Figure 4

Akap7 and Xk are targets of miR-669m. HEK293T cells were cotransfected with luciferase reporter for 3′-UTR of target genes and miRNA expression vectors, and then dual luciferase assay was performed. (A) Construction of the dual luciferase is shown. 3′-UTRs of target genes (Akap, Xk, and Slc22a4) were cloned into downstream of Renilla luciferase gene, which results in that the luciferase mRNA can be regulated by miRNAs through the 3′-UTRs. The plasmid also includes independently regulated firefly luciferase gene, which can be used as an internal control. (B) Relative luciferase activity of pMSCV/Empty and pMSCV/miR-669m were presented. Vertical axis is relative luciferase activity (fold change to pMSCV/Empty). The Renilla/firefly luciferase ratio was assessed and normalized to the control. The graphs describe mean ± SD (n = 3). Statistical analysis was performed using Student’s t test (**p < 0.01). Representative data of two independent experiments are shown.