Dual-Luciferase Reporter Assay.

The full-length SKP2 3′ UTR was PCR-amplified from human genomic DNA using the primers listed in SI Appendix, Table S7. This product was cloned into the psiCHECK-2 vector (Promega) using NotI and XhoI restriction sites and verified by Sanger sequencing. The miR-127 7mer-m8 target site in the WT SKP2 3′ UTR GGATCCG was substituted with CCGTGGC in the mutant SKP2 3′ UTR using QuikChange Lightening Site-Directed Mutagenesis Kit (Agilent) and the mutagenic primers listed in SI Appendix, Table S7. Sanger sequencing confirmed successful mutagenesis of the miR-127 7mer-m8 target site. psiCHECK-2 vector (Promega) containing either a WT or mutant 3′ UTR of SKP2 was cotransfected into LTC cells with 50 nM of miR-127 or NC mimic (Invitrogen) using the Lipofectamine 3000 Transfection Reagent (Invitrogen). The Renilla and firefly luciferase activities were measured 72 h posttransfection using the Dual-Luciferase Reporter System (Promega) following the manufacturer’s instructions. Renilla signals were normalized to an internal firefly luciferase as an internal control for transfection efficiency.