| Objective: Preeclampsia(PE)is a disease specific to pregnancy and one of the leading causes of maternal and neonatal death.To date,the pathogenesis of PE has not been understood.The literature reports that preeclampsia is a placental ischemic disease,which is due to uterine-placental blood circulation disorders and placental dysfunction,resulting in persistent placental ischemia and hypoxia and the release of a large number of cytokines into the blood circulation,ultimately triggering maternal manifestations of various diseases.Preeclampsia is closely related to the degree of trophoblast infiltration and also to the abnormal recasting of uterine spiral arterioles.Good trophoblastic soak Run,uterine spiral arteriole remodeling,so that maternal blood fully perfused in the villi space is the basis of the effective establishment of uterine-placental circulation,is the key to the success of pregnancy.Trophoblasts are the main cell types of the placenta,and their biological behavior is very similar to that of tumor cells,with the ability of normal differentiation,proliferation,apoptosis,migration and infiltration,which is the key to embryo implantation and placenta formation.The abnormal function of trophoblast cells is closely related to the pathogenesis of preeclampsia.and the changes of cell function in vivo,mainly rely on the signal conduction pathway to transmit molecular information to achieve.micro RNA(miRNA)is a group of about 18? single-stranded noncoding small RNA molecules of 23 nucleotides.In recent years,more and more studies have found that when preeclampsia occurs,multiple miRNA abnormal expression is closely related to the occurrence of trophoblast invasion,migration and other functional disorders.Our group's previous study found that Lin28 B as an RNA-binding protein,this stem cytokine showed low expression in placental tissue of preeclampsia patients.Overexpression of lin28 B in vitro cell cultures significantly increased HTR-8/svneo cell invasion.But the mechanism of lin28 B involvement in cell invasion is unclear.Access to a large number of foreign literature,found that miR-92 b is closely related to many tumors.In the study of breast tumor,it was found that overexpression of Lin28 B could upregulate the expression of miR-92 b,thus promoting the invasion and metastasis of tumor cells.Through biological information and other prediction methods,it was found that miR-92 b complemented some of the sequences in the DKK1 gene.DKK1 has been identified as the target gene of miR-92 b through dual luciferase reporter gene system.Therefore,we speculate that Lin28 may be used for DKK1 through miR-92 b to inhibit the invasion of trophoblasts and fetuses discvascular development and participate in the occurrence of preeclampsia.There are no relevant studies reported on the involvement of Lin28/miR-92 b in preeclampsia.Therefore,this study tries to explore whether the expression of Lin28/miR-92 b in preeclampsia placental tissue is reduced,whether it is related to preeclampsia condition and poor maternal and child outcomes,and whether it can participate in preeclampsia by negatively regulating the mechanism of DKK1,such as Wnt/?-catenin signaling pathway.Methods: In this study,the placental tissues of 40 preeclampsia groups(20 preeclampsia cases with early hairstyle and 20 late hairstyles)and 20 control groups(20 early hairstyle control groups with placental previa complicated with placental lineage disease,requiring early termination of pregnancy,20 late normal pregnancy)were first collected,and the clinical case data were recorded and the relevant factors were analyzed.the expression levels of placental miR-92 b in each tissue were detected by real-time PCR(q RT-PCR)method.In vitro experiments,HTR-8/svneo trophoblast lines were selected to construct Lin28 B overexpression and knock down chronic diseases.Toxic expression vector,after identifying the expression efficiency,the cell proliferation ability was detected by MTT test,the apoptosis was detected by flow cytometry,the migration ability was detected by scratch test,and the invasion ability of cells was detected by Matrigel invasion chamber experiment to judge the effect of Lin28 B on the biological behavior of trophoblast.Furthermore,the effect of Lin28 B change on miR-92 b expression was detected by q RT-PCR,and the expression efficiency of miR-92 b overexpression and knockdown lentivirus vector was determined.After co-infection of Lin28 B and miR-92 b,q RT-PCR determined whether Lin28 B expression can be reversed by miR-92 b.Finally,the expression of DKK1,Wnt/?-catenin signaling pathway was detected by q RT-PCR and Western Blot to explore the relationship between Lin28B/miR-92 b and preeclampsia.Supernatant of supernatant of trophoblast cell line HTR-8/svneo in vitro using overexpression and silencing lin28 B,respectively,acted on in vitro cultured Huvecs.the ability of tube formation of Huvecs cells was observed under microscope.Using RNA binding protein immunoprecipitation(RIP)and double luciferase reporter gene experiments identified lin28 b,mir-92 b interaction sites.Results: The pre-study of our study group has shown that the expression of Lin28 B in placental tissue of preeclampsia patients is less than that of normal placental tissue,and this study detected low expression of miR-92 b in placental tissue of preeclampsia patients(P <0.05).After constructing Lin28 B lentivirus overexpression and knockdown vector,knockdown Lin28 B could inhibit the proliferation of HTR-8/svneo trophoblast cells,whereas overexpression Lin28 B could enhance the proliferation of HTR-8/SVNeo trophoblast cells(P <0.05),Overexpression Lin28 B could enhance the migration and invasion of HTR-8/svneo trophoblast cells,but Lin28 B could inhibit the apoptosis of HTR-8/svneo trophoblast cells(P <0.05).The migration and invasion ability of the trophoblasts(P <0.05).To further explore the relevant mechanisms,the expression of miR-92 b decreased when Lin28 B knocked down,the expression of miR-92 b increased when Lin28 B overexpressed,and the trend of changes of miR-92 b was consistent with that of Lin28B(P <0.05).After constructing Lin28 B overexpression and knocking down lentivirus vector,the overexpression of Lin28 B and miR-92 b mimics and miR-92 inbithior co-transfected trophoblast cells,the expression of Lin28 B could not be miR-92 b mimics and miR-92 b inhibitor,in contrast,the expression of Lin28 B will not be knocked down by miR-92 b mimics and miR-92 b inhibitor(P <0.05).After co-transfection with knock-down Lin28 B vector.At the same time,the RNA and protein levels of dkk1,wnt/?-catenin signaling pathways will all change with the amount of mir-92 b,thus exerting biological effects.Inhibition of miR-92 b increases DKK1 expression,thereby inhibiting Wnt/?-catenin expression,and then promote the occurrence of preeclampsia.In contrast,overexpression of miR-92 b reduced the expression of DKK1,thereby increasing the expression of Wnt/?-catenin,and then promoting the invasion of trophoblasts and reducing the occurrence of preeclampsia(P <0.05).The supernatant of the supernatant of the in vitro cultured trophoblast strain HTR-8/svneo overexpressing lin28 B acted on the cultured Huvecs in vitro,and a significant increase in angiogenesis amount and length was observed.Instead,silencing Lin28 B in vitro cultured trophoblast cell line HTR-8/Svneo supernatant.The supernatant acting on HUVECs cultured in vitro observed a significant decrease in angiogenesis quantity and length(P <0.05).Lin28 B,miR-92 b interaction sites were identified using RNA-binding protein immunoprecipitation(RIP)and dual luciferase reporter gene experiments.Conclusion: This study first reported the relationship between Lin28B/miR-92 b and preeclampsia.The decrease of miR-92 b expression in preeclampsia placental tissue was consistent with the decrease of Lin28 B expression in preeclampsia placental tissue,indicating that both of them were involved in the occurrence of preeclampsia.Overexpression of lin28 B promoted HTR-8/svneo trophoblast proliferation,apoptosis,migration,and invasiveness,but silencing lin28 B reduced trophoblast proliferation,apoptosis,migration,and invasion.Lin28 B regulates miR-92 b positively in trophoblast lines,and overexpression of Lin28 B can activate the Wnt/?-catenin signaling pathway,and silencing Lin28 B can inhibit this pathway.HTR-8/Svneo co-transfected miR-92 b mimics and miR-92 b in overexpression or silencing of Lin28 B.After inbibitor,the Wnt/?-catenin signaling pathway was altered by miR-92 b and not by the change of Lin28 B,which indicated that Lin28 B played a biological effect through miR-92 b and was involved in the occurrence and development of preeclampsia.Overexpression of Lin28 B can promote angiogenesis and thus reduce the occurrence of preeclampsia,whereas silencing Lin28 B can reduce angiogenesis and promote the occurrence of preeclampsia.The action sites of miR-92 b and Lin28 B were first identified by luciferase reporter gene assay experiments. |
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