Research Paper Volume 12, Issue 3 pp 3010—3024

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

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 (AC). * 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.