Distinct MicroRNAs Expression Profile in Primary Biliary Cirrhosis and Evaluation of miR 505-3p and miR197-3p as Novel Biomarkers Masashi Ninomiya 1 , Yasuteru Kondo 1 *, Ryo Funayama 2 , Takeshi Nagashima 2 , Takayuki Kogure 1 , Eiji Kakazu 1 , Osamu Kimura 1 , Yoshiyuki Ueno 3 , Keiko Nakayama 2 , Tooru Shimosegawa 1 1 Division of Gastroenterology, Tohoku University Hospital, Sendai, Japan, 2 Division of Cell Proliferation, Tohoku University School of Medicine, Sendai, Japan, 3 Department of Gastroenterology, Yamagata University Faculty of Medicine, Yamagata, Japan Abstract Background and Aims: MicroRNAs are small endogenous RNA molecules with specific expression patterns that can serve as biomarkers for numerous diseases. However, little is known about the expression profile of serum miRNAs in PBC. Methods: First, we employed Illumina deep sequencing for the initial screening to indicate the read numbers of miRNA expression in 10 PBC, 5 CH-C, 5 CH-B patients and 5 healthy controls. Comparing the differentially expressed miRNAs in the 4 groups, analysis of variance was performed on the number of sequence reads to evaluate the statistical significance. Hierarchical clustering was performed using an R platform and we have found candidates for specific miRNAs in the PBC patients. Second, a quantitative reverse transcription PCR validation study was conducted in 10 samples in each group. The expression levels of the selected miRNAs were presented as fold-changes (2 2DDCt ). Finally, computer analysis was conducted to predict target genes and biological functions with MiRror 2.0 and DAVID v6.7. Results: We obtained about 12 million 32-mer short RNA reads on average per sample and the mapping rates to miRBase were 16.60% and 81.66% to hg19. In the statistical significance testing, the expression levels of 81 miRNAs were found to be differentially expressed in the 4 groups. The heat map and hierarchical clustering demonstrated that the miRNA profiles from PBC clustered with those of CH-B, CH-C and healthy controls. Additionally, the circulating levels of hsa-miR-505-3p, 197-3p, and 500a-3p were significantly decreased in PBC compared with healthy controls and the expression levels of hsa- miR-505-3p, 139-5p and 197-3p were significantly reduced compared with the viral hepatitis group. Conclusions: Our results indicate that sera from patients with PBC have a unique miRNA expression profile and that the down-regulated expression of hsa-miR-505-3p and miR-197-3p can serve as clinical biomarkers of PBC. Citation: Ninomiya M, Kondo Y, Funayama R, Nagashima T, Kogure T, et al. (2013) Distinct MicroRNAs Expression Profile in Primary Biliary Cirrhosis and Evaluation of miR 505-3p and miR197-3p as Novel Biomarkers. PLoS ONE 8(6): e66086. doi:10.1371/journal.pone.0066086 Editor: Aftab A. Ansari, Emory University School of Medicine, United States of America Received November 28, 2012; Accepted May 3, 2013; Published June 12, 2013 Copyright: ß 2013 Ninomiya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support for this study was provided by Grant-in-Aid for Young Scientists (B) (23790765). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yasuteru@ebony.plala.or.jp Introduction MicroRNAs (miRNAs) are small endogenous RNA molecules of 19 to 24 nucleotides that control the translation and transcription of targeting mRNAs by base-pairing to the complementary sites [1] [2] [3] [4]. The expression of miRNAs in serum is reported to be stable, reproducible and consistent among individuals of the same species [5]. So far, specific expression patterns of serum miRNAs were identified as a fingerprint for numerous diseases and cancers [6] [5]. The serum miR-122 levels are elevated in patients with liver damage due to chronic hepatitis B (CH-B) and C infection (CH-C) [7] [8]. In addition, the miR-122 and miR-34a levels are positively correlated with the disease severity in CH-C and non-alcoholic fatty-liver disease [9]. However, there are some reports that miR-122 expression in healthy controls was signifi- cantly higher than that in patients with hepatitis C virus (HCV) infection [10]. Li et. al. described that 13 miRNAs were differentially expressed in hepatitis B virus (HBV) serum and that miR-25, miR-375 and let-7f could be used as biomarkers to separate a HBV-positive hepatocellular carcinoma (HCC) group from HBV-negative HCC [11]. However, little is known about the expression profile of miRNAs in autoimmune disease such as primary biliary cirrhosis (PBC). PBC is female predominant, progressive autoimmune disease characterized by immune-mediated destruction of the intrahepatic bile ducts. The serological marker of PBC is the presence of anti- mitochondrial antibody (AMA) directed against the E2 subunit of the pyruvate dehydrogenase enzyme complexes located in the inner mitochondrial membrane [12] [13] [14]. The etiology of PBC is considered to be a combination of genetic predisposition and environmental triggers [15]. Particularly, concerning genetic predisposition, previous studies reported that common genetic variants at the HLA class II, IL12A, IL12RB2, STAT4, IRF5- PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e66086