Keap1-targeting microRNA-941 protects endometrial cells from oxygen and glucose deprivation-re-oxygenation by activating Nrf2 signaling

Background: Mimicking ischemia-reperfusion injury, oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) stimulation to endometrial cells induces significant oxidative stress and programmed necrosis, which can be inhibited by nuclear-factor-E2-related factor 2 (Nrf2) signaling activation. MicroRNA (miRNA)-induced silencing of the Nrf2 suppressor protein Keap1 is novel strategy to activate Nrf2 cascade. Methods: microRNA-941 (miR-941) expression was exogenously altered in HESC cells and primary human endometrial cells, and cells treated with OGDR. Nrf2 pathway genes were examined by Western blotting assay and real-time quantitative PCR analysis. Endometrial cell programmed necrosis and apoptosis were tested. Results: miR-941 is a novel Keap1-targeting miRNA, regulates Nrf2 signaling activation. In T-HESC cells and primary human endometrial cells, ectopic overexpression of miR-941 suppressed Keap1 3’-UTR (untranslated region) activity and downregulated its mRNA/protein expression, leading to Nrf2 cascade activation. Conversely, Keap1’s 3’-UTR activity and expression were elevated in endometrial cells with miR-941 inhibition, whereas Nrf2 activation was inhibited. miR-941 overexpression in endometrial cells largely attenuated OGDR-induced oxidative stress and programmed necrosis, both were intensified with miR-941 inhibition. Further studies show that Keap1-Nrf2 cascade activation is absolutely required for miR-941-induced endometrial cell protection. MiR-941 overexpression and inhibition were completely ineffective in Keap1-/Nrf2-KO T-HESC cells (using CRISPR/Cas9 strategy). Restoring Keap1 expression, by an UTR-depleted Keap1 construct, abolished miR-941-induced anti-OGDR activity in T-HESC cells. Conclusions: Targeting Keap1 by miR-941 activates Nrf2 cascade to protect human endometrial cells from OGDR-induced oxidative stress and programmed necrosis.