Dual light system

Luciferase plasmids (pMIR) containing 5’-UTR or 3’-UTR of Jab1 were transiently transfected with miR-24 mimics into CNE-2 cells. Cells were seeded onto 24-well plates and co-transfected 24 hours later with pMIR-5’-UTR or 3’-UTR, 30 nmol/L miR-24 mimics, or miR control and a pRL plasmid control using Plus reagent following the manufacturer’s protocol. After 48 hours, cells were washed and resuspended in the lysis buffer, and the luciferase activity was assayed with a luminometer using the Dual-Light system (Promega, Madison, WI, USA). All experiments were performed in triplicate, and the results are presented as the means of these separate experiments.

To verify predicted targets of miR-21, the pMIR-REPORT system (Ambion) was used. Briefly, 55-mer (RECK) and 57-mer (TIMP3) fragments of the wild-type 3′untranslated region (UTR) containing the putative binding site (Supplementary Table 2) were synthesized and ligated between the SpeI and HindIII restriction sites of the pMIR-REPORT Luciferase vector. Mutant 3′UTRs lacking the miR-21 binding site were also synthesized. The wild type and mutant 3′ UTRs were ligated between the SpeI and HindIII restriction sites of the pMIR-REPORT Luciferase vector to create the pLUC-targets and pLUC-mutTargets constructs. Huh7 cells were transfected with different reporter vectors (p-Luc-Empty, p-Luc-targets or p-Luc-mutTargets) and co-transfected with negative control or miR-21 inhibitor. Huh7 cells were transfected with these reporter vectors in addition to a miR-21 inhibitor or negative control. pMIR-REPORT β-gal was also transfected for use in transfection normalization. At 36 h post-transfection, luciferase and β-galactosidase activities were measured using the Dual-Light System (Promega, Madison, WI).