Association Of LncRNA SH3PXD2A-AS1 With Preeclampsia And The Mechanisms Underlying Its Inhibitory Role On Migration And Invasion Of Trophoblast Cells

BackgroundPreeclampsia(PE)is characterized by hypertension and proteinuria as a unique disease during pregnancy.The global incidence rate is 4%-5%.The pathogenesis of PE mainly includes placental shallow implantation,oxidative stress,vascular endothelial injury,abnormal immune regulation and so on,but the exact cause of preeclampsia has not been fully elucidated.Long noncoding RNA(lncRNA)is a type of RNA that is greater than 200 nucleotides in length but does not encode proteins.Previous studies have found that abnormally expressed lncRNAs are closely related to the occurrence and development of preeclampsia.It is speculated that lncRNAs may play an important role in preeclampsia by affecting the function of trophoblasts,providing a prospective diagnosis and treatment for preeclampsia.We carried out transcriptome profiling on placenta tissues from 9 early-onset severe preeclampsia(EOSPE)patients and 32 control subjects and found that the expression level of lncRNA SH3PXD2A-AS1 was upregulated in PE.Overexpression of SH3PXD2A-AS1 inhibited cell invasion,migration and proliferation,and promoted cell apoptosis in HTR8/SVneo.Knockout of SH3PXD2A-AS1 showed the opposite results.We further carried out RNA pulldown experiment coped with mass spectrometry and identified 93 SH3PXD2A-AS1-binding proteins,including 12 transcription factors(TFs).Furthermore,the target genes of TFs predicted through databases were constructed TF-Targets regulatory network and intersected with differentially expressed genes by sequencing and it was found that CTCF was most related to preeclampsia.Through RNA immunoprecipitation(RIP)experiments,we also confirmed that lncRNA SH3PXD2A-AS1 directly recruited CTCF in HTR8/SVneo.Moreover,qRT-PCR,Western blotting,chromatin immunoprecipitation(ChIP),and luciferase assays were performed to verify that lncRNA SH3PXD2A-AS1 silenced SH3PXD2A and CCR7 expression by recruiting CTCF.SH3PXD2A and CCR7 could promote the function of migration and invasion,and partially reverse the inhibition of lncRNA SH3PXD2A-AS1 in trophoblast cells.Therefore,we speculated that lncRNA SH3PXD2A-AS1 could inhibit SH3PXD2A and CCR7 expression by binding to CTCF,which affect the function of trophoblast cells and participate in the development of preeclampsia.It may become a new predictive and therapeutic biomarker for preeclampsia.ObjectiveFirstly,we identified the role of lncRNA SH3PXD2A-AS1 in preeclampsia and its clinical significance.Next,we elucidated the role of lncRNA SH3PXD2A-AS1 in the function of migration,invasion,proliferation and apoptosis in trophoblast cells.Finally,it was confirmed that lncRNA SH3PXD2A-AS1 could inhibit SH3PXD2A and CCR7 expression by recruiting CTCF,which influence the function of trophoblast cells and participate in the occurrence and development of preeclampsia.MethodsThe expression of lncRNA SH3PXD2A-AS1 in placentae was detected by qRT-PCR.Overexpression and knockout of lncRNA SH3PXD2A-AS1 were conducted to explore the role in trophoblast cells.Transwell,EdU and flow cytometry assays were applied to detected cell migration,invasion,proliferation and apoptosis.The relationship between lncRNA SH3PXD2A-AS1,CTCF and SH3PXD2A,CCR7 were investigated by bioinformatic analysis,FISH,subcellular fractionation,RNA pull-down,RIP,ChIP,luciferase reporter,qRT-PCR and Western blotting assays et al.Results1.The expression level of lncRNA SH3PXD2A-AS1 was upregulated in PE and positively correlated with blood pressure of pregnant women.2.LncRNA SH3PXD2A-AS1 overexpression inhibited cell invasion,migration and proliferation,and promoted cell apoptosis in trophpblast cells.In contrast,lncRNA SH3PXD2A-AS1 knockout had the opposite effects.3.LncRNA SH3PXD2A-AS1 was mostly located in nucleus by subcellular fractionation and FISH assays.Moreover,through RNA pulldown,mass spectrometry and RIP experiments,we found lncRNA SH3PXD2A-AS1 could directly recruit CTCF in HTR8/SVneo.4.Bioinformatic analysis,ChIP,luciferase reporter,qRT-PCR and Western blotting assays were performed to verify that lncRNA SH3PXD2A-AS1 recruited CTCF to SH3PXD2A and CCR7 promoter regions and inhbited their expression.5.SH3PXD2A and CCR7 could promote the function of migration and invasion,and partially reverse the inhibition of lncRNA SH3PXD2A-AS1 in trophoblast cells.Conclusion1.LncRNA SH3PXD2A-AS1 was highly expressed in PE placentae,and its expression level was positively correlated with blood pressure of pregnant women.2.LncRNA SH3PXD2A-AS1 inhibited cell invasion,migration and proliferation,and promoted cell apoptosis in trophoblast cells.3.LncRNA SH3PXD2A-AS1 could silence SH3PXD2A and CCR7 expression by recruiting CTCF,which ihibite the function of migration and invasion in trophoblast cells and participate in the occurrence and development of preeclampsia.