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. 2021 Dec 22;11(1):12. doi: 10.3390/cells11010012

Figure 2.

Figure 2

Nrf2 is a direct target of miR-140-5p. (A,B) The mRNA and (C,D) protein levels of Nrf2 and its downstream genes were determined by qRT-PCR and Western blotting, respectively, in MDA-MB-231 cells with miR-140-5p-KD under normoxia or miR-140-5p-OE under hypoxia. Quantification of Western blot images was done by image J software. (E) MDA-MB-231 cells with stable Nrf2-KD were subjected to hypoxia, and relative Nrf2 mRNA and miR-140-5p levels were measured by qRT-PCR. (F) miR-140-5p and its complementary sites in the 3′-UTR of Nrf2 mRNA with WT and MUT sequence are shown. MUT sequence was generated by changing their complementary sites. (G) The luciferase assay in MDA-MB-231 cells with either miR-140-5p-OE (left panel) or miR-140-5p-KD (right panel) co-transfected with WT or MUT Nrf2 3′-UTR along with EFGP. EGFP (Renilla luciferase) was used for the normalization of fluorescence intensity. Error bars indicate mean ± SEM (n = 3). Student’s t-tests were used to compare the means of two groups. ns: not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to EV.