Fig. 4

Analysis of miR-155 binding sites on kras 3’UTR and Gli1/2 binding sites on mir155hg promotor by dual-luciferase reporter assays. A Conservation of the miR-155 sequence among different species (upper panel), and conservation of the miR-155 target sequence in KRAS among different species (lower panel). Human, Homo sapiens; Mouse, Mus musculus; Rat, Rattus norvegicus. B The miRNA response elements (MREs) of miR-155 were shown on the sequence of kras 3′-UTR, and mutations were introduced on these MREs. Both wild-type and mutated sequences were cloned into the psiCHECK-2 plasmid. C dual-luciferase reporter assay testing miR-155 binding with kras 3’UTR. HEK-293 T cells were co-transfected with has-miR-155 mimics or mimics NC as control oligonucleotide (final concentration at 50 nM) together with the wild-type (KRAS-WT-3’UTR) or mutated (KRAS-Mut-3’UTR) kras 3′-UTR luciferase reporter plasmids. The renilla luciferase activity was measured and normalized to firefly luciferase activity after 36 h. **p < 0.01. The assays were performed in triplicates, and results are presented as mean ± SEM. D Schematic of the three predicted Gli1/2 binding sites on mir155 hg promotor (upper panel) and their binding sequences accordingly (lower panel). The binding sites were located at − 1088 to − 1077 (site 1), − 461 to − 447 (site 2), and − 407 to − 393 (site 3) of the mir155hg promotor. The gli2 luciferase activities were tested by applying a series of truncations (E) as well as site-targeted mutations (F) on the mir155hg promoter, along with pcDNA3.1-Gli1 and pcDNA3.1-Gli2 and pRL-TK plasmids. The specific constructs used in the truncation assays (E) included pGL3-basic vector, pGL3-mir155hg-promo-WT (containing promotor region from − 1440 to + 226), pGL3-mir155hg-promo-truncation1 (from − 913 to + 226) and pGL3-mir155hg-promo-truncation2 (from − 339 to + 226). The specific constructs used in the site-mutation assays (F) included pGL3-basic vector, pGL3- mir155hg-promo-WT (containing all 3 sites), pGL3-mir155hg-promo-mut1 (lack of site 1), pGL3-mir155hg-promo-mut2 (lack of site 2) and pGL3-mir155hg-promo-mut3 (lack of site 3). The luciferase activities were determined and presented as the ratio of firefly and renilla luciferase activity. The assays were performed with three replicates and data are presented as mean ± SEM. **p < 0.01. G Schematic of the Gli1 and Gli2 binding to the mir155hg promotor at around − 1088 in hBMECs