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 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 CypDmRNA (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).