microRNA-1203 targets and silences cyclophilin D to protect human endometrial cells from oxygen and glucose deprivation-re-oxygenation

Abstract

Oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) stimulation to the human endometrial cells mimics ischemia-reperfusion injury. Cyclophilin D (CypD)-dependent programmed necrosis pathway mediates OGDR-induced cytotoxicity to human endometrial cells. We here identified a novel CypD-targeting miRNA, microRNA-1203 (miR-1203). In T-HESC and primary human endometrial cells, ectopic overexpression of miR-1203, using a lentiviral construct, potently downregulated the CypD 3'-untranslated region (3'-UTR) activity and its expression. Both were however upregulated in endometrial cells with forced miR-1203 inhibition by its anti-sense sequence. Functional studies demonstrated that ectopic miR-1203 overexpression in endometrial cells alleviated OGDR-induced programmed necrosis, inhibiting mitochondrial CypD-p53-adenine nucleotide translocator 1 association, mitochondrial depolarization, reactive oxygen species production, and medium lactate dehydrogenase release. Contrarily OGDR-induced programmed necrosis and cytotoxicity were intensified with forced miR-1203 inhibition in endometrial cells. Significantly, ectopic miR-1203 overexpression or inhibition failed to change OGDR-induced cytotoxicity in CypD-knockout T-HESC cells. Furthermore, ectopic miR-1203 overexpression was unable to protect T-HESC endometrial cells from OGDR when CypD was restored by an UTR-depleted CypD construct. Collectively, these results show that miR-1203 targets and silences CypD to protect human endometrial cells from OGDR.

Keywords: cyclophilin D; human endometrial cells; miR-1203; oxygen and glucose deprivation-re-oxygenation (OGDR); programmed necrosis.

Figure 1

miR-1203 targets and silences CypD in human endometrial cells. The wild-type (WT) microRNA-1203 (miR-1203) targets CypD 3’-UTR (3’-untranslated region) at position 806-813 (A). T-HESC endometrial cells were infected with pre-miR-1203-encoding lentivirus (“lv-pre-miR-1203”), following puromycin selection three stable cell lines were established: “sL1/sL2/sL3”. Control T-HESC cells were infected with microRNA control lentivirus (“lv-miRC”); Expression of mature miR-1203 and CypD mRNA was tested by qPCR assays (B and D); The relative CypD 3’-UTR luciferase reporter activity was examined (C), with CypD protein expression tested by Western blotting assays (E). T-HESC cells were transfected with 500 nM of control microRNA mimic (“miRC”), the wild-type (“WT-”) or the mutant (“Mut1/2”, see sequences in A) miR-1203 mimics for 48h, the relative CypD 3’-UTR luciferase reporter activity (F), CypD mRNA (G) and protein (H) levels were tested. The primary human endometrial cells (“Endometrial cells”, same for all Figures) were infected with lv-pre-miR-1203 or lv-miRC lentivirus for 48h, expression of mature miR-1203 (I), CypD mRNA (J) and protein (K) was shown. CypD protein expression was quantified and normalized to the loading control (E, H and K). “MW” stands for molecular weight (same for all Figures). “Vec” stands for the empty vector control (same for all Figures). Data were presented as mean ± SD (n=5). * P <0.05 vs. “Vec”/“miRC”/“lv-miRC” cells. Experiments in this figure were repeated three times with similar results obtained.

Figure 2

miR-1203 inhibition can elevate CypD expression in human endometrial cells. T-HESC endometrial cells were infected with pre-miR-1203 anti-sense lentivirus (“lv-antagomiR-1203”), following puromycin selection two stable cell lines were established: “L1/L2”. Control T-HESC cells were infected with microRNA anti-sense control lentivirus (“antaC”); Expression of mature miR-1203 and CypD mRNA was tested by qPCR assays (A and C); The relative CypD 3’-UTR luciferase reporter activity was examined (B), with CypD protein expression tested by Western blotting (D). The primary human endometrial cells were infected with lv-antagomiR-1203 or antaC for 48h, expression of mature miR-1203 (E), CypD mRNA (F) and protein (G) was shown. CypD protein expression was quantified and normalized to the loading control (D and G). Data were presented as mean ± SD (n=5), and results were normalized. * P <0.05 vs. “Vec”/“antaC” cells. Experiments in this figure were repeated five times with similar results obtained.

Figure 3

Forced miR-1203 overexpression protects human endometrial cells from OGDR-induced programmed necrosis. The stable T-HESC cells, with the pre-miR-1203-encoding lentivirus (“lv-pre-miR-1203-sL1/2/3”) or the control T-HESC cells with microRNA control lentivirus (“lv-miRC”), were subjected to OGD exposure for 4h, followed by re-oxygenation (“OGDR”) for applied time periods, ROS production (DCF-DA intensity, (A) mitochondrial depolarization (JC-1 green fluorescence accumulation, (B) cytochrome C release (C) testing cytosol proteins) were tested by the assays mentioned in the text; Cell survival and necrosis were tested by CCK-8 (D) and LDH release (E) assays, respectively. The parental control T-HESC cells were treated with the OGDR procedure for applied time periods, expression of mature miR-1203 (F) and CypD mRNA (G) was tested by qPCR assays. The primary human endometrial cells were infected with lv-pre-miR-1203 or lv-miRC lentivirus for 48h, followed by OGDR procedure for the applied time periods, ROS production (H), mitochondrial depolarization (I), cytochrome C release (J, testing cytosol proteins) and cell necrosis (K) were tested similarly. For the cytochrome C release assay, relative cytosol cytochrome C level (vs. Tubulin) was quantified (C and J). Data were presented as mean ± SD (n=5). “Mock” stands for non-OGDR treatment (same for all Figures). * P <0.05 vs. “Mock” treatment in “lv-miRC” cells. ^# P <0.05 vs. OGDR treatment in “lv-miRC” cells. Experiments in this figure were repeated three times with similar results obtained. Bar= 50 μm (B and I).

Figure 4

miR-1203 inhibition can exacerbate OGDR-induced cytotoxicity in human endometrial cells. Stable T-HESC cells with the pre-miR-1203 anti-sense lentivirus (“lv-antagomiR-1203-L1/L2”, two lines) or the microRNA anti-sense control lentivirus (“anta-C”) were subjected to OGDR for applied time periods, ROS production (DCF-DA intensity, (A) mitochondrial depolarization (JC-1 green fluorescence accumulation, (B) cytochrome C release (C) testing cytosol proteins) were tested by the assays mentioned in the text; Cell survival and necrosis were tested by CCK-8 and medium LDH release assays (D). Stable T-HESC cells with the pre-miR-1203 anti-sense lentivirus (“lv-antagomiR-1203-L1”) were pretreated with cyclosporin A (CsA, 10 μM) for 1h, followed by the OGDR stimulation for 24h, cell viability and necrosis were tested similarly (E). The primary human endometrial cells were infected with lv-antagomiR-1203 or anta-C lentivirus for 48h, followed by OGDR procedure for the applied time periods, ROS production (F), mitochondrial depolarization (G), cytochrome C release (H, testing cytosol proteins) and cell necrosis (I) were tested. For the cytochrome C release assay, relative cytosol cytochrome C level (vs. Tubulin) was quantified (C and H). Data were presented as mean ± SD (n=5). * P <0.05 vs. “Mock” treatment in “anta-C” cells. ^# P <0.05 vs. OGDR treatment in “anta-C” cells. ^$ P <0.05 (E). Experiments in this figure were repeated five times with similar results obtained. Bar= 50 μm (B and G).

Figure 5

Forced miR-1203 protects human endometrial cells from OGDR via silencing CypD. The stable T-HESC cells with the CRISPR/Cas9-CypD-KO construct (“CypD-KO” cells) were infected with microRNA control lentivirus (“lv-miRC”), pre-miR-1203-encoding lentivirus (“lv-pre-miR-1203”), or the pre-miR-1203 anti-sense lentivirus (“lv-antagomiR-1203”), with puromycin selection the stable cells established. These cells and the CRISPR/Cas9 vector control cells (“Cas9-C”) were subjected to OGDR for 24h, cell survival and necrosis were tested by CCK-8 assay (A) and LDH release assay (B), respectively, with miR-1203 (C) and CypD protein (D) expression respectively examined by qPCR and Western blotting assays. The lv-pre-miR-1203-expression stable T-HESC cells were further transfected with or without the UTR-depleted CypD construct (“+UTR-null CypD”), after 48h CypD mRNA and protein expression in these cells and also in lv-miRC-expressing control cells was shown (E); Cells were subjected to OGDR for 24h, cell survival and necrosis were respectively tested by CCK-8 (F) and LDH release (G) assays, with miR-1203 (H) expression examined by qPCR. The primary human endometrial cells, with/without cyclosporin A (CsA, 10 μM) pre-treatment, were infected with lv-miRC, lv-pre-miR-1203, or lv-antagomiR-1203. After 48h cells were treated with OGDR for indicated time periods, cell necrosis and miR-1203 expression were tested by LDH (I) and qPCR (J) assays, respectively. Data were presented as mean ± SD (n=5), and results were normalized. * P <0.05 vs. OGDR treatment in “Cas9-C” cells (A–C). * P <0.05 vs. “lv-miRC” cells (E). ^# P <0.05 (F and G). * P <0.05 vs. “Mock” treatment (H). * P <0.05 vs. OGDR treatment with DMSO (vehicle control) pretreatment (I and J). Experiments in this figure were repeated four times with similar results obtained.