microRNA Expression profiles of parental Panc1 cell lines and Gemcitabine resistance Panc1 cell lines

Gemcitabine (GEM) is a key drug for treating PDAC, and it is commonly used for adjuvant chemotherapy. Although the majority of PDAC is sensitive to GEM at first, GEM cannot control PDAC for very long, suggesting that PDAC develops resistance to GEM after prolonged exposure. No reliable predictors of susceptibility to gemcitabine chemotherapy exist in pancreatic ductal adenocarcinoma. MicroRNAs (miR) are epigenetic gene regulators with tumorsuppressive or oncogenic roles in various carcinomas. This study assesses gemcitabine resistant PDAC for its specific miR expression pattern. Gemcitabine resistant variants of Panc1, a human pancreatic adenocarcinoma cell line, were established. MicroRNA screening was investigated by microarray. GEM-resistant cells were generated by exposure to gradually increasing concentrations of the reagent for 2 months. Parental Panc1cells (Panc1-Pt) were exposed to GEM at an initial concentration of 2 ng/ml. When the cells adapted, the GEM concentration was increased. The final GEM concentration was 30 ng/ml. Limiting the dilution of the established cells allowed the cloning of the GEM-resistant Panc1cells, and the three independent clones (Panc1-GRs: Panc1-GR1, -GR3, and –GR4) were used in the present experiments. Extracted total RNA was labeled with Hy5 using the miRCURY LNA Array miR labeling kit (Exiqon, Vedbaek, Denmark). Hybridized for 16 h at 37 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc.. The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’signal intensities of 95% confidence intervals. Measurements of both duplicate spots with the signal intensities greater than 2 standard deviations (SD) of the background signal intensity were considered to be valid. A relative expression level of a given miRNA was calculated by comparing the signal intensities of the averaged valid spots with their mean value throughout the microarray experiments. 3D-Gene Scanner ((Toray Industries Inc., Tokyo, Japan) was used for scanning. Images were quantified using Extraction(Toray Industries Inc., Tokyo, Japan).