The Role Of MiR-7641-mediated Trophoblast Cell Ferroptosis And Biological Behavior In The Pathogenesis Of Preeclampsia

Backgroundpreeclampsia（PE）is a pregnancy-specific hypertension disease that endangers the health of mothers and infants.Poor placentation caused by insufficient remodeling of spiral arteries and excessive oxidative stress are considered key events in the pathogenesis of PE.Ferroptosis is a newly discovered programmed cell death form,characterized by iron-dependent lipid peroxidation.Previous studies have reported the involvement of trophoblast ferroptosis in the pathogenesis of PE,but the specific mechanism is still unclear.micro RNA（miRNA）is an important post-transcriptional regulatory factor widely involved in the regulation of many pathophysiological processes including ferroptosis.Whether miRNA participates in the pathogenesis of PE by regulating trophoblast ferroptosis remains elucidated.In this dissertation,we aim to address this issue.ObjectiveThe study aimed to substantiate the association between ferroptosis and early-onset severe preeclampsia（EO-SPE）;screen miRNAs that are featured in ferroptosis and explore their potential roles in the pathogenesis of PE.The study provided a new perspective for the study on the pathogenesis of PE.Methods1.Molecules featured in ferroptosis pathway were detected.First,the level of malondialdehyde（MDA）and the activity of glutathione peroxidase（GPx）were detected using assays;the expression levels of key molecular proteins in ferroptosis were detected by western blotting and immunohistochemical stainingand then were compared between placentae of normal pregnancy and EO-SPE.2.In order to screen trophoblast cell lines sensitive to ferroptosis for further study,Erastin-induced cell death was constructed,with CCK8 assay confirming the cell viability.Differentially expressed miRNAs in the selected cell line were screened by miRNA sequencing,and were subsequently verified by q RT-PCR.Next,the expression of ferroptosis-related miRNAs in placenta tissue were detected by q RT-PCR.The intersected differentially expressed miRNAs were deemed potential key molecules that may be involved in the pathogenesis of PE.3.Cell model of trophoblast oxidative stress was constructed by H₂O₂.Subsequent cell death type was confirmed by inhibitors targeting different types of cell death.CCK8 assay was used to detect the reversal effects of inhibitors on trophoblast activity.Next,in order to evaluate ferroptosis in cell model of trophoblast oxidative stress induced by H₂O₂,MDA kit and C11-BODIPY staining were used to detect the level of lipid peroxidation,mitochondrial membrane potential was labeled by Mito Tracker^TM,and the expression level of key molecular proteins in ferroptosis was detected by western blotting.4.Next,we sought to evaluate the regulatory effect of miR-7641 on ferroptosis.Modifications of miR-7641 was achieved by transfection of mimic and inhibitor in the selected cell line.The cell viability was detected by CCK8;the level of lipid peroxidation was detected using MDA kit and C11-BODIPY staining;western blotting was used to check the expression level of key molecular proteins in ferroptosis.5.Target Scan was applied to predict the target gene of mir-7641;dual luciferase reporter gene assay was used to verify the targeting relationship between miR-7641 and the target gene;q RT-PCR and western blotting were used to detect the expression change of target gene after miR-7641 intervention.Transfection of over-expression plasmid was intended to block the regulation of miR-7641 on target gene.In order to substantiate the regulatory effect of miR-7641 on ferroptosis that is supposed to be mediated by the target gene,cell viability was detected by CCK8 assay;the level of lipid peroxidation was detected using MDA kit and C11-BODIPY staining;the expression levels of key molecular proteins in ferroptosis were checked by western blotting.6.After Modifications of miR-7641 through mimic and inhibitor transfection,cell proliferation was detected using CCK8 assay,apoptosis by FITC Annexin V/PI staining,and migration and invasion by transwell to assess the regulatory effect of miR-7641 on the biological behavior of trophoblast.Result1.The level of ferroptosis was higher in placentae of EO-SPE than those of control group.2.JAR cell line was sensitive to Erastin,an ferroptosis inducer.Differentially expressed miRNAs featured in Erastin-induced ferroptosis were miR-3178,miR-3195 and miR-7641,among which the level of miR-7641 was decreased in EO-SPE placentae.3.There was excessive ferroptosis in cell model of trophoblast oxidative stress induced by H₂O₂.4.miR-7641 mimic inhibited trophoblast ferroptosis induced by H₂O₂,whereas miR-7641 inhibitor aggravated trophoblast ferroptosis induced by H₂O₂.5.Cullin3（CUL3）was the predicted target gene of miR-7641.Co-transfection of CUL3 over-expression plasmid and miR-7641 mimic reversed the inhibitory effect of miR-7641 mimic on CUL3 expression and ferroptosis,as well as the up-regulation of miR-7641 mimic on nuclear factor erythroid 2-like 2（NFE2L2/Nrf2）expression.6.miR-7641 mimic inhibited trophoblast apoptosis,but promoted its invasion and migration;miR-7641 inhibitor promoted trophoblast apoptosis,but inhibited its invasion and migration.ConclusionmiR-7641 negatively regulates trophoblast ferroptosis by targeting CUL3/Nrf2.In addition,miR-7641 plays a protective role in the pathogenesis of PE by inhibiting trophoblast apoptosis and promoting its migration and invasion.To recap,low expression of miR-7641 in placentae might promote the onset and development of EO-SPE by inducing ferroptosis of trophoblast and negatively regulating its biological function.