Fig. 3

SIRT1 is a novel miR-124a and miR-155 target. Depiction of the genomic structure of the human SIRT1 gene on chromosome 10 and location of the putative miR-124a and miR-155 binding sites within its 3′-UTR (a). Target prediction algorithm identified a putative miR-124a binding site and three putative miR-155 binding sites, indicated by the red bars. Positions and seed sequences of the putative binding sites are listed in the adjacent table (a). A reporter vector containing the SIRT1 3′-UTR was co-transfected with pre-miR-124a or pre-miR-155 into HEK-293 cells, and hRLuc reporter activity was determined relative to a vector construct containing the SIRT1 3′-UTR co-transfected with pre-miR-scrambled control (b). Control constructs lacking either the miR-124a (Mut 124a) or the miR-155 binding sites (Mut 155) were generated by site-directed mutagenesis. Both mutant vectors were co-transfected with the respective miRNA or with scrambled control into HEK-293 cells, and hRLuc reporter activity was determined; luciferase activity relative to scrambled control is given. Data are means ± SD; ns not significant, *p < 0.01, n = 8. c CD4⁺ T cells of healthy donors were transiently transfected with miR-124a, miR-155, or scrambled control, respectively, and stimulated with anti-CD3/CD28 Dynabeads for 36 h. Relative SIRT1 mRNA was detected by qPCR, n = 6, *p < 0,01 (d, left panel), and SIRT1 protein expression was determined by Western Blot analysis (d, right panel). One blot is representative of n = 3