LncRNA XIST Regulates Trophoblast Cell Biological Behavior Through The MiR-135b-5p/FOXO1 Axis

Objective:Preeclampsia(PE)is a pregnant specific disease,which is one of the main causes of maternal and fetal morbidity and mortality.Its main clinical manifestation is that after 20 weeks of gestation,there is a systemic multi-system syndrome characterized by hypertension and proteinuria.The incidence of PE is 5%-8% of pregnant women.There are many hypotheses and theories about the etiology of preeclampsia,such as abnormal immune response,oxidative stress,abnormal placental implantation,placental function,and genetic factors.Although the etiology of preeclampsia is not yet fully clear,the dominant theory tends to be the "two-stage" theory.In the first stage,excessive or atypical maternal immune response of trophoblasts during early pregnancy,and insufficient decidualization or failure of uterine preparation lead to insufficient spiral artery remodeling,thereby impairing placental function.Reduced uterine and placental blood flow and poor placental perfusion.In the second stage,hypoxia and reoxygenation caused by poor placental perfusion lead to further disorders of placental function and oxidative stress of the placenta.Then trophoblastic cell apoptosis and necrosis,systemic inflammatory response factors released into the maternal circulation.Excessive systemic inflammation results in endothelial injury and hyperactivation of blood vessels,followed by a series of multi-system clinical symptoms.Therefore,preeclampsia is a disease with impaired interaction between two independent organs of different genetic origins.So far,there are a lot of studies on the etiology and pathogenesis of preeclampsia,but scholars have not yet obtained satisfactory theory.Therefore,we are still facing difficulties in studying the pathogenesis of preeclampsia.Long non-coding RNAs(Lnc RNAs)are a series of RNA molecules that are widely transcribed from human and other mammalian genomes with length exceeding 200 nt.They were first discovered by Okazaki from mouse DNA transcripts in 2002.They participate in the biological functions of diseases mainly through the expression of genes regulated by transcription and post-transcriptional level and epigenetics.The position of relative protein coding genes,including intergenic RNA,antisense transcripts,introns,promoter-related long-chain non-coding RNA,etc.It has been reported to affect various steps of gene expression,including chromosome inactivation,gene imprinting,epigenetic regulation,nuclear plasma transport,transcriptional regulation,gene splicing and translation.At the cellular level,lnc RNA is involved in cell proliferation,differentiation,cell cycle,apoptosis and autophagy.Although the research progress of lnc RNA is rapid,the understanding of the mechanism of lnc RNA is still limited.Especially how these RNA events occur and how they target and control other RNA.Lnc RNA can be epigenetic regulated by summoning chromosome reconstitution complexes to specific gene loci.Some lnc RNAs can participate in transcription,and some of them act as antisense transcripts to regulate the dynamic balance of RNA at post-transcriptional level.In addition,many lnc RNAs can produce small RNAs,which regulate their target molecules.In recent years,there have been many studies on the abnormal expression of lnc RNA and how they participate in the occurrence and development of preeclampsia.A total of 738 differentially expressed lnc RNAs were detected in placenta of preeclampsia patients and normal pregnant women by using lnc RNA gene chip,suggesting that abnormal expression of lnc RNA may be involved in the occurrence and development of preeclampsia.In our previous study,DNA microarray technique was used to detect the difference of gene expression between placenta of normal pregnant women and placenta of preeclampsia patients.The results showed that X chromosome inactivation specific transcript(XIST)was low expressed in placental trophoblasts of preeclampsia patients.However,XIST was localized and localized in placental trophoblasts of preeclampsia patients.The amount is still unclear.Micro RNAs are highly conserved non-coding sequences,usually 18-24 nucleotides in length.Mature micro RNAs can bind to other related proteins to form active RNA-induced silencing complexes(RISC)and participate in the regulation of downstream target genes.Many studies have found that many micro RNAs are involved in the pathogenesis of preeclampsia.During normal pregnancy,trophoblasts can migrate through epithelial basement membrane to uterine spiral arterioles,and then invade vascular endothelium and even uterine myometrium.This behavior is similar to that of tumor cells.Epithelial-mesenchymal transition(EMT)refers to the transformation of epithelial cells into mesenchymal cells.It is characterized by the loss of polarity of epithelial cells into spindles,the reconstruction of cytoskeleton,the loss of epithelial cell markers(such as cytokeratin and E-cadherin),and the expression of interstitial cell markers(such as vimentin and cadherin).When the tumor cells invade and metastasize,the down-regulation of E-cadherin protein weakens the adhesion between the cells,and makes the tumor cells easy to fall off and metastasize from the tumor body.The purpose of this study was to investigate whether XIST plays a role in the pathogenesis of PE and regulates the proliferation and invasion of trophoblasts,binding to micro RNAs and then negatively regulating target proteins,thus participating in the process of trophoblastic EMT.This study aims to explore the possible molecular mechanism of XIST and downstream genes affecting the biological behavior of trophoblasts.MethodsPart one: Differential expression of XIST in placental trophoblast of preeclampsia and its effect on trophoblast function.15 placentas of preeclampsia patients delivered in obstetrics department of the First Affiliated Hospital of China Medical University were collected,and 15 healthy pregnant women in the same period were served as normal control group.The expression of XIST in placenta was detected by Real-time PCR.CRISPR/Cas9 was used to up-regulate XIST gene expression and package lentivirus.Simultaneously,XIST si RNA was designed and synthesized.Detection of the proliferation of HTR-8/SVneo cells after transfection and infection by Ed U method and the migration and invasion ability of HTR-8/SVneo were detected by Transwell method.In situ immunofluorescence hybridization was used to locate XIST in placenta.Part TwoXIST binds with miR-135 b,and regulates trophoblast biological behavior through miR-135 b.Using DIANA bioinformatics software to predict a binding point in XIST with miR-135 b.To investigate the expression of miR-135 b.Dual Luciferase Reporter detects its binding effect.Ed U and transwell investigate the biological behavior of trophoblast when it was transfected with miR-135 b mimic and its inhibitor.Part threeBy using western blot,we found out that FOXO1 protein is remarkably down-regulated in preemlampsia placenta.And FOXO1 can possibly promote the proliferation and invasion of trophoblast.Dual Luciferase Reporter detects the binding effect of miR-135 b and FOXO1 3’UTR area.And FOXO1 protein downregulated when trophoblast was transfected with miR-135 b can verify this too.Co-transfection of XIST and FOXO1 investigated that XIST may regulate trophoblast biological behavior through FOXO1.Further more,co-transfection of XIST and miR-135 b resulting in the change in the expression level of FOXO1 protein suggested that XIST could regulate FOXO1 expression through miR-135 b.Using FOXO1 knock-in vector to transfect trophoblast investigated that,the level of EMT related proteins,including E-cadherin,β-catenin,N-cadherin and vimentin changes,suggesting that FOXO1 may participate in the EMT process in trophoblast.Conclusion1.The expression of m RNA of long-chain non-coding RNA XIST in preeclampsia placenta was lower than that in normal pregnancy placentas.XIST promotes the trophoblast HTR-8/SVneo proliferation and invasion,and XIST may promote the proliferation of trophoblast HTR-8/SVneo by reducing apoptosis of trophoblast and increasing the proportion of G2/S phase cells.2.micro RNA-135 b expression in preeclampsia placenta was higher than that in normal pregnancy placenta.Micro RNA-135 b may play an important role in inhibiting the trophoblast HTR-8/SVneo proliferation and invasion.XIST and micro RNA-135 b can bind to each other.XIST can regulate the proliferation and invasion of trophoblast HTR-8/SVneo through micro RNA-135 b.3.FOXO1 expression in preeclampsia placenta was lower than that in normal pregnancy placenta.FOXO1 may promote the trophoblast HTR-8/SVneo proliferation and invasion ability,and the invasion may be related to the enhancement of EMT by FOXO1.Mir-135 b can target and bind to the FOXO1 3’UTR region.XIST can regulate the proliferation and invasiveness of HTR-8/SVneo trophoblasts through micror-135b/FOXO1.