic function. By this method, we performed xenogeneic direct hemoperfusion experiment using resected whole swine liver for treatment of a canine liver failure model, and compared the contribution of each adsorbent column both by blood analysis and from the histological point of view. MATERIALS AND METHODS Canine liver failure model was produced by portocaval shunting and ligating the entire hepatoduodenal ligament. The xenogeneic direct hemoperfusion system was constructed using a roller pump, a leukocyte-adsorbent column, an immunoglobulin-adsorbent column, a combined device of oxygenator and warmer, the resected whole swine liver accommodated in a chamber, and a dissolved oxygen meter through which canine whole blood leaving the external jugular vein circulated in this order. RESULTS Xenogeneic direct hemoperfusion was successfully performed for 3 h without hyperacute rejection occurring. Adequate ammonia detoxification and bile juice secretion were exhibited, and no findings of hepatocyte destruction by immunological cells and proteins were detected. Blood data showed that the immunoglobulin adsorbent were more effective than the leukocyte adsorbent in avoiding hyperacute rejection. This result indicates that hyperacute rejection has a closer relation to humoral immune responses, especially regarding removal of complements than to cellular immune responses. CONCLUSIONS We successfully performed xenogeneic direct hemoperfusion of the whole swine liver without hyperacute rejection using our method.

Sa1097 Analysis of Weekend Admission on Upper Gastrointestinal Bleeding; Is Weekend Effect Demonstrated in Japan?

Background and Aims: Whether weekend admission influences the mortality of upper gastrointestinal bleeding(UGIB) remains unclear in Japan. In Europe and North-America, some previous studies identified worse outcomes of weekend admission, however the opposite results were reported from Hong-Kong, Korea and Taiwan. The outcomes may vary considerably between each nation. This study was conducted to assess the weekend effect in patients with UGIB on the basis of a 10-year single-center experience in Japan. Methods and Patients:595 hospitalized patients, who were diagnosed with UGIB between January 2004 and March 2014, were enrolled. We divided those patients into two groups, variceal and nonvariceal UGIB. Bivariate analyses and multivariate logistic regression models were used to evaluate predicting variables of the in-hospital mortality, included patient characteristics, waiting time for endoscopy, and weekend admission. Results: 595 patients consisted of 484 nonvariceal and 111 variceal UGIB. 169 out of 484 Patients with nonvariceal UGIB were weekend admission and had similar mortality rates compared to the weekday group (2.96% vs 2.54%;odds ratio[OR],1.17;confidence interval[CI],0.38-3.63; P=0.79). The waiting time for endoscopy was also not different (9.97 hours versus 9.19 hours, P=0.55). There was no statistical difference in baseline characteristics. In the multivariate logistic regression model, mortality was correlated with Blatchford score ([OR],1.32 per point;[CI],1.05-1.72;P= 0.015) and waiting time to endoscopy ([OR],1.04 per hour;[CI],1.009-1.07; P=0.014). 34 out of 111 Patients with variceal UGIB were weekend admission and had relatively higher mortality rates compared to the weekday group (17.7% vs 10.4%;[OR],1.85;[CI],0.595.81;P=0.29), but not significant. The waiting time for endoscopy was not different (3.35 hours versus 5.3 hours, P =0.09). There was no statistical difference in baseline characteristics, except Child-Pugh scores. Child-Pugh scores of patients with variceal UGIB on weekend admission were significantly higher than those on weekday admission (9.29 vs 8.3; P=0.01). In the multivariate logistic regression model, mortality was correlated with Child-Pugh scores ([OR],1.83 per point;[CI],1.16-3.22; P=0.008). Conclusion:In Japan, quality of care did not appear to differ between week/weekend admission of patients with UGIB and weekend effect was not demonstrated in this study. Patient characteristics and waiting time for endoscopy were correlated with the risk of the in-hospital mortality.

Abstract 3978: DDX20, a suppressor of hepatocarcinogenesis, controls NF-κB activity through regulating the function of miRNA-22 and miRNA-140-3p targeting transcriptional coactivators

MiRNA expression profile of human cancers has been characterized by an overall miRNA downregulation. While the causes include a transcriptional silencing and a failure of miRNA maturation, another possibility is that the deregulation of the machinery components for executing miRNA function also may cause similar consequences. Then, we screened the protein expression levels of the established members of the miRNA-containing ribonucleoproteins (Dicer, Ago2, TRBP2, DDX20 also known as Gemin3, and Gemin4) in various organ cancers by immunohisotchemistry. We detected reduced expression of DDX20 only in hepatocellular carcinoma tissues, while the levels of other genes were not drastically changed. We show that DDX20 suppresses NF-κB activity through regulating a subset of miRNA function. A microRNA library screen revealed that miR-140-3p and miR-22 suppress NF-κB activation by targeting the expression of NRIP1 and NCOA1, both are coactivators of NF-κB. However, in DDX20 deficient cells, this suppressive effect was lost, leading to NF-κB activation, a key regulator of hepatocarcinogenesis. In addition, DDX20 knockdown indeed induced cell transformation, revealed by a sphere-forming assay. These results indicate that DDX20 deficiency enhances NF-κB activity by impairing the NF-κB-suppressive action of microRNAs, and suggest that deregulation of the microRNA machinery may be one of the mechanisms of oncogenesis in liver cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2011-3978