| Pre-eclampsia(PE)is a severe complication of pregnancy.It is defined as new-onset hypertension after 20 weeks of gestation,with or without proteinuria.Pre-eclampsia is one of the leading causes of increased maternal and fetal morbidity and mortality during pregnancy,with an annual incidence of 5% to 8% worldwide.This systemic syndrome of pregnancy affects multiple systems in the body in severe cases,including the nervous system,circulatory system,respiratory system,digestive system,urinary system,and blood system.Clinically,hypertension and proteinuria are the main manifestations.Cardiovascular and cerebrovascular accidents,pulmonary edema,liver and kidney dysfunction,Hellp syndrome,coagulation dysfunction,and DIC can occur in severe cases.Patients with severe pre-eclampsia have severe clinical symptoms and are prone to pre-or postpartum cardiovascular and cerebrovascular accidents,placental abruption,postpartum hemorrhage,eclampsia,or intrauterine stillbirth.However,the current clinical treatments for pre-eclampsia are minimal,and the only effective method is to control blood pressure and terminate pregnancy in time.These approaches may lead to multiple adverse outcomes for mother and child,including preterm birth,neonatal wet lung,neonatal multiple organ dysplasia,and even death.The high cost of treatment and poor prognosis result in severe family and social burdens.Understanding the pathogenesis of pre-eclampsia,exploring effective clinical treatment methods,and formulating prevention and treatment strategies are enormous challenges faced by obstetrics and cannot be delayed.To date,the exact pathogenesis of pre-eclampsia remains unclear.The academic community believes that the onset of pre-eclampsia is multifactorial and multi-mechanisms co-occur or staggered.The dysfunction of placental trophoblast cells and defective spiral artery remodeling is the primary pathological basis of pre-eclampsia.Understanding the differentially expressed molecules in the placenta of pre-eclampsia and normal control groups and exploring their correlation with pre-eclampsia’s clinical pathogenesis will help us find early diagnosis methods and treatment strategies for pre-eclampsia.Circular RNAs have been a research hotspot in the field of non-coding RNAs in recent years.High stability and exemplary conservation are the main characteristics of such non-coding RNAs,suitable for use as diagnostic markers and therapeutic targets for diseases.According to the pre-placental circular RNA microarray data,circ DLG1 was screened.circ DLG1 was significantly reduced in pre-eclampsia and localized in the basal plate of the maternal-fetal interface.In vitro experiments interfered with the expression of circ DLG1,and the invasion,migration,and epithelialmesenchymalization levels of trophoblast cells were significantly changed.It is suggested that there is an essential link between circ DLG1 and the pathogenesis of pre-eclampsia.We propose the circ DLG1/miR-421/m TOR hypothesis based on competing for endogenous RNAs’ mechanism.Through a series of experiments,it is proved that circ DLG1 is involved in one of the pathogenesis of pre-eclampsia by affecting the biological behavior of trophoblast cells.The preliminary clinical diagnosis basis and prevention strategies provide new ideas.Aim:Through circular RNA microarray analysis,the differentially expressed circular RNAhsa＿circ＿0068697(circ DLG1)in the placenta of preeclampsia patients and the placenta of normal pregnant women was screened;the circular structure of circ DLG1 was identified and clarified;the expression location of circ DLG1 in the placenta was clarified and subcellular localization in trophoblasts;collect clinical specimens to analyze the clinical correlation between circ DLG1 and the onset of preeclampsia;in vitro experiments explore the effects of circ DLG1 on trophoblast migration and invasion ability,epithelial-mesenchymalization;bioinformatics predict circ The mechanism of DLG1 affecting the biological behavior of trophoblast cells;to explore the mechanism of circ DLG1 affecting trophoblast cell migration and invasion ability and epithelial-mesenchymalization.Methods:In this study,placental tissue of severe preeclampsia pregnant women who underwent cesarean section was selected(a control group was also set).Microarray analysis was performed;PCR,q RT-PCR,and Sanger-aeq were used to determine the circular structure of circ DLG1;in situ hybridization was used to determine the expression localization in the placenta and subcellular localization in trophoblasts;q RT-PCR was used to determine the role of circ DLG1 in eclampsia.Expression level in the placenta of patients with preeclampsia;clinical data were collected,and univariate and multivariate regression analysis was used to identify the clinical correlation between circ DLG1 and the onset of preeclampsia;HTR-8/Svneo human trophoblast cells were cultured;overexpression plasmid and short hairpin RNA transfected HTR-8/Svneo cells;Western blotting to determine the expression levels of epithelialmesenchymalization-related proteins;transwell chamber and scratch experiments to determine the effect of circ DLG1 on trophoblast migration and invasion;bioinformatics analysis of the downstream factors of circ DLG1,and a network map;Bioinformatics analysis,dual luciferase reporter gene,RNA pull down experiment to explore the binding relationship between circular RNA and downstream targeted micro(miR-421),micro RNA and m RNA;using plasmid transfection/co-transfection HTR-8/Svneo cells;q RT-PCR cells to detect the expression of related genes;Western blotting to detect the expression levels of epithelial-mesenchymalization-related indicators;transwell chamber and scratch experiments to determine the effect on trophoblast migration and invasion;SPSS,Graphpad Prism are used for data analyze.Results:According to the microarray data,the FC ratio was greater than 2 and the p was less than 0.01 as the screening criteria to screen circ DLG1;circ DLG1 is a circular RNA formed by back-splicing of exons 15-20 of DLG1;The expression level in the placenta of patients with preeclampsia was significantly lower than that in the control group;circ DLG1 was associated with the onset of preeclampsia;circ DLG1 was expressed in the cytoplasm of trophoblasts;in trophoblasts,overexpression of circ DLG1,HTR-8/Svneo cells invaded,migrated and epithelialized Interstitialization was significantly enhanced;inhibition of circ DLG1,HTR-8/Svneo cell invasion,migration and epithelial-mesenchymalization were significantly attenuated;according to the "ce RNA" theory,the downstream micro RNAs and m RNAs of circ DLG1 were predicted using bioinformatics;circ DLG1 had miR-421 binding site,overexpression of circ DLG1,the expression level of miR-421 was significantly reduced;inhibition of circ DLG1,the expression level of miR-421 was significantly increased; overexpression of miR-421,HTR-8/Svneo cells migration and invasion ability,epithelial-mesenchymal Compared with sh-circ DLG1 group,the migration,invasion and epithelial-mesenchymalization of HTR-8/Svneo cells were rescued after cotransfection of sh-circ DLG1 and miR-421 inhibitor;compared with sh-circ DLG1 group,After co-transfection of sh-circ DLG1 and m TOR,the migration,invasion and epithelial-mesenchymalization of HTR-8/Svneo cells were rescued;after inhibition of circ DLG1,m TOR phosphorylation level was significantly reduced,and cotransfection of sh-circ DLG1 and miR-421 This effect was partially rescued after inhibitor or sh-circ DLG1 and m TOR.Conclusion:The expression level of circ DLG1 was significantly reduced in the placenta of preeclampsia,which may be directly related to the pathogenesis of preeclampsia.As a competitive endogenous RNA,circ DLG1 can regulate the expression of m TOR by binding to miR-421,thereby promoting the migration,invasion and epithelialmesenchymalization of trophoblast cells. |
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