Miridian microrna hairpin inhibitor

Targeting peptide-PEG(2000)-poly-L-lysine with a degree of polymerization of 30 (Peptide-PEG-K30) was synthesized using standard fluorenylmethyloxycarbonyl (FMOC) solid phase synthesis methods³⁵ on an automated PS3 peptide synthesizer from Protein Technologies Inc (Tucson, AZ, USA). FMOC protected amino acids were also purchased from Protein Technologies Inc. The heterobifunctional molecule, FMOC-PEG2000-COOH was purchased from JenKem Technology USA (Allen, TX, USA). Two different targeting peptides were used in this study, REKA, which is named after the four amino acids in its sequence and a peptide targeting VCAM1 which has the sequence: VHPKQHR. The targeting peptide-PEG-K30 molecule was subsequently purified using reverse phase high performance liquid chromatography (HPLC, Shimadzu Corporation, Japan) and confirmed using a Bruker UltrafleXtreme (Fremont, CA, USA) matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). The miRNA inhibitor molecules (miRIDIAN microRNA Hairpin Inhibitors, Dharmacon, USA) are single-stranded, chemically enhanced oligonucleotides diluted to a working stock solution of 100 µM.

PC3-N and LNCaP cells were reverse transfected with siPORT NeoFX Transfection Agent (Ambion) and 50 nM mirVana miRNA mimics (Ambion) or 50 nM mirVana Negative Control No. 1 mimics (Cat. #4464058, Ambion). To repress miRNA activity, PC3-ML cells were transfected with DharmaFECT Transfection Reagent 2 (Dharmacon) and 25 nM miRIDIAN MicroRNA Hairpin Inhibitors (Dharmacon) or 25 nM miRIDIAN Negative Control No. 1 (IN-001005-01-05, Dharmacon) (Suppl. Table S1). Negative control mimics and inhibitors were referred to as SCR (scrambled) controls. Cells were reverse transfected using Lipofectamine 3000 (Invitrogen) with pCMV6-Entry-TrueORF vectors for RBL1 and TIMP2 (Origene) to overexpress these proteins or reverse transfected using siPORT NeoFX Transfection Agent (Ambion) and 20 nM Silencer Select Pre-Designed siRNAs for RBL1, TIMP2 and Negative Control No. 1 (s11854, s656; 4390843 respectively, Ambion) to block their gene expression.

3×10⁵ reporter cells were seeded per well in six-well dishes. Cells were transfected the following day with a mixture of inhibitors for miR-19a and miR-19b at 5 nM each (MiRIDIAN microRNA Hairpin Inhibitors, GE Dharmacon) using Lipofectamine RNAiMAX (Thermo Fisher). Fluorescence was assessed by flow cytometry 48 hours after transfection.