Sing a Microplate Reader (VersaMAx, Molecular Devices). All experiments were repeated
Sing a Microplate Reader (VersaMAx, Molecular Devices). All experiments were repeated three times. Values are presented as the mean ?standard deviation (SD).Invasion assayCells were seeded into the top chamber of a 96-well matrigel-coated plate with 8-m-pore polyethylene terephthalate membrane inserts (Corning). MiR-21 inhibitor and negative control oligonucleotides were transfected at a final concentration of 200nM.The bottom chamber was filled with 0.75 mL Ham F-12 medium with 10 FBS as a chemoattractant. The inserts were filled with 0.5 mL Ham F-12 medium with 1 FBS. After incubation for 48 h, the filter membrane was fixed with 100 methanol and stained with hematoxylin and eosin. The degree of invasiveness was quantified by counting the number of cells in 4 random fields of view per filter using 400?magnification. Data obtained from three separate inserts are shown as mean values.Statistical purchase AZD-8055 analysisTwenty four hours before transfection, cells were seeded in plates and grown to 50 confluence. For inhibition of miR-21, RMG-II cells were transfected with mirVana miRNA Inhibitors or a control (Ambion). Transfections were performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol.Dual luciferase reporter assayAll statistical analyses were performed using StatMate III software (ATMS, Tokyo, Japan). Comparisons between parameters were made using Fisher’s exact test. For survival analysis, PFS and OS distributions were determined using the Kaplan eier method, and the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26795252 resulting curves were compared using the log-rank test. P <0.05 was considered statistically significant.ResultsChromosome 17q23-25 amplification, miR-21 expression, and PTEN protein expression in CCCpGL3 wild-type PTEN 3-UTR and pGL3 mutant-type PTEN 3-UTR luciferase plasmids were obtained from Addgene (Cambridge, MA). RMG-II cells were seeded in 6-well plates (5?05 cells/well). After 24 h, the cells were transfected with pGL3 control vector (Promega), pGL3 wild-type PTEN 3-UTR vectors, or pGL3 mutant-type PTEN 3-UTR vectors using Lipofectamine 2000 reagent. Luciferase activities were measured using the DualLuciferase Reporter Assay system (Promega) 24 h after transfection. Firefly luciferase activity was normalized to renilla activity for each sample. All the experiments were performed in triplicate.MTS assayMTS assay was performed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) following the manufacturer's protocol. Briefly, miR-21 inhibitor and negative control oligonucleotides were transfected at a final concentration of 200nM. After 24 hours transfection, RMG-II cells were seeded into 96-well plates at a density of 1 ?104 cells per well. MTSCGH array profiles of chromosome 17 in 28 primary CCCs revealed that 9 out of 28 patients (32 ) showed 17q23-25 amplification that included miR-21 (Figure 1). MiR-21 and PPM1D mRNA expression were then measured by real-time RT-PCR analysis (Additional file 1: Figure S1). We defined standardized value as each median value of miR-21 and PPM1D expression without 17q23-25 amplification. Overexpression of miR-21 and PPM1D were found in 60 and 57 of these tumors, respectively. Seven of 9 tumors (77.7 ) with 17q23-25 amplification showed miR-21 overexpression, and 10 of 19 tumors (52.6 ) without 17q23-25 amplification also showed miR-21 overexpression. In addition, 6 of 9 tumors (66.6 ) with 17q23-25 amplification showed PPM1D overexpression, and 10 of.