Fig. 2

Identification of HES1 as a target of miR-182 in MTC. a Schematic overview of in silico analyses for identification of potential miR-182 targets. Venn diagram displays number of genes from different sources narrowed down through cross comparison. Left circle represents a cumulative list of miR-182 targets from three different prediction algorithms (TargetScan, PicTar, DIANA Lab). Right circle shows the amount of genes related to cancer according to gene ontology terms. Bottom circle depicts a list of differentially regulated genes in MTC received from the Oncomine database. A set of ten targets was identified and their expression examined preliminary from Oncomine obtained data comparing primary (PS) versus metastatic (M) MTC tissues displaying a significant downregulation of HES1 (right graph). b Validation of HES1 expression in MTC samples compared to healthy donor tissue. Values are normalized to RNU6B. Mann-Whitney test was used for statistical calculation. c-e HES1 expression values with activated RET and miR-182 status in different cell line models. TT cells infected with Ad.RET∆TK and control Ad.GFP (c, left) or transfected with miRZIP-182 and control miRZIP-scramble (c, right), RETM918T (d, left) and miR-182 (e, left) or miR-scr control transfected NThy-ori 3.1 cells, as well as NThy.RETM918T (d, right) and NThy.miR-182 cells (e, right) transfected with miRZIP-182 or miRZIP-scramble were harvested 24 h after treatment and analyzed for HES1 expression on RNA and protein level. For qRT-PCR values were normalized to actin and compared relative to the controls Ad.GFP (c) or ZIP-scr (d-e). Statistical significance was calculated by Student’s t-test. Actin served as loading control in immunoblots. f Scheme of the HES1 3′UTR with a putative miR-182 binding site and the mutated sequence (top). Luciferase activities were measured 24 h after cotransfection of NThy-ori 3.1 cells with HES1 3′UTR and increasing amounts (0.5, 1.0, 2.0 μg) of miR-182 or mutHES1 3′UTR (right) and 1 μg of miR-182 or miR-scr (left). Stable miR-182 NThy-ori 3.1 were transfected with HES1 3′UTR as positive control (center). Results are shown as fold increase relative to scramble control which was set as 1; * p < 0.05, ** p < 0.01, *** p < 0.001