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. Author manuscript; available in PMC: 2015 Feb 21.
Published in final edited form as: ACS Chem Biol. 2013 Nov 25;9(2):334–338. doi: 10.1021/cb400704n

Figure 2. miR-126 regulates cd97 by directly targeting its 3’-UTR.

Figure 2

(a) pMIR-REPORT™ constructs used for the luciferase assay. Specific miRNA binding in the 3’-UTR (untranslated region) should suppress reporter expression. (b) Human embryonic kidney (HEK293) cells were co-transfected with pMIR-REPORT™ carrying the indicated 3’-UTRs and pcDNA3.1™(+)-miR126 (miR-126) or pcDNA3.1™(+) (empty vector control). Relative luciferase activity for the construct bearing the cd97 3’-UTR decreased by 40% upon expression of miR-126, indicating that cd97 is a direct target of miR126. 2miR contains two miR-126 binding sites; Irs1 is a known target of miR-126. (c) Predicted miR-126 interaction sites in the cd97 3’-UTR and in cd97 3’-UTR mutants. (d) Either removing the predicted binding site or mutating three nucleotides within the binding site abolished miR-126-dependent suppression of the luciferase activity of the cd97 3’-UTR construct, confirming the miR-126 binding site within the cd97 3’-UTR. Mutations at a random site within the cd97 3’-UTR had no effect on suppression of luciferase activity. miR-ex control was taken as 100%. (e) The most favorable miR-126* interaction site in the cd97 3’-UTR as predicted by RNAhybrid algorithm, and the cd97 3’-UTR mutant used for luciferase assay. (f) Neither mutations at the predicted binding site for miR-126* nor at any other random position within the cd97 3’-UTR reversed suppression of luciferase activity, suggesting that miR-126* does not target the cd97 3’-UTR. miR-ex control was taken as 100%. P values were obtained using one-sided Student’s t-tests. *P < 0.01.