Figure 3: Functional analysis of the GATA4 c.* 84C>T variant located in the 3´ UTR. (a) The location and Sanger sequencing result of the 3´ UTR variant are shown in the diagrams. (b) In the dual-luciferase activity assay, HEK293 cells were cotransfected with luciferase reporters containing the 3´ UTR fragment along with hsa-miR-3194 mimics. (c) In the dual-luciferase activity assay, HEK293 cells were cotransfected with luciferase reporters containing the 3´ UTR fragment along with hsa-miR-1225-3p mimics. The luciferase reporter targets include two types of 3´ UTR fragments of GATA4 with a 307-bp fragment of the 3´ UTR sequence containing either C or T at the variation site. They are wild-type 3´ UTR of GATA4 with original base C (labeled WT 3´ UTR) and mutant 3´ UTR of GATA4 with variation base T (labeled mut 3´ UTR), respectively. Cotransfection with miRNA mimics (b) hsa-miR-3194 or (c) hsa-miR-1225-3p resulted in no significant difference in luciferase activity between cells transfected with mut 3´ UTR and those with wt 3´ UTR. Results are the mean ± standard deviation of three independent experiments. NS: not significant between two groups. GATA4: GATA-binding protein 4; WT: wild-type; MT: mutant; UTR: untranslated region; CDS: coding sequence.