Mir 17 5p mimic

The reporter plasmids (pGL3-Firefly Luciferase-Renilla_Luciferase Reporter carrying the PAUPAR sequence or a mutant sequence) were designed by Genechem Co., Ltd. (Shanghai, China). The reporter plasmid and miR-17-5p mimic or negative control mimic (RiboBio, Guangzhou, China) were co-transfected into HEK-293 T cells and the cells were cultured for 48 h. The luciferase activity was then examined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Rat INS-1 pancreatic β-cell line (ATCC, Manassas, VA, USA) were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (Gibco, USA), 2.5 mmol/L mercaptoethanols, 10 mmol/L HEPES, 100 U/ml penicillin-streptomycin, and 11.1 mmol/L glucose, and incubated at 37°C in a 5% carbon dioxide incubator. INS-1 cells were transfected with miR-17-5p mimic (RiboBio Co., Ltd., Guangzhou, China) according to standard protocol in the presence of different concentrations of glucose (5 and 25 mmol/L). In some experiments, INS-1 cells were treated, respectively, with different concentrations of glucose (5, 25, and 50 mmol/L) for 72 h. To investigate whether miR-17-5p inhibited the expression of TXNIP, INS-1 cells were transfected with miR-17-5p mimic or miR-17-5p inhibitor in the presence of high glucose (25 mmol/L).