BACKGROUND Mimicking ischemia-reperfusion injury, oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) applied to endometrial cells produces significant oxidative stress and programmed necrosis, which can be inhibited by nuclear-factor-E2-related factor 2 (Nrf2) signaling. MicroRNA (miRNA)-induced repression of Keap1, a Nrf2 suppressor protein that facilitates Nrf2 degradation, is novel strategy to activate Nrf2 cascade. METHODS MicroRNA-941 (miR-941) was exogenously expressed in HESC and primary human endometrial cells, and the Nrf2 pathway examined by Western blotting and real-time quantitative PCR analysis. The endometrial cells were treated with OGDR, cell programmed necrosis and apoptosis were tested. RESULTS MiR-941 is a novel Keap1-targeting miRNA that regulates Nrf2 activity. In T-HESC cells and primary human endometrial cells, ectopic overexpression of miR-941 suppressed Keap1 3'-UTR (untranslated region) expression and downregulated its mRNA/protein expression, leading to activation of the Nrf2 cascade. Conversely, inhibition of miR-941 elevated Keap1 expression and activity in endometrial cells, resulting in suppression of Nrf2 activation. MiR-941 overexpression in endometrial cells attenuated OGDR-induced oxidative stress and programmed necrosis, whereas miR-941 inhibition enhanced oxidative stress and programmed necrosis. MiR-941 overexpression and inhibition were completely ineffective in Keap1-/Nrf2-KO T-HESC cells (using CRISPR/Cas9 strategy). Restoring Keap1 expression, using an UTR-depleted Keap1 construct, abolished miR-941-induced anti-OGDR activity in T-HESC cells. Thus Keap1-Nrf2 cascade activation is required for miR-941-induced endometrial cell protection. CONCLUSIONS Targeting Keap1 by miR-941 activates Nrf2 cascade to protect human endometrial cells from OGDR-induced oxidative stress and programmed necrosis. Video Abstract.

中文翻译：

---

靶向 Keap1 的 microRNA-941 通过激活 Nrf2 信号保护子宫内膜细胞免受氧气和葡萄糖剥夺-再氧化。

背景 模拟缺血再灌注损伤，氧气和葡萄糖剥夺 (OGD)-再氧合 (OGDR) 应用于子宫内膜细胞会产生显着的氧化应激和程序性坏死，这可以被核因子 E2 相关因子 2 (Nrf2) 抑制信号。MicroRNA (miRNA) 诱导的 Keap1 抑制是一种促进 Nrf2 降解的 Nrf2 抑制蛋白，是激活 Nrf2 级联的新策略。方法 在 HESC 和原代人子宫内膜细胞中外源表达 MicroRNA-941 (miR-941)，并通过蛋白质印迹和实时定量 PCR 分析检查 Nrf2 通路。用OGDR处理子宫内膜细胞，检测细胞程序性坏死和凋亡。结果 MiR-941 是一种新型的靶向 Keap1 的 miRNA，可调节 Nrf2 活性。在 T-HESC 细胞和原代人子宫内膜细胞中，miR-941 的异位过表达抑制 Keap1 3'-UTR（非翻译区）表达并下调其 mRNA/蛋白质表达，导致 Nrf2 级联激活。相反，抑制 miR-941 可提高子宫内膜细胞中 Keap1 的表达和活性，从而抑制 Nrf2 的激活。子宫内膜细胞中的 MiR-941 过表达减弱了 OGDR 诱导的氧化应激和程序性坏死，而 miR-941 抑制增强了氧化应激和程序性坏死。MiR-941 过表达和抑制在 Keap1-/Nrf2-KO T-HESC 细胞中完全无效（使用 CRISPR/Cas9 策略）。使用 UTR 耗尽的 Keap1 构建体恢复 Keap1 表达，消除了 T-HESC 细胞中 miR-941 诱导的抗 OGDR 活性。因此，miR-941 诱导的子宫内膜细胞保护需要 Keap1-Nrf2 级联激活。结论 miR-941 靶向 Keap1 可激活 Nrf2 级联反应，以保护人类子宫内膜细胞免受 OGDR 诱导的氧化应激和程序性坏死。视频摘要。