Rensp luciferase reporter plasmid

Rat LA7 cells (obtained directly from ATCC, catalog #: CRL-2283), which were originally derived from a DMBA-induced mammary tumor in a Sprague Dawley rat, and human MCF7 cells (obtained directly from ATCC, catalog #: HTB-22) were used in in vitro experiments. MicroRNA-200a inhibition was performed using 5nM miR-200a-targeting and non-targeting control power inhibitors (Exiqon), transfected in tandem with a synthetic miR-200a target RenSP luciferase reporter plasmid (Switchgear Genomics, Carlsbad, CA) at a ratio of 1:5 (ng:nL) with Dharmafect Duo transfection reagent (GE Healthcare, Pittsburgh, PA). Twenty-four hours after transfection, cells were seeded into two separate 96-well plates, one for luciferase readout to verify miRNA inhibition and one for proliferation analysis. Luciferase and proliferation analyses were done 24 hours after reseeding, for a total of 48 hours after miRNA inhibition. RenSP luciferase signal was analyzed using LightSwitch Assay Reagent (Switchgear Genomics) according to manufacturer protocol. Cellular proliferation was measured using a BrdU ELISA kit (Roche, Basel, Switzerland) following manufacturer protocol.

The cell lines MDA-MB-231 and M6 were co-transfected in tandem with 10 nM miR-15b mimic or control mimic (Exiqon) and a synthetic miR-15b target RenSP luciferase reporter plasmid (Switchgear Genomics, Carlsbad, CA, USA) at a ratio of 1:5 (ng:nL) with Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Twenty-four hours after transfection, cells were seeded into two separate 96-well plates, one for luciferase readout to verify miR-15b overexpression and one for proliferation analysis. Luciferase and proliferation analyses were conducted 48 h after reseeding, for a total of 72 h after miR transfection. RenSP luciferase signal was analyzed using LightSwitch Assay Reagent (Switchgear Genomics, Menlo Park, CA, USA) according to the manufacturer’s protocol. Overexpression of miR-15b was achieved by reverse transfection using Lipofectamine RNAimax (Thermo Fisher Scientific) and 10 nM of miR-15b mimic, which are artificial double-stranded RNAs consisting of the guide strand that mimics the function of the endogenous miRNA or miR-15b inhibitor. The miR-15b inhibitor was designed to be an exact antisense to mir15b forming a duplex with the miRNA-15b guide strand, preventing it from binding to its intended targets and promoting its degradation (Exiqon) [26 ]. The expression of miR-15b and target genes was evaluated 24 h after transfection.