Figure 4.

SLC8A2 Gene Is a New Target of miR-206

(A) Schematic representation of human SLC8A2 gene structure on chromosome 19. The rectangles represent coding (black) and non-coding (white) exons. Sequence alignment among human, rat, and mouse miR-206 seeds (underlined), showing a high percentage of conservation, was zoomed in. The nucleotides mutated in pmiR-GLO+3′UTR-NCX2(1-840ΔmiR-206) were indicated with a star. (B) Real-time PCR for NCX2 mRNA in PC12 cells transfected with 50 nM of NT-miR (white bars), miR-206 (gray), or anti-miR-206 (black) showing NCX2 mRNA reduction after miR-206 transfection. (C and D) Western blot analysis of NCX2 protein in PC12 cells (C) and primary neurons (D) transfected with 50 nM of NT-miR, miR-206, or anti-miR-206 showing NCX2 protein reduction after miR-206 transfection. (E) Luciferase assay in PC12 cells co-transfect with pmiR-GLO+3′UTR-NCX2(1-840) or pmiR-GLO+3′UTR-NCX2(1-840ΔmiR-206) plus NT-miR, miR-206, or anti-miR-206 showing luciferase decrease in cells co-transfected with the wild-type construct and miR-206 molecules. *p < 0.05; **p < 0.005; ***p < 0.001 versus NT-miR by one-way ANOVA analysis followed by Bonferroni test. Each column represents the mean ± SEM (n = 3/6).