NRF2 is a direct regulatory target of miR-144. (A) Sequence alignment and evolutionary conservation between miR-144 and its 2 putative binding sites in the 3′UTR of NRF2 mRNA from several indicated species, with sequences recognized by miR-144 seed sequence shown in bold. (B) NRF2 3′UTR luciferase reporters (upper panel) were cotransfected with empty vector, miR-144, or miR-320 expression constructs (detailed in supplemental Figure 3). The overexpression of miR-144 significantly repressed the luciferase activity of a NRF2 3′UTR reporter construct. While mutation of the first miR-144 binding site (mut1) only modestly decreased the miR-144–mediated repression, mutation of the second site (mut 2) abolished this effect. MiR-320 does not contain a NRF2 3′UTR binding site and did not significantly alter the luciferase activity of any of the 3 reporters compared with empty vector control. *Significantly different by Student t test: *P ≤ .05, ***P ≤ .0001.