miR-193b regulates the expression of KRAS by directly targeting its 3′-UTR.

<p>(A) The TargetScan online database (<a href="http://www.targetscan.org" target="_blank">http://www.targetscan.org</a>) predicted KRAS as an miR-193b target. The site between 1074 and 1080 (boxed region), one of the two predicted miR-193b binding sites in the 3′-UTR of KRAS, is shown, highlighting the evolutionarily conservation among humans (hsa), the chimpanzees (Ptr), rhesus monkey (Mml), bushbaby (Oga), and treeshrew (Tbe). (B) The two miR-193b binding sites at positions 303 to 309 and 1074–1080 in the KRAS 3′-UTR, identified using the TargetScan online database (<a href="http://www.targetscan.org" target="_blank">http://www.targetscan.org</a>), are shown. Seed sequences were mutated as shown in the boxed regions. The wild-type or mutant constructs were inserted into the pGL3 vector directly downstream of the luciferase gene. (C) 293A cells were co-transfected with miR-193b or scrambled oligonucleotide, wild-type or mutant firefly luciferase constructs of the KRAS 3′-UTR segment containing miR-193b binding sites, and Renilla luciferase (endogenous control). Luciferase activity was measured 24 hours after transfection using the Dual-Luciferase Reporter Assay Systems. Renilla-normalized luciferase activity is expressed relative to that obtained for the scrambled oligonucleotide under each condition. WT, wild-type; Mut, mutant. (D) KRAS and CCND1 protein levels were assessed by Western blotting 48 hours after transfection. (E) The protein levels of p-ERK and p-Akt were evaluated by Western blotting 48 hours after miR-193b transfection.