Mechanism By Which CircDNAJB6 Affects The Biological Behavior Of Pre-eclamptic Trophoblast Cells

Objective:Preeclampsia（PE）is a disease of pregnancy with hypertension and urinary protein as the main diagnostic factors.It is one of the leading causes of perinatal maternal and child mortality with a prevalence of 3-5%and can lead to severe multisystem organ damage and threaten maternal life.There is a lack of truly effective treatment for this disease,and maintaining maternal-fetal stability until termination of pregnancy at the appropriate time is the current management strategy for this disease.Studies have shown that the development of preeclampsia during pregnancy may lead to impaired maternal cardiovascular function in later life.In order to ensure a healthy prognosis for the mother and fetus and even the mother,it is crucial to explore in depth the pathogenesis of preeclampsia and to find truly effective molecular predictions and treatments based on its pathogenesis^[1].It has been reported that preeclampsia is a human-specific disease that is less likely to occur in other species,which stems from the fact that in humans,the mother and fetus require 60%of nutrient exchange,while other mammals require only 20%of nutrient exchange^[2].Pre-eclampsia is considered a multifactorial,multi-mechanism and multi-pathway pathogenic disease by national and international scholars.At present,researchers widely accept the"two-stage theory",that is,the first stage is preclinical,with defective trophoblast invasion and migration,resulting in impaired uterine spiral artery recasting and the release of multiple placental factors from placental ischemia and hypoxia;the second stage is the entry of placental factors into the maternal circulation,promoting the activation of systemic inflammatory response and vascular endothelial damage that causes diverse clinical manifestations of pre-eclampsia^[3].Defects in trophoblast migration and invasion are recognized as the initiating factors of preeclampsia disease.The presence of epigenetic changes in preeclampsia characterized by abnormal placental formation suggests a role for epigenetics in pregnancy complications^[4].Epigenetics are heritable genetic information changes in gene function without altering the DNA sequence of genes and ultimately lead to phenotypic changes,including DNA methylation,RNA modifications,histone modifications,nucleosome remodeling,and non-coding RNAs.There is growing evidence that epigenetics plays a crucial role in the development and progression of preeclampsia.m6A modification is an epigenetic modification modality,one of the post-transcriptional regulatory marks,and has been shown to play an important role in regulating RNA splicing,translation,stability,and translocation.Most m6A sites are located in the conserved motif DRACH（D=G or A or U,R=G or A,H=A or U or C）^[5].Insulin-like growth factor 2 m RNA binding proteins（IGF2BP1/2/3,IGF2BPs）are cytoplasmic m⁶A reading proteins that can enhance m RNA stability by binding to target transcripts via GG（m⁶A）C,a typical m⁶A motif^[6],and are involved in many disease progressions in an m⁶A-dependent manner.The m⁶A-related roles that reading proteins can play are complex and cannot exclude the role of other proteins in m RNA stability and expression regulation.IGF2BPs may interact with other m RNA-binding proteins after recognition of m⁶A sites to form large protein complexes to accomplish functional regulation of m RNA^[7].The differential expression of noncoding RNAs in preeclampsia is associated with a variety of cellular phenotypes,and this study focuses on the mechanisms underlying the role of m6A modifications and circular RNA（circ RNA）in abnormal trophoblast function in preeclampsia.Although circ RNAs are usually expressed at lower levels than their linear counterparts,circ RNAs are major transcripts for many genes,and there may be competition between classical and reverse splicing for most circ RNA-producing motifs.Whether circular properties and stability of circ RNAs are associated with m⁶A modifications and whether they are involved in the pathogenesis of preeclampsia is also This paper is also a study.Recent studies on circ RNAs and preeclampsia have focused on"circ RNAs affect trophoblast function and are involved in the development of preeclampsia".In this study,we screened the circ RNA microarray dataset of preeclampsia,and circ DNAJB6 was selected by co-analysis.The aim of this experiment was to investigate the effect of circ DNAJB6 on trophoblast cell function,and to explore its role in the development of preeclampsia.Methods:I.Expression and identification of circ DNAJB6 in placenta of preeclampsia1.Download circ RNA microarray datasets of different placental tissues of preeclampsia patients and control normal tissues from GEO database,use bioinformatics analysis,screen differentially expressed genes（DEGs）,jointly analyze differential genes,and perform parental genes of differentially expressed circ RNAs for GO functional enrichment and KEGG pathway enrichment,and prediction of potential sponge-binding mi RNAs.2.Collected pre-eclamptic and control normal placental tissues to validate the expression levels of circ RNA and its host genes in pre-eclamptic placental tissues screened from the microarray dataset.3.PCR and Sanger sequencing to validate the circular structure of circ DNAJB6.4.RNase R assay and actinomycin D assay for stability analysis of circ DNAJB6.5.FISH to detect the cellular and tissue localization of circ DNAJB6.6.Nucleoplasmic isolation assay to detect the expression of circ DNAJB6 in different cellular components.II.m⁶A reading protein IGF2BP1 mediates circ DNAJB6 stability enhancement affects trophoblast function1.Construct and synthesize circ DNAJB6 si RNA.2.Cultivate HTR8/SVneo cells,transfect circ DNAJB6 si RNA-1/2/3 and control group into HTR8/SVneo cells,and verify the construction of circ DNAJB6 interference trophoblast cell model by PCR.3.The Cell migration and invasion function of circ DNAJB6 si RNA trophoblasts were detected by Transwell and cell scratch test.4.Western Blot assay was used to detect epithelial stromal phenotype related proteins.5.Construct and synthesize DNAJB6 si RNA.6.Cultivate HTR8/SVneo cells,transfect DNAJB6 si RNA and control group into HTR8/SVneo cells,and verify the construction of DNAJB6 interference trophoblast cell model by PCR and Western Blot.7.The Cell migration function of DNAJB6 si RNA trophoblasts was detected by cell scratch test.8.Western Blot assay was used to detect epithelial mesenchymal phenotype related proteins in DNAJB6 si RNA trophoblast cells.9.Predict the m6A site of circ DNAJB6.10.Detect the overall m6A level of preeclampsia diseases.11.RNA pull down and mass spectrometry were used to detect the possible binding proteins of circ DNAJB6,verifying the binding relationship between IGF2BP1 and circ DNAJB6.12.Me RIP and luciferase Reporter gene experiments verified that circ DNAJB6 has m⁶A modification,and may combine with IGF2BP1 in the form of m⁶A.13.After Dactinomycin D treatment,the circ DNAJB6 level in HTR8/svneo cells interfering with IGF2BP1 was detected by PCR.14.Construct circ DNAJB6 si RNA,circ DNAJB6 si RNA+mi R-1290 mimic,and circ DNAJB6 si RNA+mi R-1290 inhibitor HTR8/SVneo cell models,and detect the expression level of mi R-1290 by PCR.15.Pull down and luciferase Reporter gene tests verified the binding relationship between circ DNAJB6 and mi R-1290.16.FISH detection for mi R-1290 and circ DNAJB6 localization.17.Database and bioinformatics prediction of potential target genes that may bind to mi R-1290.Establish a mi R-1290 mimic HTR8/SVneo cell model,and detect HIGD2A RNA and protein levels using PCR and Western Blot assays.18.Construct and synthesize the overexpression plasmid HIGD2A.19.Co transfection of mi R-1290 mimic and overexpression plasmid HIGD2A into HTR8/SVneo cells,Transwell and cell scratch test were used to detect Cell migration and invasion function,and Western Blot was used to detect the level of epithelial mesenchymal phenotype protein.20.Western blot and PCR were used to detect the expression of HIGD2A m RNA and protein levels in circ DNAJB6 si RNA cells and control group cells.21.Construct circ DNAJB6 si RNA,mi R-1290 inhibitor,and HIGD2A overexpression plasmids to transfect HTR8/SVneo cells alone or jointly.Transwell and cell scratch tests were used to detect Cell migration and invasion functions,and Western blot tests were used to detect changes in epithelial mesenchymal phenotype related proteins.Results.I.Expression and identification of circ DNAJB6 in placenta of preeclampsia1.The pre-eclampsia placenta tissue microarray dataset GSE102897 and GSE96984 were selected.After correcting the batch differences of the two datasets,Conjoint analysis was carried out.Circ DNAJB6 was screened according to the log FC value and the corrected P value.It is an up-regulated gene in pre-eclampsia placenta tissue,mainly distributed on human chromosome 7.It is formed by reverse splicing of Exon 3,4,5 of DNAJB6 located on Chromosome 7（281nt）.The parental genes of the screened differential cyclic RNA were functionally enriched.GO analysis closely related to preeclampsia placenta was mainly enriched in the structural composition of extracellular matrix and adhesion molecule binding force;KEGG pathways highly associated with preeclampsia placenta include extracellular matrix receptors,local adhesion,and PI3K/AKT pathways.Circ RNA can serve as endogenous competitive RNA and bind to proteins to function,predicting potential binding mi RNAs and proteins.The MRE elements of circ DNAJB6 can be predicted through targetscan,mi Randa,and circbank websites.2.Design specific primers that span the reverse splicing junction region of circ DNAJB6,and detect its expression in the placenta of preeclampsia through q RT PCR.The results are consistent with the microarray,and the mother gene DNAJB6 has decreased m RNA expression in the placenta tissue of preeclampsia.3.The agarose gel electrophoresis and Sanger sequencing results were consistent with the predicted reverse splicing sites of circ RNA.4.The RNase R experiment results suggest that the m RNA of the linear parent gene DNAJB6 of circ DNAJB6 is significantly reduced,while circ DNAJB6 is not digested and its m RNA level remains unchanged,confirming that circular RNA is more stable than linear parent genes;After treatment with Dactinomycin D（a transcription inhibitor）,the half-life of circ DNAJB6 is longer than that of its linear transcript,and circ DNAJB6 m RNA is more stable than that of linear DNAJB6 m RNA.5.Nuclear cytoplasmic separation experiments and FISH showed that circ DNAJB6 was expressed in the placenta and mainly localized in the cytoplasm of placental trophoblasts.II.m⁶A reading protein IGF2BP1 mediates circ DNAJB6 stability enhancement affects trophoblast function1.Cultivate HTR8/SVneo cells,transfect circ DNAJB6 si RNA-1/2/3 and control group into HTR8/SVneo cells,and verify the successful construction of circ DNAJB6 si RNA interference cell model through PCR detection.2.The Transwell experiment confirmed that circ DNAJB6 si RNA cells exhibit enhanced cellular invasion function.3.Scratch test confirmed that circ DNAJB6 si RNA cells had enhanced Cell migration ability.4.Western blot confirmed that circ DNAJB6 si RNA reduced the phenotypic proteins of epithelial cells and increased the phenotypic proteins of interstitial cells in trophoblastic cells.5.Design targeted si RNA for DNAJB6,transfect HTR8/SVneo cells,and validate the construction of DNAJB6 interference model through PCR and Western Blot.6.Scratch and invasion experiments confirmed that the Cell migration and invasion ability of DNAJB6 si RNA cells decreased.7.Western blot confirmed that the expression level of DNAJB6 si RNA trophoblast epithelial cell phenotype protein increased,while the expression level of interstitial phenotype protein decreased.8.The m⁶A locus of circ DNAJB6 was predicted,and the m⁶A modified classical RRACH motif was found in the fourth and fifth Exon.9.Detect the total m⁶A methylation level changes of circ RNA in preeclampsia samples and control placental tissues.10.Pull down and mass spectrometry were used to detect the m⁶A related protein that circ DNAJB6 may bind to,and IGF2BP1 was found to serve as the m⁶A"reader"and primarily stabilize RNA.11.Western Blot confirmed that the pull-down product of circ DNAJB6 increased the enrichment of IGF2BP1 compared to the control group.Me RIP and luciferase confirmed that IGF2BP1 binds to circ DNAJB6 in a m⁶A manner.12.Blocking cell RNA transcription with Dactinomycin D showed that knockout of IGF2BP1 in HTR-8/SVneo cells significantly shortened the half-life of circ DNAJB6.13.Predicting mi RNAs that may bind to circ DNAJB6,mi R-1290 was found to be closely related to preeclampsia and can regulate cell proliferation,migration,apoptosis,and other functions,suggesting that circ DNAJB6 and mi R-1290 may jointly play a synergistic role in the pathogenesis of preeclampsia.14.PCR experiments suggest an increase in mi R-1290 expression levels in circ DNAJB6si RNA cells.The Pull-down experiment confirmed that the circ DNAJB6 probe can specifically enrich mi R-1290 in cell lysate.The luciferase Reporter gene test showed that the luciferase activity of wild-type circ DNAJB6 was significantly inhibited in the mi R-1290 overexpression group,but the luciferase activity of circ DNAJB6 mutant had no significant difference between the mi R-1290 overexpression group and the control group.15.FISH detection of mi R-1290 and circ DNAJB6 co-located in the trophoblast cytoplasm of placental tissue.16.Mi RTar Base and other websites predict that mi R-1290 may bind to the target gene HIGD2A.17.Transfection of mi R-1290 mimic into HTR8/SVneo cells resulted in a decrease in HIGD2A RNA and protein levels detected by PCR and Western Blot assays.18.The overexpression plasmid HIGD2A was constructed and synthesized,and co transfected with mi R-1290 mimic and the overexpression plasmid HIGD2A entered HTR8/SVneo cells.Transwell and cell scratch experiments confirmed that the migration and invasion ability of mi R-1290 mimic transfected Cell migration increased,and the overexpression plasmid HIGD2A reversed this change after entering cells.19.Western blot confirmed that after transfection with mi R-1290 mimic,the epithelial mesenchymal changes in trophoblasts were altered,while overexpression of plasmid HIGD2A partially reversed the changes in EMT in trophoblasts.20.PCR and Western blot analysis showed a decrease in the expression of HIGD2A m RNA and protein levels in circ DNAJB6 si RNA cells and control group cells.21.The overexpression plasmids circ DNAJB6 si RNA,mi R-1290 inhibitor,and HIGD2A were transfected alone or co transfected into HTR8/SVneo cells.Compared with the circ DNAJB6 si RNA group,the migration,invasion,and EMT of trophoblastic cells were partially reversed after transfection with circ DNAJB6 si RNA and HIGD2A.Conclusion:DNAJB6 forms circ DNAJB6 through variable splicing in the placenta of preeclampsia,and IGF2BP1 dependent m⁶A modification enhances the stability of circ DNAJB6.Upregulation of circ DNAJB6 leads to reduced invasion and migration of preeclampsia trophoblasts,as well as changes in EMT phenotype.At the same time,downregulation of DNAJB6 inhibits trophoblast function and affects epithelial mesenchymal transition,jointly affecting the occurrence and development of preeclampsia diseases.The changes in cell phenotype and function produced by circ DNAJB6 acting on extracellular trophoblast cells may be a mechanism of self-regulation and adaptation of cells to the environment.This provides a new direction for the multi-target prediction and treatment of Circ DNAJB6 in preeclampsia.