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.

941-binding sites, at position of 276-283) was generated using the same protocol described previously [31], which was transfected to human endometrial cells using the Lipofectamine 2000 protocol. Afterwards, cells were subjected to the applied genetic modifications, with the Keap1 3'-UTR luciferase activity tested through the Promega kit [43].

Transfection of miR-941 mimic.
Human endometrial cells were seeded into the six-well tissue culture plates (at 1 × 10 5 cells in each well). Lipofectamine 2000 was utilized for the transfection of 500 nM of the wild-type ("WT") or the mutant ("Mut") miR-941 mimics (synthesized by Shanghai Genechem Co.). After 48h, miR-941 levels were determined by qPCR.

RNA-Pull down assay.
The RNA-Pull down assay was carried out through the previously-described protocol [44,45], testing miR-941-bound mRNA using the Pierce Magnetic RNA Pull-Down Kit, Shanghai, China). In brief, T-HESC cells were transfected with biotinylated miR-941 mimic or control mimic (100 nmol/L) for 48h, and cells were harvested using the lysis buffer described early [45]. The biotincaptured RNA complex was pulled down by incubating the cell lysates (600 μg of each treatment) with the streptavidin-coated magnetic beads [44]. The bound mRNA was purified using the RNeasy Mini Kit (QIAGEN, Shanghai, China), with expression of Keap1 mRNA, in the bound fractions examined by qPCR. Its levels were normalized to the input controls.

Cell viability.
Human endometrial cells were seeded into the 96-well tissue culture plates (3,000 cells in each well). Following the applied treatments, a cell counting kit-8 (CCK-8) kit (Dojindo Laboratories, Kumamoto, Japan) was utilized to examine cell viability [8], with CCK-8 optic density (OD) examined at testwavelength of 450 nm.
2.10. Lactate dehydrogenase (LDH) assay. The human endometrial cells were seeded into the 12-well tissue culture plates (at 0.5 × 10 5 cells in each well). LDH release to the cell medium, the quantitative marker of cell necrosis [46], was tested through a two-step LDH detection kit (Promega, Shanghai, China) [8]. LDH contents in the medium were always normalized to total LDH levels.
Cells were further cultured for applied time periods.

Western blotting.
Human endometrial cells were seeded into the six-well tissue culture plates (at 1 × 10 5 cells in each well). Following the applied treatments, cellular lysates were achieved and quantified [7,8]. The lysates proteins (40 μg per treatment into each lane) were separated by SDS-PAGE gels, and transferring to polyvinylidene difluoride (PVDF) blots [47]. The detailed protocols for Western blotting and data quantification (through the Image J software) were previously described [40,48]. Assaying of nuclear fraction proteins was described early [40].

Mitochondrial immunoprecipitation (Mito-IP). T-HESC cells were
harvested and resuspended [8], with the supernatants collected as the cytosolic fraction lysates. The pellets were resuspended to achieve mitochondrial fraction lysates and quantified [49]. Mitochondrial fraction lysates (300 μg per treatment) were pre-cleared (using anti-IgG Sepharose beads), and incubated with anti-CypD antibody (Santa Cruz Biotech). The mitochondrial complex was captured by anti-IgG.
2.14. JC-1 mitochondrial depolarization assay. The human endometrial cells were seeded into the 12-well tissue culture plates (at 0.5 × 10 5 cells in each well).

Superoxide detection.
Endometrial cells with the applied genetic treatments were initially seeded onto 96-well tissue-culturing plates (at 3 × 10 3 cells of each well). Following the applied OGDR stimulation, the superoxide colorimetric assay kit (BioVision, Shanghai, China) was applied to examine the cellular superoxide contents. In brief, the superoxide detection reagent (50 µL/well) was added for 15 min under the dark, with the superoxide's absorbance tested at the test-wavelength of 450 nm [31].

Lipid peroxidation assay.
As reported [31] endometrial cells with the applied genetic treatments were initially seeded onto the six-well tissue-culturing plates (at 1 × 10 5 cells per well). Following the applied OGDR stimulation, the lipid peroxidation assay kit (Abcam, Shanghai, China) was utilized to examine cellular lipid peroxidation levels, via the malondialdehyde method. The lipid peroxidation levels, determined by the thiobarbituric acid reactive concentration, were tested and quantified using the previously-described protocol [31,51].
2.17. NQO1 activity. The detailed protocol for testing the relative NQO1 activity in human endometrial cells has been described elsewhere [52]. In brief, the inducer potency was quantified by using the NQO1 bioassay. T-HESC cells or the primary human endometrial cells (10 4 per well of a 96-well plate), with applied genetic treatments, were cultured for 24h. The NQO1 enzyme activity was tested and quantified in cell lysates using menadione as the substrate.
The GFP-positive T-HESC cells were sorted via FACS. The selected single cells were further incubated in complete medium with puromycin for 10 days, with Nrf2/Keap1 KO verified by Western blotting and/or qPCR assays.
In the primary human endometrial cells ("Endometrial cells"), infection of lv-pre-miR-941 led to over 10 folds increase in the expression of mature miR-941 (Fig. 1M), causing downregulation of Keap1 mRNA (Fig. 1N). miR-941 overexpression similarly induced Nrf2 signaling activation, leading to Nrf2 protein (but not mRNA) accumulation ( Fig. 1O and P), HO1 and NQO1 upregulation (both mRNA and protein, Fig. 1P and Q) as well as an increase in NQO1 activity (Fig. 1R). Collectively, these results show that miR-941 targets Keap1 and activates Nrf2 signaling in human endometrial cells.
Results show that transfection of the WT-miR-941 potently inhibited OGDR-induced cytotoxicity in T-HESC cells (Fig. 2G and H), while the mutants were completely ineffective ( Fig. 2G and H).
In the primary human endometrial cells OGDR-induced ROS production (tested by superoxide accumulation, Fig. 2I), mitochondrial depolarization (Fig. 2J) and cytosol cytochrome C release (Fig. 2K) were significantly attenuated by ectopic miR-941 overexpression. The latter also inhibited alleviated OGDR-induced cytotoxicity in the human endometrial cells (Fig. 2L). These results clearly show that miR-941 overexpression inhibited OGDR-induced oxidative stress, programmed necrosis and cytotoxicity in human endometrial cells.

Discussion
The function of miR-941 is largely unknown in human cells. The study by Hu et al., has shown that miR-941 is a human-specific miRNA, highly expressed in the human brain and in other human cells [58]. It has regulatory effects on gene expression, participates in hedgehog-and insulin-signaling pathways, and thus regulating evolution of human longevity [58]. Zhang  Recent studies have implied that miRNA-induced silencing of Keap1, the Nrf2 suppressor protein, is a novel strategy to provoke Nrf2 cascade activation in human cells. In breast cancer cells miR-200a targeted Keap1 to activate Nrf2 signaling cascade [36]. In SH-SY5Y neuroblastoma cells and differentiated human neural progenitor cells, miR-7 activated Nrf2 signaling by targeting and silencing Keap1 [35]. In the retinal pigment epithelium cells (RPEs) and retinal ganglion cells (RGCs), miR-141 targeted Keap1 and activated Nrf2 signaling, protecting cells against ultra-violet (UV)-induced oxidative stress [41]. A very recent study by Xu et al., has shown that Keap1-targeting miR-626 activated Nrf2 signaling and protected RPEs from oxidative injury [31]. Therefore, Keap1-targeting miRNAs can induce significant Nrf2 cascade activation, protecting human cells from oxidative stress.
The results of this study confirm that miR-941 is a direct and specific Keap1-