| Background:As a common hypertensive disorder during pregnancy,preeclampsia(PE)poses a serious threat to maternal and infant health.Its pathogenesis is related to apoptosis mediated shallow invasion of trophoblast cells,poor remodeling of maternal spiral arteries,oxidative stress damage,and inflammatory response,etc.The placenta plays a central role in the occurrence and development of PE.Ferroptosis is a newly discovered mode of cell death driven by iron-dependent lipid peroxidation.Recent studies have shown that ferroptosis is closely related to the occurrence and development of PE.Numerous evidence suggests that miRNAs are involved in the occurrence and development of PE and are reliable biomarkers for the diagnosis and treatment of PE.In other disease models,miRNAs have been proven to regulate ferroptosis,but there is currently a lack of evidence that miRNAs regulate ferroptosis and participate in the occurrence and development of PE.In this study,we first screened miR-2115-3p associated with ferroptosis in the PE pregnant mouse model,and then clarified the role and molecular mechanism of miR-2115-3p in regulating ferroptosis in the PE at both in vivo and in vitro levels.Finally,the possibility of exosomal miR-2115-3p inhibiting ferroptosis in the PE pregnant mouse and improving the outcome of PE was explored.Chapter Ⅱ.The role of ferroptosis in the pathogenesis of PE and the screening of related miRNAsObjective:To clarify the important role of ferroptosis in PE,and to identify the potential target miRNAs that regulate ferroptosis in PE.Methods:Reduced uterine perfusion pressure(RUPP)was used to establish a rat model of PE.PE rats were treated with ferrostatin-1,a specific ferroptosis inhibitor,and the blood pressure,uterine morphology,and fetal survival rate were evaluated.At the same time,the level of urine protein was detected by Coomassie brilliant blue(CBB)method,and the level of serum sFlt-1 was detected by enzyme-linked immunosorbent assay(ELISA).The GSH level,GPXs activity and Fe2+ level in placental tissues were detected by colorimetry.The MDA level in placental tissues was detected by thibabituric acid(TBA)method.The placental tissues of PE patients and normal pregnant women were collected,and the differentially expressed miRNAs were analyzed by miRNAs sequencing.The expression levels of ferroptosis-related miRNAs in human placenta and umbilical cord blood were detected by RT-qPCR,and the expression levels of ferroptosis-related proteins were detected by western blot(WB).Results:1.Ferroptosis inhibitor reduced maternal blood pressure after PE.2.Ferroptosis inhibitor promoted fetal survival after PE.3.Ferroptosis inhibitor ameliorated maternal and placental damage after PE.4.Scatter plot showed that 317 miRNAs were up-regulated and 251 miRNAs were down-regulated in the placentas of PE patients compared with those of normal pregnant women(FC>1.5).5.The volcano plot showed that 67 miRNAs were significantly up-regulated and 53 miRNAs were significantly down-regulated in the placenta of PE patients compared with normal placental tissues(p<0.05,|log2(FC)|>1.0).6.Among all differentially down-regulated miRNAs,miR-130a-5p,miR-2115-3p,miR-3690,miR-615-3p,miR-582-3p and miR-665 might target genes related to ferroptosis.7.Compared with the normal group,the expression of miR-130a-5p was significantly increased in the umbilical cord blood and placenta tissues of the PE group,while the expressions of miR-2115-3p,miR-3690,miR-615-3p,miR-582-3p and miR-665 were decreased in varying degrees.Among them,the expression of miR-2115-3p was down-regulated most significantly.8.Compared with the control group,the expression level of GOT1 was significantly increased in the placenta of PE group,while the expression level of ACSL3 was not significantly changed.9.The expression of miR-2115-3p was negatively correlated with GOT1 expression in both umbilical cord blood and placenta,especially in placenta.Conclusions:1.Ferroptosis inhibitor ameliorates maternal,placental,and fetal damage after PE.2.The expression of miR-2115-3p,miR-3690,miR-615-3p,miR-582-3p and miR-665 is decreased in umbilical cord blood and placenta,while GOT1 expression is increased in placenta after PE.3.miR-2115-3p expression is negatively correlated with GOT1 expression in both umbilical cord blood and placenta.Chapter Ⅲ.The role and mechanism of miR-2115-3p in inhibiting ferroptosis of trophoblasts by targeting GOT1 in the pathogenesis of PEObjective:To clarify the regulatory effect and mechanism of miR-2115-3p on ferroptosis in extravillous trophoblasts(EVTs).Methods:Human EVTs cell line HTR-8/SVneo was constructed and cultured in vitro.Hypoxia stimulation was used to simulate PE.Dual luciferase reporter gene assay was used to determine the targeting relationship between miR-2115-3p and glutamic oxaloacetic transaminase 1(GOT1).miR-2115-3p mimic or miR-2115-3p inhibitor was used to over-express or knockdown miR-2115-3p in HTR-8/SVneo cells,and plasmid or interfering RNA was used to over-express or knockdown GOT1 in HTR-8/SVneo cells.CCK-8 method was used to detect the survival of HTR-8/SVneo cells.DCFH-DA probe method was used to detect the content of reactive oxygen species(ROS)in HTR-8/SVneo cells.The expression levels of ferroptosis-related proteins in HTR-8/SVneo cells were detected by WB.The GPXs activity and GSH level of HTR-8/SVneo cells were detected by colorimetry.The MDA level,total iron content,Fe2+ content and Fe3+ content of HTR-8/SVneo cells were detected by TBA method.Results:1.miR-2115-3p targeted GOT1 mRNA.2.GOT1 inhibited the proliferation of HTR-8/SVneo cells and promoted ROS synthesis.3.The expression of GPX4 in HTR-8/SVneo cells was significantly decreased after hypoxic stimulation,while the expression levels of GOT1,ACSL4 and TfRl were significantly up-regulated.After GOT1 knockdown,the expression of GPX4 in HTR-8/SVneo cells was restored.However,the expression levels of GOT1,ACSL4 and TfR1 were decreased in different degrees.4.The expression level of miR-2115-3p was significantly down-regulated and the expression level of GOT1 was significantly increased in HTR-8/SVneo cells after hypoxia stimulation.After overexpression of miR-2115-3p,the expression level of GOT1 was significantly decreased in HTR-8/SVneo cells.Overexpression of GOT1 had no effect on the expression of miR-2115-3p in HTR-8/SVneo cells.5.Overexpression of miR-2115-3p significantly enhanced the survival of HTR-8/SVneo cells,and DCFH-DA probe assay showed that ROS content was significantly reduced after up-regulation of miR-2115-3p in HTR-8/SVneo cells.6.Up-regulation of GOT1 reversed miR-2115-3p-induced HTR-8/S Vneo cell survival and ROS suppression.7.After overexpression of miR-2115-3p,the expression levels of GPX4 and SLC7A11 were significantly increased,while the expression levels of GOT1,ACSL4 and TfR1 were significantly decreased in HTR-8/SVneo cells.8.The up-regulation of GPX4 and SLC7A11 and the down-regulation of ACSL4 and TfRl induced by miR-2115-3p were significantly weakened by overexpression of GOT1.9.After overexpression of miR-2115-3p,GPXs activity and GSH expression in HTR-8/SVneo cells were significantly increased,and MDA content was significantly decreased.10.After up-regulation of miR-2115-3p,the total iron content and Fe2+ content in HTR-8/SVneo cells were significantly decreased,and Fe3+content was significantly increased.11.Overexpression of GOT1 abolished the miR-2115-3p-induced increases in GPXs activity,GSH expression and Fe3+ content,and the miR-2115-3p-induced decreases in MDA,iron content and Fe2+ content.Conclusions:1.miR-2115-3p targets and inhibits GOT 1.2.miR-2115-3p inhibits ferroptosis in trophoblasts after hypoxia by down-regulating GOT 1.Chapter Ⅳ.Explores the effect of miR-2115-3p on PE based on exosomes derived from mesenchymal stem cellsObjective:To clarify the possibility that miR-2115-3p inhibits ferroptosis and improves the outcome of PE through exosomes derived from mesenchymal stem cells(MSCs),and to provide new ideas for the treatment of PE with miR-2115-3p in the future.Methods:Human umbilical cord MSCs(hUC-MSCs)were cultured in vitro.The expression levels of MSCs markers were detected by flow cytometry,and hUC-MSCs exosomes were isolated.transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and WB were used to detect the expression of specific markers of exosome morphology and particle size.RT-qPCR was used to evaluate the expression level of miR-2115-3p in hUC-MSCs exosomes.Exosomes were collected from hUC-MSCs overexpressing or knockdown miR-2115-3p with miR-2115-3p mimic or miR-2115-3p inhibitor and infused back into RUPP rats.The blood pressure,uterine morphology and fetal rat survival rate were evaluated,and the urinary protein level was detected by CBB method.ELISA was used to measure the serum level of sFlt-1.Colorimetry was used to measure the GSH level,GPXs activity and Fe2+ level in placental tissue.HTR-8/SVneo cells were treated with exosomes collected from hUC-MSCs pre-labeled with PKH67 fluorescein,and the uptake of hUC-MSCs exosomes by HTR-8/SVneo cells was observed by fluorescence microscopy.The hUC-MSCs were transfected with miR-2115-3p mimic or miR-2115-3p inhibitor to over-express or knock down miR-2115-3p.HTR-8/SVneo cells were treated with exosomes and the expression level of miR-2115-3p was detected by RT-qPCR.CCK-8 method was used to detect the cell survival,DCFH-DA probe method was used to detect the ROS content of cells,WB was used to evaluate the expression level of ferroptosis-related proteins of cells,colorimetric method was used to detect the activity of GPXs of cells,colorimetric method was used to detect the GSH level of cells,TBA method was used to detect the level of MDA,total iron content,Fe2+ content and Fe3+ content of cells.Results:1.hUC-MSCs grew adherent and showed spindle shape.The expressions of CD29,CD44,CD90 and CD 105 in hUC-MSCs were positive,but the expression of CD34 and CD45 was negative.2.Exosomes showed typical saucer-like structure under TEM.NTA showed that the particle size of exosomes was mainly distributed between 30-150 nm.WB confirmed that the expressions of CD9,CD63,Alixl and TSG101 in hUC-MSCs exosomes were positive,while the expression of endoplasmic reticulum specific marker Calnexin was negative.3.miR-2115-3p was significantly enriched in hUC-MSCs exosomes,and the level of miR-2115-3p in hUC-MSCs exosomes was up-regulated or down-regulated accordingly after overexpression or knockdown of miR-2115-3p.4.After the infusion of hUC-MSCs exosomes,the blood pressure of PE maternal rats decreased,the uterus of PE rats increased significantly,the number of embryos increased significantly,and the number of dead fetuses decreased significantly.The hUC-MSCs exosomes significantly improved the survival rate of fetal rats and decreased the levels of urine protein and serum sFlt-1 in PE maternal rats.It increased GSH content and GPXs activity in placenta,and decreased Fe2+ and MDA content in placenta.5.After overexpression of hUC-MSCs exosomal miR-2115-3p,the protective effect of hUC-MSCs exosomes against PE was enhanced,and after knockdown of hUC-MSCs exosomal miR-2115-3p,the protective effect of hUC-MSCs exosomes against PE was weakened.6.Green fluorescence appeared in the cytoplasm of HTR-8/SVneo cells after treatment with hUC-MSCs pre-labeled with PKH67 fluorescein.7.The expression of miR-2115-3p in HTR-8/SVneo cells is decreased after hypoxia stimulation,and hUC-MSCs exosomes can increase the level of miR-2115-3p in HTR-8/SVneo cells.The level of miR-2115-3p in HTR-8/SVneo cells was correspondingly increased/decreased.8.Exosomes derived from hUC-MSCs can increase the survival rate of HTR-8/SVneo cells and inhibit the expression of ROS in HTR-8/SVneo cells after hypoxic stimulation.9.Up-regulation of miR-2115-3p further enhanced the effects of hUC-MSCs exosomes on promoting HTR-8/SVneo cell proliferation and inhibiting ROS expression,while down-regulation of miR-2115-3p attenuated the proliferation promotion and ROS inhibition mediated by hUC-MSCs exosomes.10.hUC-MSCs exosomes can up-regulate the expression levels of GPX4 and SLC7A11 and down-regulate the expression levels of GOT1,ACSL4 and TfR1 in HTR-8/SVneo cells under hypoxia stimulation.11.hUC-MSCs exosomes promoted GPXs activity,GSH and Fe3+contents,and inhibited MDA,total iron and Fe2+ contents in HTR-8/SVneo cells.12.Up-regulation of miR-2115-3p further enhanced the above effects of hUC-MSCs exosomes,and down-regulation of miR-2115-3p weakened the above effects of hUC-MSCs exosomes.Conclusions:1.miR-2115-3p is significantly enriched in hUC-MSCs exosomes.2.Exosomal miR-2115-3p derived from hUC-MSCs can improve the progression of PE and inhibit ferroptosis of trophoblasts after PE,and hUC-MSCs exosomal miR-2115-3p is a potential target for the treatment of PE. |
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