The Mechanism Of Circullar RNA VRK1 In Modulating Trophoblast Cell Biological Progression In Preeclampsia

Preeclampsia refers to new hypertension after 20 weeks of pregnancy,with or without proteinuria,headache,vertigo,nausea,vomiting,epigastric discomfort,and other symptoms.It is a serious worldwide obstetric emergency,with an annual incidence rate of 2%-8% worldwide,which is the main cause of the increase in morbidity and mortality in pregnant women and newborns.There is currently no effective treatment for preeclampsia,and rapid delivery is the only treatment.The consequences of this treatment include premature fetal birth,fetal development failure,and damage to multiple organs of the mother and fetus.The pathogenesis of preeclampsia is still unclear,and there is a lack of ideal biomarkers and therapeutic targets in diagnosis and treatment.Therefore,screening the pathogenic genes in preeclampsia,and studying its mechanism is very important for the early diagnosis and treatment of preeclampsia.Circular RNA has a high degree of conservation and specificity in the mammal.It has a high abundance of expression in tissues and is suitable as a biomarker for the diagnosis of preeclampsia.Circular RNA is believed to be involved in the occurrence of preeclampsia.Therefore,it is of great significance to further understand the role of circular RNA in preeclampsia.This study screened out a novel circular RNA through the circular RNA microarray,and explored its influence on the biological behavior of trophoblasts and its relationship with preeclampsia,hoping to help the diagnosis and treatment of preeclampsia.Part ?Analysis of circular RNA microarray in preeclampsia Aim: Through circular RNA microarray analysis,the differentially expressed circular RNAs in the placenta of preeclampsia and normal pregnancy are screened out.Expression profiles are drawn,quality control of microarray data,analysis of expression levels between samples,and biological informatics analysis including circular RNA structure analysis and classification are performed.GO function enrichment and KEGG pathway enrichment provide new biomarkers and therapeutic targets for the diagnosis and treatment of preeclampsia.Methods: In this study,we selected 10 placentas of pregnant women who underwent cesarean section in the First Affiliated Hospital of China Medical University's Obstetrics Department,and divided them into early-onset preeclampsia group and normal pregnancy group.Microarray analysis was performed to screen out significantly differentially expressed circular RNAs;Quality control analysis,cluster analysis,and expression mapping analysis on microarray data;GO function enrichment analysis and KEGG pathway enrichment were performed to analyze these differentially expressed circular RNA.Results: 1.According to the microarray data,273 differentially expressed circular RNAs were screened out based on the FC ratio greater than 2 and p less than 0.01.2.GO function enrichment shows that differentially expressed genes are mainly related to cell metabolism,cell binding,and nucleic acid.3.Enrichment of the KEGG pathway shows that differentially expressed genes are mainly related to cell adhesion and cell metabolism.Conclusion: The circular RNA microarray data screened out possible early biomarkers and therapeutic targets for the diagnosis of preeclampsia.Cluster analysis,GO function enrichment,and KEGG pathway enrichment analysis help to screen out circular RNA molecules related to the onset of preeclampsia.Part ? The influence of circVRK1 on trophoblast cell biological behavior Aim: In the differentially expressed circ RNA,combining with the FC ratio and pvalue,we aim to screen a new type of circular RNA molecule,namely circVRK1.Next,we aim to explore the ring structure of circVRK1 and its expression in the placenta of preeclampsia and analyze its correlation with the onset of preeclampsia.In vitro experiments,we explore the expression and location of circVRK1 in trophoblasts and its influence on the biological behavior of trophoblasts.Bioinformatics predicts the mechanism by which circVRK1 affects the biological behavior of trophoblast cells.Methods: qPCR was performed to confirm the expression pattern of circVRK1 in the placenta of patients with preeclampsia;HTR-8/Svneo human trophoblast cell culture;PCR,q PCR,Sanger sequencing to confirm the ring structure of circVRK1;immunofluorescence to verify the expression location of circVRK1 in trophoblast cells;small interfering RNA and short hairpin RNA transfection of HTR-8/Svneo cells;invasion experiments ane scratch experiments to clarify the influence of circVRK1 on the biological behavior of trophoblast cells;western blot to clarify the expression level of related proteins;Targetscan,mi Randa,and circinteractome analyze downstream factors are used to predict downstream molecules;SPSS and Graphpad Prism for data statistics and charting.Results: 1.The expression level of circVRK1 in the placenta of preeclampsia patients was significantly higher than that of normal pregnant women.2.circVRK1 is a circular RNA that is back spliced by exons 2-11 ofVRK1.3.circVRK1 is widely expressed in the cytoplasm of trophoblasts.4.Knockdown the expression of circVRK1,the migratory and invasive ability of HTR-8/Svneo cells was significantly enhanced.5.Knockdown the expression of circVRK1,and HTR-8/Svneo cells transform from epithelial cell phenotype to mesenchymal cell phenotype(EMT).6.According to the "ce RNA" theory,bioinformatics is used to predict the downstream micro RNA and m RNA of circVRK1.Conclusion: The expression level of circVRK1 in the placenta of preeclampsia is significantly increased,which may be directly related to the onset of preeclampsia.circVRK1 is an exon-type circular RNA expressed in the cytoplasm.circVRK1 inhibits the migration,invasion,and EMT of trophoblasts.Part ?: The mechanism of circVRK1 modulating the biological behavior of trophoblasts Aim: To explore the mechanism of circVRK1 affecting the progression of preeclampsia.Methods: Bioinformatics analysis,RNA pull-down experiment,dual-luciferase reporter gene to verify the downstream targeted micro RNA;small interfering RNA,short hairpin RNA,miRNA mimics,miRNA inhibitor,plasmid to co-transfection HTR-8/Svneo cells;q PCR to detect the expression of the related genes;immunofluorescence experiment to clarify the expression location of circVRK1 and downstream micro RNA;invasion experiment and scratch experiment to clarify the influence on the biological behavior of trophoblast;western blot to detect the expression level of related proteins.Results: 1.circVRK1 has a mi R-221-3p binding site,knockdown circVRK1,the expression level of mi R-221-3p is significantly increased.2.After the overexpression of mi R-221-3p,the migratory and invasive ability,and EMT of HTR-8/Svneo cells are significantly elevated.3.circVRK1 and mi R-221-3p are widely expressed in the cytoplasm of trophoblasts.4.After co-transfection with circVRK1 knockdown and mi R-221-3p inhibitor,compared with circVRK1 knockdown group,the migration,invasion,and EMT of HTR-8/Svneo cells were reversed.5.After co-transfection with circVRK1 knockdown and PTEN,the migratory and invasive ability,and EMT of HTR-8/Svneo cells were partially reversed compared with the circVRK1 knockdown group.6.After knocking down circVRK1,the Akt phosphorylation level was significantly increased.After co-transfection with circVRK1 knockdown and mi R-221-3p inhibitor or circVRK1 knockdown and PTEN,this effect was partially reversed.Conclusion: circVRK1,as a competitive endogenous RNA,can inhibit the expression of PTEN and phosphorylation of Akt by binding mi R-221-3p,modulating the migration,invasion,and EMT process of trophoblasts.