Figure 4

Reporter studies of the direct VKORC1 mRNA-miRNA interaction. (A) pMIR-REPORT with the VKORC1 3′UTR binding site for miR-133a (wild-type (WT) or mutated for the miR-133a binding site (MUT)) were cotransfected with pRL-TK vector and scrambled control or pre-miR-133a. Each combination of pMIR-REPORT (WT and MUT) and pRL-TK was tested in quadruplicate in four independent experiments. Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well. The normalized data were expressed as changes relative to the data of the cells transfected with SCR and pre-miR and set as 100%. (B) Western blot analysis of myc-VKORC1 expression levels in HEK293T cells after cotransfection with pCMV6-VKORC1 3′UTR WT or MUT and scrambled control or pre-miR-133a. Fusion protein was detected with an anti-c-myc antibody. Densitometric analysis was performed with Quantity One 1-D analysis software. A representative Western blot is shown. Statistical significance was taken as P< 0.05. Results are represented as mean ± SD from four (A) and six (B) experiments performed in triplicate. t-Student was calculated in scrambled control versus pre-miR (^*P< 0.05; ^**P< 0.01).