Mutating miR144, miR153, miR27a and miR142-5p binding sites in Nrf2 3′ UTR confirms Nrf2 as a direct target of miR144/153/27a/142-5p.

<p>(A) Schematic representation of different Nrf2 3′ UTR luciferase reporters used in the transfection experiments is depicted. Constructs a, and e were generated in Dr. Jen-Tsan Chi’s laboratory and constructs b, c, d were generated in our laboratory and sequence verified for incorporation of mutagenized nucleotides (data not shown). (B) SH-SY5Y cells were co-transfected with either miR144 or scramble miR and 200 ng pGL3 reporter construct containing wild type or miR144 site mutated 3′ UTR of Nrf2. The relative firefly luciferase activity normalized with renilla luciferase was measured 48 h post-transfection and results are plotted as percentage change over respective control. (C) Cells were co-transfected with both the reporter gene (either wild type or miR 153 site mutated Nrf2 3′ UTR) constructs and scramble miR mimic/miR153 mimic. 48 h following transfection, firefly luciferase activity normalized to renilla activity was determined. The results are represented as percentage change over respective controls. (D) 100 nM of pre-miR scramble control (or) pre-miR27a was transfected into SH-SY5Y cells together with a wild-type or miR27a binding site mutated Nrf2 3′ UTR construct. Cells were lysed 48 h post-transfection and luciferase signal was measured. Firefly luciferase signals were normalized to renilla luciferase signal and percentage change over corresponding controls was represented. (E) Luciferase reporters containing wild-type or miR142-5p site mutant of human Nrf2 3′ UTR were co-transfected with scramble control miRNA or miR142-5p precursors into SH-SY5Y cells. 48 h after transfection, dual luciferase activity was measured. After normalization for renilla luciferase activity, the results were plotted as percentage change and compared to the corresponding controls. In (B-E), values are expressed as means±s.e.m from 3 independent samples and * indicate significantly (p<0.05) different when compared with wild-type Nrf2 3′ UTR construct by one way ANOVA/Newman Keuls post-test; ns-not significant when compared with the respective miRNA target site mutated Nrf2 3′ UTR constructs.