ISLET1 Participates In The Pathogenesis Of Preeclampsia By Regulating Trophoblasts Invasion And Angiogenesis

Objective1.Detection of m RNA expression profiles in severe preeclampsia(SPE)and normal pregnancy(NP)placental tissues;2.To screen out differentially expressed m RNAs(DE-m RNAs)in SPE placental tissues.MethodsPlacental samples were collected from six paired SPE and NP women.m RNA expression profiles were identified by high throughput RNA sequencing(RNA-Seq)and validated in another 40 SPE and NP samples by quantitative reverse transcript PCR(q RT-PCR).Several bioinformatic tools were performed to analysis the potential function of DE-m RNAs and to predict target genes.Results1.The m RNA expression profiles in the placental tissues of the SPE were significantly different from that in the NP.A total of 60 DE-m RNAs were detected,of which 46 were up-regulated and 14 were down-regulated m RNAs.2.GO analysis results suggest that DE-m RNAs are mainly enriched in immune response regulation,cell migration,adhesion,antigen processing and presentation;KEGG pathway analysis is mainly enriched in cell adhesion molecules,asthma,autoimmune bowel disease,tuberculosis autoimmune diseases such as systemic lupus erythematosus.Which suggests that the occurrence of preeclampsia is closely related to autoimmunity.3.qRT-PCR and IHC results showed that the m RNA and protein expression of ISL1 in SPE placenta was significantly lower than that in NP placentae.Conclusions: A total of 60 DE-m RNAs were detected,of which 46 were up-regulated and 14 were down-regulated m RNAs.One crucial m RNA,ISL1 was identified to be associated with occurrence of SPE.Objective1.To Define the distribution of ISL1 expression in human placenta;2.To study the effect of ISL1 on the biological behavior of human trophoblasts;3.To explore the downstream target genes of ISL1.Methods1.IHC,IF were used to verify the distribution of ISL1 in human placentae;2.Specific Si RNAs were transfected into human trophoblast cell line HTR-8/SVneo by Lipo RNAi MAX to achieve an effective knockdown of ISL1 expression.Then,loss function assays were conducted to explore the effect of ISL1,such as scratch healing test,angiogenesis experiment,transwell migration and invasion tests.3.Predicting the target genes of ISL1 by an online transcription factor prediction site in conjunction with the existing CHIP-Seq databases.And then,q RT-PCR were used to verified the correlation between ISL1 and its predicted downstream target genes.Results1.ISL1 was expressed in vascular endothelial cells and various trophoblast subtypes in human placenta tissue,and the expression of ISL1 in extracellular trophoblast cells is the highest;2.ISL1-Si RNA-1 achieves an effective knockdown of ISL1 m RNA and protein levels with a knockdown efficiency of 84.7%.Cell loss function assays showed that after mitigation of ISL1 expression levels,trophoblast migration,invasion and angiogenesis significantly increase;3.The online transcription factor prediction website combined with the existing CHIP-Seq database predicted that ZEB1,TGF-β,Wnt5 a,GATA3,etc.may be the target genes of ISL1.q RT-PCR verified that after knocking down the expression of ISL1,the expression level of ZEB1 and TGF-β are significantly down-regulated,and the expression of Wnt5 a and GATA3 do not change significantly.ConclusionISL1 is expressed in vascular endothelial cells and various trophoblast subtypes in human placenta tissue.Among them,the expression of the extravillous trophoblasts is the highest.After knocking down the expression of ISL1,it significantly promoted the migration,invasion and angiogenesis of human trophoblasts.Moreover,the expression level of ISL1 was significantly correlated with the expression of ZEB1 and TGF-β.