Title Hepatic microRNA expression is associated with the responseto interferon treatment of chronic hepatitis C

Background: HCV infection frequently induces chronic liver diseases. The current standard treatment for chronic hepatitis (CH) C combines pegylated interferon (IFN) and ribavirin, and is less than ideal due to undesirable effects. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that control gene expression by degrading or suppressing the translation of target mRNAs. In this study we administered the standard combination treatment to CHC patients. We then examined their miRNA expression profiles in order to identify the miRNAs that were associated with each patient’s drug response. Methods: 99 CHC patients with no anti-viral therapy history were enrolled. The expression level of 470 mature miRNAs found their biopsy specimen, obtained prior to the combination therapy, were quantified using microarray analysis. The miRNA expression pattern was classified based on the final virological response to the combination therapy. Monte Carlo Cross Validation (MCCV) was used to validate the outcome of the prediction based on the miRNA expression profile. Results: We found that the expression level of 9 miRNAs were significantly different in the sustained virological response (SVR) and non-responder (NR) groups. MCCV revealed an accuracy, sensitivity, and specificity of 70.5%, 76.5% and 63.3% in SVR and non-SVR and 70.0%, 67.5%, and 73.7% in relapse (R) and NR, respectively. Conclusions: The hepatic miRNA expression pattern that exists in CHC patients before combination therapy is associated with their therapeutic outcome. This information can be utilized as a novel biomarker to predict drug response and can also be applied to developing novel anti-viral therapy for CHC patients. Background Hepatitis C virus (HCV) infection affects more than 3% of the world population. HCV infection frequently induces chronic liver diseases ranging from chronic hepatitis (CH) C, to liver cirrhosis (LC) and hepatocellular carcinoma (HCC) [1]. The current standard treatment for CHC combines pegylated interferon (Peg-IFN) and ribavirin, and has been found to be effective in only 50% of HCV genotype 1b infection. Furthermore this form of therapy is often accompanied by adverse effects; therefore, there is a pressing need to develop alternative strategies to treat CHC and to identify patients that will not be responsive to treatment [2]. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that control gene expression by degrading or suppressing the translation of target mRNAs [3,4]. There are currently 940 identifiable human miRNAs (The miRBase Sequence Database – Release 15.0). These miRNAs can recognize hundreds of target genes with incomplete complementary over one third of human genes appear to be conserved miRNA targets [5,6]. miRNA can associate not only several pathophysiologic events but also cell proliferation and differentiation. However, there are many miRNAs whose functions are still unclear. Examples include miR-122 which is an abundant liver-specific miRNA that is said to constitute * Correspondence: ymurakami@genome.med.kyoto-u.ac.jp Center for Genomic Medicine, Kyoto University Graduate School of Medicine, 53 Shogoinkawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan Full list of author information is available at the end of the article Murakami et al. BMC Medical Genomics 2010, 3:48 http://www.biomedcentral.com/1755-8794/3/48 © 2010 Murakami et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. up to 70% of all miRNA molecules in hepatocytes [7]. The expression level of miR-122 was reportedly associated with early response to IFN treatment, while others like miR-26 have expression status that is associated with HCC survival and response to adjuvant therapy with IFN [8,9]. IFN beta (IFNb) on the other hand, has been shown to rapidly modulate the expression of numerous cellular miRNAs, and it has been demonstrated that 8 IFNb-induced miRNAs have sequencepredicted targets within the hepatitis C virus (HCV) genomic RNA [10]. Finally several miRNAs have been recognized as having target sites in the HCV genome that inhibits viral replication [10-12]. To date, various parameters have been examined in an attempt to confirm the effects of the IFN-related treatment for CHC. In patients with chronic HCV genotype 1b infection, there is a substantial correlation between responses to IFN and mutation in the interferon sensitivity determining region (ISDR) of the viral genome [13]. Substitutions of amino acid in the HCV core region (aa 70 and aa 91) were identified as predictors of early HCV-RNA negativity and several virological responses, including sustained response to standard combination therapy [14]. In order to assess the drug response to combination therapy for CHC using gene expression signatures, several researchers cataloged the IFN related gene expression profile from liver tissue or peripheral blood mononuclear cells (PBMC) [15,16]. It was found that failed combination therapy was associated with up-regulation of a specific set of IFN-responsive genes in the liver before treatment [17]. Additional reports have indicated that two SNPs near the gene IL28B on chromosome 19 may also be associated with a patient’s lack of response to combination therapy [18]. These reports suggest that gene expression during the early phase of anti-HCV therapy may elucidate important molecular pathways for achieving virological response [19]. Our aim in this study was to identify gene related factors that contribute to poor treatment response to combination therapy for CHC. In order to achieve this we studied the miRNA expression profile of CHC patients before treatment with CHC combination therapy and tried to determine the miRNAs that were associated with their drug response. Knowing patients’ expression profile is expected to provide a clearer understanding of how aberrant expression of miRNAs can contribute to the development of chronic liver disease as well as aid in the development of more effective and safer therapeutic strategies for CHC. Methods Patients and sample preparation Ninety-nine CHC patients with HCV genotype 1b were enrolled (Table 1). Patients with autoimmune hepatitis, or alcohol-induced liver injury, or hepatitis B virus-associated antigen/antibody or anti-human immunodeficiency virus antibody were excluded. There were no patients who received IFN therapy or immunomodulatory therapy before enrollment in the study. Serum HCV RNA was quantified before IFN treatment using Amplicor-HCV Monitor Assay (Roche Molecular Diagnostics Co., Tokyo, Japan). Liver biopsy specimen was collected from each patient up to one week prior to administering combination therapy. Histological grading and staging of liver biopsy specimens from the CHC patients were performed according to the Metavir classification system. Pretreatment blood tests were conducted to determine each patient’s level of aspartate aminotransferase, alanine aminotransferase, total bilirubin, alkaline phosphatase, gamma-glutamyl transpeptidase, white blood cell (WBC), platelets, and hemoglobin. Written informed consent was obtained from all of the patients or their guardians and provided to the Ethics Committee of the Graduate School of Kyoto University, who approved the conduct of this study in accordance with the Helsinki Declaration. Table 1 Clinical characteristics of patients Characteristics SVR (n = 46) R (n = 28) NR (n = 25) p-value Age (years) 57.0 ± 9.8 61.2 ± 8.3 60.6 ± 7.6 0.09† Male (%) 28 (61%) 11 (39%) 9 (36%) 0.08§ Weight (kg) 59.5 ± 9.0 56.6 ± 9.9 56.0 ± 7.7 0.13† HCV RNA (×10 copies/ml) 1.90 ± 1.95 1.83 ± 1.04 1.58 ± 0.93 0.62†