MicroRNA-1203 Targets And Silences Cyclophilin D To Protect Human Endometrial Cells From Oxygen And Glucose Deprivation/re-oxygenation

Background and aims.In obstetric clinic practice,postpartum hemorrhage is one of the common complications,which can cause moderate to severe ischemic damage of endometrium.However,ischemia of endometrium is often accompanied by reperfusion,which can lead to obvious oxidative damage of human endometrial cells.At the molecular level,ischemia-reperfusion can lead to the production of ROS and the over oxidative damage of endometrial cells,resulting in the production of circulating lipid peroxides and the reduction of various antioxidants.These factors will lead to further DNA breakage,protein damage and mitochondrial dysfunction,and eventually lead to endometrial cell and tissue death.It has been confirmed that cyclophilin D(Cyp D)-dependent programmed necrosis pathway mediates the cytotoxic effect of OGDR on human endometrial cells.In vitro experiments,oxygen and glucose deprivation(OGD)and subsequent reoxygenation(OGDR)are often used to act on cultured endometrial cell models to simulate ischemia-reperfusion and oxidative damage.Micro RNAs(mi RNAs)are endogenous non coding RNAs(nc RNAs)with about 22 nucleotides(NT).Mi RNA has been proved to be widely involved in the occurrence and development of various human diseases.Mi RNAs can inhibit the translation and / or expression of target m RNA by binding directly to the 3'-untranslated region(3'-UTR).In this paper,we will describe a new identified Cyp D targeting mi RNA,microrna-1203(mi R-1203).Our results further show that mi R-1203 targets and silences Cyp D to protect human endometrial cells from OGDR induced programmed necrosis.Methods.The binding sites of mi R-1203 and OGDR 3'-UTR were predicted and confirmed by three kinds of bioinformatics prediction softwares(targetscan,mi RBase and mir DB),and the wild type(WT)or two mutants(Mut1 / 2)mi R-1203 and OGDR were transfected by human endometrial cell line T-HESC luciferase reporter gene 3'-UTR binding;construction of pre-mi R-1203 lentivirus overexpression and interference vectors(lv-pre-mi R-1203 / lv-mirc and lv-antago mi R-1203 / antac)in vitro,transfection of T-HESC and human primary endometrial cells,real-time quantitative polymerase chain reaction(q PCR)to verify the expression level of mi R-1203 and Cyp D m RNA,The protein expression of Cyp D was detected by Western blotting.The level of ROS was measured by flow cytometry(FCM)with DCFH-DA(2 ',7'-dichlorofluorescein acetate),mitochondrial depolarization with JC-1 green fluorescent probe,and CCK-8(cell counting kit-8)after OGDR induced overexpression of mi R-1203 and inhibition of T-HESC cell lines and human primary endometrial(cellscountingkit-8),Western blotting and CCK-8 were used to detect the release of cytochrome C and LDH.At the same time,the expression of mi R-1203 and Cyp D m RNA was detected by OGDR at different time points(6h,12 h,18h,24h);T-HESC cells were pretreated with ROS scavenger NAC(N-acetylcystein,500 ? m,1H)and LV antago CCK-8 was used to detect cell viability and LDH release in culture medium to detect cell necrosis.CRISPR / cas9 was used to knock out Cyp D(Cyp D-KO)in T-HESC cells completely,then overexpression or inhibition of mi R-1203 expression and construction of UTR-deleted Cyp D vector(UTR-null Cyp D)to transfect mi R-1203 overexpression T-HESC After 24 hours of OGDR treatment,CCK-8 was used to detect cell viability,LDH release was used to detect cell necrosis,mi R-1203 expression was detected by q PCR,and Cyp D protein expression was detected by Western blotting;in primary human endometrial cells,after overexpression and inhibition of mi R-1203 expression,Cs A pretreatment,OGDR 24 hours induction,LDH release was used to detect cell necrosis,and mi R-1203 expression was detected by q PCR.Results.Through biogenic prediction and luciferase reporter gene,we found that mi R-1203 targeting the 3'-UTR of Cyp D(position 806-813),T-HESC cell line and human primary endometrial cells overexpression of mi R-1203 significantly inhibited the m RNA and protein expression levels of Cyp D,on the contrary,inhibition of mi R-1203 significantly increased the expression level of Cyp D.Overexpression of mi R-1203 in endometrial cells alleviates OGDR-induced programmed necrosis,inhibits the association of mitochondrial Cyp D-p53-ant1(Adenine nucleotide translocator),mitochondrial depolarization,ROS production and lactate dehydrogenase release.In contrast,OGDR-induced programmed necrosis and cytotoxicity increased with the suppression of mi R-1203 in endometrial cells.In addition,overexpression or inhibition of mi R-1203 did not alter the OGDR-induced T-HESC cytotoxicity of Cyp D knockout or inhibition.Finally,when transfected with UTR-deficient Cyp D overexpression vectors,mi R-1203 overexpression did not protect OGDR-induced cell damage.Conclusion.These results suggest that mi R-1203 protects endometrial cells from OGDR by targeting and silencing Cyp D,and that exogenous drive of mi R-1203-Cyp D cascade may be a novel strategy for protecting endometrial cells from OGDR.