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Higashihiroshima, Japan

Ogawa T.,Osaka City University | Ogawa T.,Kinki University | Enomoto M.,Osaka City University | Fujii H.,Osaka City University | And 5 more authors.

Background: MicroRNAs (miRNAs) are important in hepatic pathophysiology and the development of liver cancer. Objective: To explore miRNAs that are regulated with the progression of liver fibrosis caused by chronic liver disease. Design: The regulated miRNAs in human livers infected with hepatitis C virus were identified by microarray analysis. Their expression in human livers with non-alcoholic steatohepatitis, mouse livers from two fibrosis models and cultured stellate cells was validated by realtime RT-PCR. The regulation of miR-222 expression in stellate cells by nuclear factor kappa B (NF-κB) was assayed. Finally, the effects of an miR-222 precursor or inhibitor on the expression of cyclin-dependent kinase inhibitor 1B (CDKN1B) and the growth of LX-2 cells were determined. Results: It was found that miR-199a-5p/199a-3p and miR-221/222 were upregulated in the human liver in a fibrosis progression-dependent manner. Among these miRNAs, miR-221/222 were upregulated in LX-2 cells and increased during the course of culture-dependent activation of mouse primary stellate cells, in a manner similar to the expression of α 1(I) collagen and α-smooth muscle actin mRNAs. The expression of miR-221/222 increased in mouse models of liver fibrosis. In contrast, an NF-κB inhibitor significantly suppressed the miR-222 induction that was stimulated in culture by transforming growth factor α or tumour necrosis factor α. Although overexpression or downregulation of miR-222 failed to regulate the growth of LX-2 cells, miR-222 bound to the CDKN1B 3′UTR and regulated the expression of the corresponding protein. Conclusion: miR-221/222 may be new markers for stellate cell activation and liver fibrosis progression. Source

Ogawa T.,Osaka City University | Fujii H.,Osaka City University | Yoshizato K.,Osaka City University | Yoshizato K.,PhoenixBio Co. | Kawada N.,Osaka City University
American Journal of Pathology

Nonalcoholic steatohepatitis (NASH) progresses to liver fibrosis and cirrhosis, which can lead to life-threatening liver failure and the development of hepatocellular carcinoma. The aim of the present study was to create a rabbit model of NASH with advanced fibrosis (almost cirrhosis) by feeding the animals a diet supplemented with 0.75% cholesterol and 12% corn oil. After 9 months of feeding with this diet, the rabbits showed high total cholesterol levels in serum and liver tissues in the absence of insulin resistance. The livers became whitish and nodular. In addition, the number of rabbit macrophage antigen-positive cells and the expression of mRNAs for inflammatory cytokines showed a significant increase. Moreover, fibrotic septa composed of collagens and α-smooth muscle actin-positive cells were found between the central and portal veins, indicating alteration of the parenchymal architecture. There was also a marked increase of mRNAs for transforming growth factor-β1 and collagen 1A1. Comprehensive analysis of protein and gene expression revealed an imbalance of the antioxidant system and methionine metabolism. We also found that ezetimibe attenuated steatohepatitis in this model. In conclusion, the present rabbit model of NASH features advanced fibrosis that is close to cirrhosis and may be useful for analyzing the molecular mechanisms of human NASH. Ezetimibe blunted the development of NASH in this model, suggesting its potential clinical usefulness for human steatohepatitis. Copyright © American Society for Investigative Pathology. Source

Sekiya Y.,Osaka City University | Ogawa T.,Osaka City University | Yoshizato K.,Osaka City University | Yoshizato K.,PhoenixBio Co. | And 2 more authors.
Biochemical and Biophysical Research Communications

MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has been previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3′UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of α-SMA, DDR2, FN1, ITGB1, and PDGFR-β, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis. © 2011 Elsevier Inc. Source

Phoenixbio Co. and Tottori University | Date: 2012-10-12

A chimeric non-human animal having an in vivo human hepatocyte population, wherein the effects of non-human animal cells on drug metabolism are suppressed or deleted is provided. A method for producing a chimeric non-human animal that lacks a drug-metabolizing system or has a suppressed drug-metabolizing system and is provided with a drug-metabolizing system driven by human hepatocytes, is provided. The method comprises transplanting human hepatocytes into a non-human animal characterized by (i) being immunodeficient, (ii) having liver damage, and (iii) lacking the functions of an endogenous Cyp3a gene.

Sato S.,Hokkaido University | Li K.,Hokkaido University | Kameyama T.,Hokkaido University | Hayashi T.,Hokkaido University | And 15 more authors.

Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but nottype I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes. © 2015 Elsevier Inc. Source

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