Sample collection was approved by the Institutional Review Board of the corresponding institutes and recorded by the National Institutes of Health (NIH) Office of Human Subjects Research. find more A total of 23 ICC and CHC cases were used to build mRNA and microRNA signatures. The initial diagnosis was made based on serological test and imaging, and was confirmed histopathologically by pathologists. The characteristics of 68 Caucasian ICC patients from an independent cohort were described recently.23 HuCCT1 and HUH28 cell lines were used
for miR-200c functional studies. These cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank and were cultured in RPMI supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mmol/L L-glutamine. An immortalized human cholangiocyte-derived cell line, H69, kindly provided by Dr. Gregory Gores (Mayo Clinic), was cultured as described.24 A selleck chemical luciferase reporter
containing an upstream 0.9-kb fragment of pri-miR-200c was kindly provided by Dr. Li Wang (University of Utah School of Medicine).25 A detailed description of other transfection reagents, methodologies such as cell culture, cell proliferation and apoptosis assays, luciferase assay, immunohistochemical analysis, and cell migration and invasion assays can be found in the Supporting Materials. Total RNA was extracted from frozen tissue using Trizol (Invitrogen) according to the manufacturer’s protocol. Only RNA samples with good RNA quality as confirmed with the Agilent 2100 Bioanalyzer (Agilent Technologies) were
included for array study. Gene expression profiling of 23 tumor samples (16 ICC, 7 CHC), as well as seven paired noncancerous liver tissues from ICC patients and seven benign liver lesions (five focal nodular hyperplasia [FNH], two adenoma) was carried out on Affymetrix GeneChip Human Gene-ST 1.0 arrays according to the manufacturer’s protocol and processed as described.26 Affymetrix gene expression arrays obtained from different platforms were combined selleck screening library with the match probes package in R. Raw gene expression data were normalized using the robust multi-array average (RMA) method and global median centering. For genes with more than one probe set, the mean gene expression was calculated. Total RNA was used for the nCounter microRNA platform. All sample preparation and hybridization was performed according to the manufacturer’s instructions. All hybridization reactions were incubated at 65°C for a minimum of 12 hours. Hybridized probes were purified and counted on the nCounter Prep Station and Digital Analyzer (NanoString) following the manufacturer’s instructions. For each assay a high-density scan was performed.