The Mechanism Of HOTAIR Targeting Mir-152-3p To Regulate HLA-G In Preeclampsia Placenta

Objective:To study HOTAIR regulating the expression of HLA-G by miR-152-3p,detect the changes of trophoblast function after overexpression and inhibition of HOTAIR,miR-152-3p and HLA-G,and explore the downstream pathways related to placental function.To study the mechanism of HOTAIR and miR-152 regulating HLA-G to affect the signaling pathway of placental function and lead to the occurrence of PE,So as to provide evidence for the development of drugs to prevent PE and the formulation of treatment plans.Methods:1.RNA sequencing analysis of placental tissues,bioinformatics analysis,dual-luciferase reporter gene experiment,acquired or lost function experiment,to study the mutual regulation relationship and mechanism of HOTAIR,miR-152-3p and HLA-G.2.Constructed high and inhibition expression of HOTAIR lentiviral vector and transfected HTR-8/Svneo cells,detected the expression levels of HOTAIR,miR-152-3p and HLA-G by qRT-PCR assay.Detected the expression of HLA-G protein by Western Blot assay,immunofluorescence assay and ELISA assay.The proliferation,migration and invasion functions were detected by CCK8,EDU,cell cycle assay,apoptosis and Transwell assay.Detected EMT-related proteins by WB.3.After trophoblast cell transfections with miR-152-3p mimics and miR-152-3p inhibitor,the expression of HLA-G was detected by qRT-PCR assay.Detected the migration and invasion function by Transwell assay.Detected the expression of HLA-G and EMT-related proteins by WB.4.After trophoblast cells were co-transfected with HOTAIR for high expression and inhibition of lentivirus and miR-152-3p mimics and miR-152-3p inhibitor.Detected the migration and invasion functions by Transwell assay.Detected the expressions of HLA-G and EMT-related proteins by WB5.The expression of HLA-G was detected by qRT-PCR after over-expression and inhibition of HLA-G.The expression of HLA-G protein and EMT-related proteins was detected by WB.Transwell assay was used to detect the migration and invasion functional,and used NK cytotoxicity assay to detect the cytotoxic reaction of NK cells to the trophoblast cells after overexpression and inhibition of HLA-G vector transfection.Results:1.RNA-seq sequencing results showed that there were 6059 differentially expressed lncRNA,931 miRNA and 9798 mRNA in patients with severe preeclampsia and normal maternal placental tissues.The expression of HLA-G was down-regulated in the placenta of preeclampsia.Enrichment analysis of differences lncRNA,miRNA candidate target gene and mRNAKEGG showed that it was related to the regulation of inflammatory mediators such as T cell receptor signaling pathway,cell adhesion molecule,apoptosis,B cell receptor signaling pathway,regulation of inflammatory mediators TRP channels,Ras signaling pathways,TGF-? signaling pathways,allograft rejection,graft versus host disease,autoimmune thyroid disease,rheumatoid arthritis,antigen processing and rendering,cytokines and cytokine receptors pathway.It imply us that inflammatory pathways may be one of the PE's pathogenesis.2.The results of double luciferase experiment showed that the luciferase activities of HOTAIR and HLA-G wild type after transfection with miR-152-3p mimics were significantly reduced(P<0.05).But the luciferase expression activities of mutant HOTAIR and HLA-G vectors was no significant inhibition(P>0.05).3.qRT-PCR results showed that miR-152-3p decreased and HLA-G increased by high expresse HOTAIR.While the result were reversed when inhibite the expression level of HOTAIR.The expression of HLA-G decreased by high expression miR-152-3p.And the expression of HLA-G increased by inhibite miR-152-3p.4.WB results showed that the HLA-G protein increased when high expressied HOTAIR.While inhibited HOTAIR expression can reversed it.The HLA-G protein decreased by high expression miR-152-3p.And inhibited miR-152-3p the expression of HLA-G was decreased.5.Immunofluorescence experimental results showed that HTR-8/Svneo cell cytoplasm(soluble type)and cell membrane surface(membrane-bound type)HLA-G fluorescence signal was enhanced after HOTAIR expression was highly expressed,while HLA-G fluorescence signal was weakened after HOTAIR expression inhibition.6.ELISA results showed that the expression level of HLA-G in the superscript of cells in the HOTAIR hyperexpression group was increased,and the result of HOTAIR inhibition was opposite.The expression of HLA-G decreased in the supergene of high expression miR-152-3p.The expression of HLA-G in the supergene of cells decreased,and the expression level of HLA-G in the supergene of cells increased after transfection with miR-152-3p inhibitor,which could be partially reversed after co-transfection.7.The proliferation function changes of HTR-8/Svneo cells transfected with HOTAIR after overexpression and inhibition of lentivirus were detected by CCK8,EDU,cell cycle experiment and apoptosis staining.After overexpression or inhibition of HOTAIR the proliferation function have no significant change.8.Transwell results showed that the migration and invasion functions were enhanced by highly expresse HOTAIR.While the functions of migration and invasion were weakened by inhibite HOTAIR.After high expressed miR-152-3p the function of migration and invasion were induced.The results were inversed by inhibite miR-152-3p.This effect was partially reversed after cotransfection.The functions of migration and invasion were enhanced by high expresse HLA-G.The results were inversed by inhibite HLA-G.9.WB results showed that the mesenchymal markers of Fibronectin,N-cadherin,MMP9 and MMP2 were up regulated and the Vimentin have no significantly change when HOTAIR hyperexpression.The epithelial markers of E-cadherin was down-regulated.After HOTAIR expression inhibition,the expression levels of Fibronectin,N-cadherin,MMP9 and MMP2 in the mesenchymal markers were down-regulated,the expression levels of Vimentin were not significantly changed,and the expression of E-cadherin in the epithelial markers was up-regulated.10.WB results showed that the mesenchymal markers of Fibronectin,Vimentin and N-cadherin were down-regulated and MMP9 and MMP2 were have no significant change after miR-152-3p hyperexpression.The epithelial markers of E-cadherin was up-regulated.After inhibited miR-152-3p the interstitial markers of Fibronectin,Vimentin and N-cadherin were up-regulated,the MMP9 and MMP2 were not significantly changed,and the epithelial markers of E-cadherin were down-regulated.11.WB results showed that the mesenchymal markers of Fibronectin,N-cadherin and MMP9 were up-regulated and the Vimentin and MMP2 were have not significantly change after HLA-G hyperexpression,while the epithelial markers of E-cadherin was down-regulated.After the inhibition of HLA-G expression,the mesenchymal markers of Fibronectin,N-cadherin and MMP9 were down-regulated and the Vimentin and MMP2 were have't significantly change,and the epithelial markers of E-cadherin was up-regulated.12.The WB results after co-transfection showed that the expression of Fibronectin in the interstitial marker of HOTAIR+miR-152-3p mimics was down-regulated compared with that of the HOTAIR group after co-transfection.Expression of N-cadherin in interstitial markers after co-transfection with HOTAIR+miR-152-3p inhibitor was compared with that of the HOTAIR group.After co-transfection with HOTAIR+miR-152-3p inhibitor,compared with the HOTAIR+miR-152-3p mimics group,the expression of Fibronectin and N-cadherin in the interstitial markers was up-regulated,and there was no significant change in the expression levels of MMP2 and Vimentin.After the shHOTAIR+miR-152-3p mimics co-transfected,the epithelial marker E-cadherin was up-regulated.While the interstitial marker Fibronectin and N-cadherin was down-regulated,and the MMP2 was up-regulated.After co-transfection with shHOTAIR+miR-152-3p inhibitor,there were no significant abnormalities of EMT-related proteins compared with the shHOTAIR group.After co-transfection with shHOTAIR+miR-152-3p inhibitor,compared with the shHOTAIR+miR-152-3p mimics group,the expression of the superior cortical marker E-cadherin was down-regulated,the expression of the interstitial marker N-cadherin was up-regulated,and the expression of MMP2 was down-regulated.13.The results of NK cytotoxicity test showed that the cells dissolution rate decreased after HLA-G hyperexpression.While the dissolution rate increased by inhibite HLA-G expression.Conclusions:1.Aberrant expression of lncRNA miRNA and mRNA in preeclampsia placental tissues,HLA-G is low in preeclampsia placenta,and immune related pathway,cell adhesion,apoptosis and other pathways may be related to the pathogenesis of severe preeclampsia.2.HOTAIR regulates the expression of HLA-G by targeting miR-152-3p.3.Hyperexpression or low expression of HOTAIR had no significant change in the function of proliferation.4.HOTAIR hyperexpression and inhibite HLA-G and inhibite miR-152-3p can enhance the function of migration and invasion by promoting the EMT process,while inhibition of the expression of HOTAIR,HLA-G and promotion of the expression of miR-152-3p can inhibit the migration and invasion function of HTR-8/Svneo cells by weakening the EMT process.HOTAIR regulated the expression of HLA-G by miR-152-3p to promote the EMT process and increase the function of trophoblast cell migration and invasion.5.High expression of HLA-G can protect HTR-8/Svneo cells from killing by NK cells,while inhibition of HLA-G increases the dissolution rate of HTR-8/Svneo cells.6.HOTAIR regulates the pathogenesis of preeclampsia by differential expression of HLA-G through miR-152-3p.