High throughput screening identifies microRNAs regulating PCSK9 and LDL receptor expression

Using a high throughput human microRNA library screen, we identified microRNAs targeting the 3’untranslated region of human PCSK9 The high throughput human miRNA screen was conducted with the NYU RNAi Core using a human microRNA library from Ambion mirVana, Life Technologies. HEK293T cells were seeded in a 384-well, white solid bottom polystyrene TC-treated microplates and co-transfected with 1.5 pmol of miRNA mimic, and 10 ng of total DNA plasmids composed of a 2:1 ratio of PCSK9 3’-UTR firefly luciferase reporter plasmid (Switchgear Genomics) to renilla luciferase control plasmid using Lipofectamine 2000 (Invitrogen). The cells and transfection mix and were dispensed into the 384-well plates using a Wellmate/BioTek Dispenser (ThermoScientific) outfitted with small-bore tubing. The plates were centrifuged at 1,000x g for 1 min to settle the contents and then incubated in a humidified incubator at 37°C and 5% CO2 for 48 hours. To measure PCSK9 3’UTR-luciferase activity, media was aspirated and the wells were washed with 1xPBS using a EL406 Microplate Washer (BioTek). Luciferase activity was assayed with the Dual-Glo Luciferase Assay® (Promega) according to the manufacturer’s instructions and read on a spectrophotometer (Envision). Positive (miR-224) and negative control miRNAs were included on each screen plate, and the miRNA library was assayed in quadruplicate. PCSK9 3’UTR firefly luciferase values were normalized to those of control renilla luciferase values for each replicate.