Fig. 3.

Functional characterization of SHOX2 3′UTR variant. A SHOX2 3′UTR with the c.*28C allele creates a novel miR-92b-5p binding site (pairing of the seed region is shown). B SHOX2 mRNA (left) and miR-92b-5p (right) expression levels in human and murine heart tissues (human fetal and adult heart, mouse embryonic total heart and right atrium (RA) E11.5) and HL-1 cells (derived from mouse atrial cardiomyocytes). SHOX2 mRNA and miRNA (miR-92 b-5p) are co-expressed in the same tissues. C Luciferase activity of wild type (c.*28T) or mutant (c.*28C) psiCHECK2-SHOX2 3′UTR constructs, co-expressed with miR-92b-5p (indicated in light grey) or negative control miRNA (miR-ctrl, indicated in dark grey) in HEK293 and HL-1 cells, determined 48 h after transfection. miR-92b-5p significantly reduces the activity of the mutant but not the wild type SHOX2 3′UTR reporter in both cell lines. D HEK293 cells were transiently co-transfected with mutant (c.*28C) psiCHECK2-SHOX2 3′UTR reporter and miR-92b-5p together with either miR-92b-5p inhibitor or negative control inhibitor (ctrl-inhibitor) and luciferase activity was determined after 48 h. The allele-specific effect of miR-92b-5p on the luciferase activity of the mutant SHOX2 3′UTR reporter is reversed by a specific miR-92b-5p inhibitor, but not altered using the control inhibitor. Data are expressed as mean ± SEM of 3–4 independent experiments. For each experiment triplicates were measured. p values were determined by a paired t test (*p < 0.05; **p < 0.01, ns not significant)