The micelle components, DMSO and Tween 80 were used as negative controls

RNA from the cells was extracted using a mirVana miRNA Isolation kit, according to the manufacturer’s instructions. For quantitative realtime RT-PCR, total RNA was reverse-transcribed to cDNA in a 20 L reaction volume using a High Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions. TaqMan probes for DR5 and 2-microglobulin were purchased from Applied Biosystems. The expression levels of mRNAs were quantified using the Applied Biosystems 7300 Real-Time PCR system according to the manufacturer’s instructions. The expression level of DR5 mRNA was normalized against the level of 2MG mRNA in the same sample. microRNA expression analysis was performed by using TaqMan miRNA assays to evaluate miR-221. Total RNA was reverse-transcribed to cDNA in a 15 L reaction volume using each specific primers and TaqMan MicroRNA Reverse Transcription kit. The expression levels of microRNAs were quantified using the 7300 Real-Time PCR system according to the manufacturer’s instructions. The results were normalized relative to the RNA gene RNU48. microRNA PG-490 Mimics transfection PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786614 Cells were transfected with 5 nM miRIDIAN microRNA Mimics using Lipofectamine PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786681 RNAiMAX according to the manufacturer’s instructions. After 24 hours of transfection, cells were treated with or without 40 mM metformin. The cells were harvested 24 hours after the treatment for FACS analysis and Western blotting. Statistical Analysis Data are the means S.D. of three determinations. Data was analyzed using the Student’s t-test and differences were considered significant at P <0.05. Results Metformin suppresses the cell growth of pancreatic cancer cells To investigate the effect of metformin on human pancreatic cancer cells, we examined whether metformin suppressed the cell growth using trypan blue dye exclusion assay. PANC-1, MIA PaCa-2, and AsPC-1 cells were treated with indicated concentrations of metformin for 72 hours, and viable cells were counted by trypan blue dye exclusion assay. As shown in Fig 1, metformin decreased the growth of these cancer cells in a dose-dependent manner. 4 / 15 Metformin Suppresses MiR-221 and Sensitizes TRAIL Fig 1. Metformin suppresses the cell growth of pancreatic cancer cells. PANC-1, MIA PaCa-2, and AsPC-1 cells were treated with the indicated concentrations of metformin. After incubation for 72 hours, cells were counted by trypan blue dye exclusion assay. Data are the means SD of 3 determinations. P<0.05, P<0.01. doi:10.1371/journal.pone.0125779.g001 Metformin induces G1-phase arrest in pancreatic cancer cells through down-regulation of miR-221 We next performed cell cycle analysis using flow cytometry after treatment with the indicated concentrations of metformin for 48 hours in human pancreatic cancer cells. As shown in Fig 2A, and S1, S2 and S4 Figs, metformin caused G1-phase arrest in a dose-dependent manner. Moreover, we examined the effect of metformin on the expression of G1 phase-related proteins. As a result, metformin at 40 mM increased the expression of p27 protein. Lee et al. previously identified a number of microRNAs with increased expressions, including miR21, -221, -301, -376a, -155, as well as others in human pancreatic cancer cells. In addition, p27 protein was down-regulated by miR-221 in human pancreatic cancer cells. Therefore, we examined the effect of metformin on the expression of miR-221. As shown in Fig 2C, metformin reduced the expression of miR-221 in a dose-dependent manner. 5 / 15 Metformin Su