Figure 5. Nrf2 is a direct target of miR-450b-5p on renal IRI in male rats. (A) A schematic diagram used to search the target mRNAs of miR-450b-5p in three databases (MiRDB, StarBase and TargetScan). (B) Validation of the four differently expressed mRNAs in rats caused by IRI injury based on qRT-PCR assay. (C) Western Blotting analysis showed the expression level of Nrf2 from renal tissues in SHAM, FLX, IRI and IRI+FLX groups. (D) The construction diagram of the target genes (Nrf2) double-luciferase reporter genes. (E) The relative luciferase expression with Nrf2 3′-UTR after co-transfection with miR-450b-5p mimic or NC mimic in HK-2. (F) Nrf2 could bind to miR-450b-5p transcript in vivo in HK-2 cells. HK-2 cell lysates were immunoprecipitated with Nrf2 antibody or control IgG followed by qRT-PCR. (G) Western Blotting analysis showed the expression level of NF-κB, Nrf2 and Nrf2-dependent antioxidant enzymes, including HO-1, NQO1 and GST from renal tissues in IRI group and IRI+FLX group after the injection of NC agomir and miR-450b-5p agomir. Data were presented as Mean ± SD, *significant difference (P < 0.05).