Dual-Luciferase Gene Reporter Assay

For the dual-luciferase gene reporter assay between H19 and miR-877-3p, the full length of wild-type H19 was amplified by PCR, and then the PCR products were subcloned into psiCHECK-2 luciferase reporter vector (Luc-H19-WT; Promega, WI, USA). Luc-H19 mutant (Luc-H19-Mut) was also constructed, which contained a mutated H19 at the binding site of miR-877-3p (5′-AGAGGACA-3′ mutated to 5′-CACAACTC-3′). For the dual-luciferase gene reporter assay between miR-877-3p and Bcl-2, the 3′ UTR of Bcl-2 was obtained by PCR. Two critical binding sites between Bcl-2 3′ UTR and miR-877-3p were mutated from 5′-AGAGGAC-3′ to 5′-CACAACT-3′ and from 5′-TGAGGAC-3′ to 5′-AACAACT-3′. The wild-type and mutant 3′ UTR of Bcl-2 mRNAs were subcloned into the psiCHECK-2 luciferase reporter vectors. HEK293 cells were cultured in 24-well plates and transfected with the corresponding plasmids, miR-877-3p mimics, AMO-miR-877-3p, or their respective NC. Forty-eight hours later, Renilla and firefly luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Roche, Mannheim, Germany) and GloMax Luminometry System (Promega, WI, USA).