[13] A previous study on miRNA expression in PBC patients has shown increased expressions of miR-299-5p and miR-328 in liver tissue.[14] Meanwhile, no study has examined the expression of miRNAs in Japanese patients with PBC. The aim Alectinib supplier of this study was to examine the relationship between miRNA expression in peripheral blood

mononuclear cells (PBMCs) and clinical presentation in Japanese patients with PBC. This study involved 58 patients diagnosed as having PBC at Fukushima Medical University Hospital between 2000 and 2012, patients with control diseases including 25 patients with autoimmune hepatitis (AIH), six patients with PBC-AIH overlap syndrome, and 23 patients with SLE, and 30 healthy controls. Patients were diagnosed as having PBC if they met at least two of three criteria: (i) chronic elevation of cholestatic liver enzymes, alkaline phosphatase (ALP) and gamma-glutamyltranspeptidase (GGT), (ii) presence of serum AMA detected by either indirect immunofluorescence or ELISA using commercially available kits, and (iii) typical histological findings of biopsied liver specimens.[11] AIH was diagnosed according to the revised scoring system proposed by the international autoimmune hepatitis group for diagnosis of AIH.[15] PBC-AIH overlap was diagnosed based on the Paris criteria proposed by Chazouilleres et al.[16] More specifically, PBC-AIH overlap was defined as meeting

at least Rapamycin solubility dmso two of three criteria for PBC, that is, (i) ALP level ≥ 2 × upper limit of normal (ULN) or GGT level ≥ 5 × ULN; (ii) positive for AMA; and (iii) a liver biopsy specimen showing florid bile duct lesions on, and at least two of three criteria for AIH, that is, (i) serum alanine aminotransferase (ALT) level ≥ 5 × ULN; (ii) serum immunoglobulin

(Ig) G level ≥ 2 × ULN or positive for anti-smooth muscle antibody (ASMA); and (iii) a liver biopsy showing moderate or severe periportal or periseptal lymphocytic piecemeal necrosis. SLE was diagnosed based on the criteria of the American College of Rheumatology.[17, 18] The present study was conducted with the approval of the ethics committee of Fukushima Medical University, and all patients provided consent before participating in the study. Peripheral blood was drawn from each patient and volunteer into a tube containing EDTA-2Na and centrifuged to separate PBMCs. Carnitine palmitoyltransferase II Total RNA was then isolated from the PBMCs using a mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer’s protocol to extract miRNA. Quantitative real-time PCR (qRT-PCR) was performed using 20 μL each of the samples containing a fixed concentration of RNA. For miRNA qRT-PCR, TaqMan MicroRNA Reverse Transcription Kit, TaqMan Universal Master Mix II, and TaqMan MicroRNA Assay primers (Applied Biosystems, Foster City, CA, USA) were used to determine the expression of previously-described miRNAs, including miR-26a, miR-328, miR-299-5p, miR-146a, miR-155, miR-16, miR-132 and let7a.