Role And Regulatory Mechanism Of LncRNAs In Trophoblastic Cell Dysfunction Induced By BaP/BPDE Exposure And Recurrent Spontaneous Abortio

Objective The etiology of recurrent abortion is complex,and half of the etiology mechanism is still unclear.Benzo pyrene(Ba P)and its metabolite BPDE have been reported to be associated with recurrent abortion,but the specific causal relationship and regulatory mechanism are unclear.Trophoblast cells play an important role in embryonic development,and their proliferation,invasion and migration functions are crucial for normal pregnancy.Cuproptosis,as a new cell death mode,may be involved in the occurrence of abortion,but the mechanism of inducing recurrent abortion remains to be improved.Therefore,this paper explores the mechanism of BPDE inducing recurrent abortion through trophoblast cells.As an important epigenetic regulatory factor,noncoding RNA may be involved in many biological processes such as embryonic development.Therefore,this study studied the mechanism of lnc RNA in BPDE’s regulation of recurrent abortion(RM)induced by trophoblast dysfunction.To provide theoretical basis for clinical detection and treatment of unexplained recurrent abortion.Methods Part Ⅰ Lnc-HZ05 regulates BPDE-inhibited human trophoblast cell proliferation and affects the occurrence of miscarriage by directly binding with mi R-hz05 1.MTS,cloning and cell cycle assays were detected the proliferation and cycle function of trophoblast cells;2.Western blot and RT-q PCR were detected the expression level of pathway proteins and the m RNA stability of genes;3.Dual-Luciferase Reporter,M2S-RIP and RNA pull down assays were detected the interaction between mi R-hz05,Lnc C-Hz05 and FOXO3 a.Part II Lnc-HZ11/NF-κB negative feedback loop regulates BPDE-inhibited human trophoblast cell invasion and migration and induces miscarriage 1.According to the inclusion and exclusion criteria,12 patients with normal abortion(HC)and 12 patients with recurrent abortion(RM)were collected from the chorionic tissues.RNA and protein were extracted to verify the m RNA and protein expression levels of NC-HZ11 and EGR1 signaling pathway,respectively.The invasion and migration function of chorionic membrane was verified by Trans-well;2.In trophoblast cells with knockdown or overexpression of lnc-HZ11 and EGR1 were to verify the RNA and protein expression levels of EGR1 signaling pathway and invasion and migration function;The binding levels of NF-κB to CXCL12 and lncHZ11 promoter and DNMT1 to lnc-HZ11 promoter were verified by Ch IP and double luciferase assay;The binding level of NF-κB with EGR1 and the ubiquitination level of EGR1 were verified by IP assay.The nucleation process of EGR1/NF-κB was verified by nuclear plasma separation experiment.The binding level of lnc-HZ11 and EGR1 was verified by RIP and RNA pulldown experiments.Trans-well assay was used to verify the invasion and migration function.Methylation test was used to verify the methylation level of lnc-HZ11 promoter region.The morphology and particle size of EVs were verified by TEM and NTA;3.In BPDE-exposed trophoblast cells with knockdown or overexpression of lncHZ11 and EGR1 were to verify the expression levels of EGR1 signaling pathway and the invasion and migration function;The binding levels of NF-κB with CXCL12 and lnc-HZ11 promoter and DNMT1 with lnc-HZ11 promoter were verified by Ch IP assay;The binding level of NF-κB with EGR1 and the ubiquitination level of EGR1 were verified by IP assay;The nucleation process of EGR1/NF-κB was verified by nuclear plasma separation experiment;RIP experiment was used to verify the binding level of lnc-HZ11 and EGR1;Trans-well assay was used to verify the invasion and migration function of cells;Methylation test was used to verify the methylation level of lnc-HZ11 promoter region;4.The binding level of NF-κB to EGR1 and the ubiquitination level of EGR1 were verified by IP assay in HC and RM chorionic tissues(n = 12);The binding levels of NF-κB in CXCL12 and lnc-HZ11 promoter and DNMT1 in lnc-HZ11 promoter were verified by Ch IP assay;Methylation test was used to verify the methylation level of lncHZ11 promoter region;Pearson correlation analysis was used to verify the correlation between lnc-HZ11 and EGR1 signaling pathway;5.In Bap-abortion mouse model the RNA and protein expression levels of lncHz11 and Egr1/Nf-κb/Cxcl12 in embryonic tissue were verified by RT-q PCR and WB;6.In the Bap-abortion model with AS-Hz11 intervention,RNA and protein expression levels of lnc-Hz11 and Egr1 signaling pathway in embryonic tissues were verified by RT-q PCR and WB assay;7.In the Bap-abortion model with EV-AS-Hz11 intervention,the RNA and protein expression levels of lnc-Hz11 and Egr1 signaling pathway in embryonic tissue were verified by RT-q PCR and WB assay;8.According to the inclusion and exclusion criteria,12 sera from patients with HC and 12 sera from patients with RM were collected and EV-RNA was extracted to verify the expression level of EV-HZ11.Part Ⅲ Lnc-HZ11 regulates BPDE-promoted human trophoblast cell cuproptosis and induces miscarriage 1.According to the inclusion and exclusion criteria,12 patients with HC and 12 patients with RM were collected from the chorionic tissues.RT-q PCR and WB assays were used to verify the m RNA and protein expression levels of Fe-S cluster,Mitochondria,Lipids and copper ion input and output genes;2.In trophoblastic cells with knockdown or overexpression of lnc-HZ11 and SLC31A1,and the m RNA and protein expression levels of Fe-S cluster,Mitochondria,Lipids and copper ion input and output genes were verified by RT-q PCR and WB assays;The ubiquitination level of SLC31A1 were verified by IP assay;The cell membrane localization of SLC31A1 was detected by membrane separation assay;Copper ion levels inside and outside cells were detected by copper ion level test.The cell proliferation function was verified by CCK8 assay;3.In Cu:ES-exposed trophoblastic cells with knockdown or overexpression of lncHZ11 and SLC31A1,and the m RNA and protein expression levels of Fe-S cluster,Mitochondria,Lipids and copper ion input and output genes were verified by RT-q PCR and WB assays;Copper ion levels inside and outside cells were detected by copper ion level test.The cell proliferation function was verified by CCK8 assay;4.In BPDE-exposed trophoblastic cells with knockdown or overexpression of lncHZ11 and SLC31A1,and the m RNA and protein expression levels of Fe-S cluster,Mitochondria,Lipids and copper ion input and output genes were verified by RT-q PCR and WB assays.The ubiquitination level of SLC31A1 were verified by IP assay.The cell membrane localization of SLC31A1 was detected by membrane separation assay.Copper ion levels inside and outside cells were detected by copper ion level test.The cell proliferation function was verified by CCK8 assay;5.In the Bap-abortion model with AS-Hz11 intervention,RT-q PCR and WB assays were used to verify the m RNA and protein expression levels of Fe-S cluster,Mitochondria,Lipids and copper ion input and output genes;6.According to the inclusion and exclusion criteria,12 sera from patients with HC and 12 sera from patients with RM were collected,and copper ion level test was used to detect the serum copper ion level.Results Part Ⅰ Lnc-HZ05 regulates BPDE-inhibited human trophoblast cell proliferation and affects the occurrence of miscarriage by directly binding with mi R-hz05 1.Lnc-HZ05 was highly expressed and mi R-hz05 and FOXO3a/P21/CDK2 pathways were lowly expressed in BPDE-exposed trophoblast cells;2.The upregulated lnc-HZ05 inhibited FOXO3a/P21/CDK2 pathway and cell proliferation in BPDE-exposed trophoblast cells;3.The downregulated mi R-hz05 inhibited FOXO3a/P21/CDK2 pathway and cell proliferation in BPDE-exposed trophoblast cells;4.Lnc-HZ05 upregulated FOXO3 a m RNA by directly and specifically binding as a ce RNA with mi R-hz05;5.The upregulated lnc-HZ05 and downregulated mi R-hz05 were associated with the downregulated FOXO3a/P21/CDK2 pathway in RM tissues;6.In the abortion model of mice exposed to Ba P,the abortion rate of pregnant mice was significantly increased,and the expressions of mi R-hz05,Foxo3 a and P21 were down-regulated,and the expressions of Cdk2 were down-regulated.Part II Lnc-HZ11/NF-κB negative feedback loop regulates BPDE-inhibited human trophoblast cell invasion and migration and induces miscarriage 1.In RM chorionic tissue,the invasion and migration function was significantly downregulated,the expression of lnc-HZ11 was highly expressed,and the expression of EGR1 signaling pathway was decreased;2.In trophoblast cells,lnc-HZ11 promoted ubiquitination of EGR1 and inhibited the expression of EGR1,EGR1 signaling pathway,finally inhibited cell invasion and migration;3.There exists NLS region in EGR1-F4,which promotes the nuclear entry process of NF-κB,and then promotes the transcriptional activation of NF-κB on CXCL12;4.Lnc-HZ11-S4 interacted with EGR1-F4,inhibiting the interaction between EGR1-S4 and NF-κB,then inhibiting the nuclear entry of NF-κB and the transcriptional activation of CXCL12;5.DNMT1 inhibits the transcriptional activation of NF-κB on lnc-HZ11 by promoting the methylation level of lnc-HZ11 promoter;6.Lnc-HZ11 and NF-κB form a negative feedback regulation,lnc-HZ11 inhibits the expression level of NF-κB,and NF-κB can further promote the transcription level of lnc-HZ11,and form a balanced and stable mechanism in vivo to maintain the normal physiological environment;7.In RM chorionic tissue,EGR1-Ub level was significantly increased,the interaction between EGR1 and NF-κB was decreased,the binding level of NF-κB to CXCL12 promoter was significantly decreased,and the binding level of DNMT1 to lncHZ11 promoter was significantly decreased.However,the binding level of NF-κB to lnc-HZ11 promoter region was significantly increased.8.Pearson correlation analysis showed that lnc-HZ11 was negatively correlated with EGR1,NF-κB and CXCL12 proteins in RM chorionic tissue.9.In BPDE-exposed trophoblastic cells,the expression of lnc-HZ11 is upregulated,the expression of EGR1/NF-κB/CXCL12 pathway is significantly downregulated,the level of EGR1-Ub is increased,the interaction between EGR1 and NF-κB is decreased,and the entry of EGR1/NF-κB is decreased.Then the transcriptional activation of CXCL12 decreased,finally the invasion and migration function of cells was downregulated;10.The trophoblast cells secrete EV-HZ11,and BPDE and lnc-HZ11 promote the secretion of EV-HZ11,thus inhibiting the invasion and migration function of receptor trophoblast cells;11.In the Bap-aborted mouse model with AS-Hz11 and EV-AS-Hz11 intervention,the abortion rate of pregnant mice decreased,the expression level of lnc-HZ11 decreased,and the expression level of Egr1 signaling pathway increased;12.In the serum of RM population,EV-HZ11 content increased significantly,which was a stable predictor of early abortion.The area under the subject working curve AUC = 0.973.Part Ⅲ Lnc-HZ11 regulates BPDE-promoted human trophoblast cell cuproptosis and induces miscarriage 1.In RM chorionic tissue,the expressions of Fe-S cluster,Mitochondria,and Lipids genes decreased,the expressions of FDX1,SLC31A1 and Hsp70 increased,finally promoted copper death of cells;2.In trophoblast cells,lnc-HZ11 promoted intracellular copper ion level,inhibited the expression levels of Fe-S cluster,Mitochondria,and Lipids genes,promoted the expression of FDX1,SLC31A1 and Hsp70,promoted copper death,and inhibited cell proliferation;3.In Cu:ES-exposed trophoblastic cells with knockdown of lnc-HZ11 and Cuchelating agent TEPA/DC inhibited intracellular copper ion levels,promoted the expression levels of Fe-S cluster,Mitochondria,and Lipids genes,inhibited the expressions of FDX1,SLC31A1 and Hsp70,and promoted cell proliferation;4.In trophoblast cells with overexpressed of lnc-HZ11,the copper chelating agent TEPA/DC inhibited the intracellular copper ion level,promoted the expression level of Fe-S cluster,Mitochondria,and Lipids genes,and inhibited the expression of FDX1,SLC31A1 and Hsp70,and inhibited cell proliferation;5.In trophoblastic cells,lnc-HZ11 promoted the expression level of SLC31A1 protein by inhibiting the ubiquitination degradation,and promoted the cell membrane localization of SLC31A1,and then inhibited the expression levels of Fe-S cluster,Mitochondria,and Lipids genes,promoted the expression of FDX1,SLC31A1 and Hsp70,promoted cell cuproptosis,and inhibited cell proliferation;6.In BPDE-exposed trophoblastic cells,BPDE inhibits the expression levels of Fe-S cluster,Mitochondria,and Lipids genes,promotes the expression of FDX1,SLC31A1 and Hsp70 by up-regulating the lnc-HZ11-SLC31A1,finally promoted cell cuproptosis,and inhibited cell proliferation;7.In the mouse abortion model with AS-Hz11 intervention,after AS-Hz11 intervention,the abortion rate of pregnant mice decreased,Fe-S cluster,Mitochondria,and Lipids genes increased,and the expressions of FDX1,SLC31A1 and Hsp70 decreased.Conclusions Part Ⅰ Lnc-HZ05 regulates BPDE-inhibited human trophoblast cell proliferation and affects the occurrence of miscarriage by directly binding with mi R-hz05 1.Lnc-HZ05 was highly expressed and mi R-hz05 and FOXO3a/P21/CDK2 pathways were lowly expressed in trophoblast cells exposed with BPDE and in RM tissues;2.Lnc-HZ05 promotes the expression level of FOXO3 a m RNA by specifically binding to mi R-hz05;3.Mi R-hz05/Foxo3a/P21/Cdk2 axis may induce abortion in mice through cell proliferation function.Part II Lnc-HZ11/NF-κB negative feedback loop regulates BPDE-inhibited human trophoblast cell invasion and migration and induces miscarriage 1.A novel lnc-HZ11 highly expressed in RM tissues,BPDE exposed trophoblast cells,and placenta of Bap-aborted mice,and suppressed migration and invasion of trophoblast cells;2.Lnc-HZ11-S4 inhibits the interaction between NLS region of EGR1 and NF-κB by interacting with EGR1-S4,and then inhibited the transcription level of CXCL12;3.DNMT1 promoted the DNA methylation level of lnc-HZ11 promoter region and suppressed NF-κB-mediated lnc-HZ11 transcription;4.EVs-packaged lnc-HZ11(EV-HZ11)suppressed migration and invasion of the normal trophoblast cells,and can be used as a detection indicator of recurrent abortion;5.The lnc-HZ11/EGR1 axis can induce disturbance of trophoblast cell invasion and migration,which is a potential therapeutic target for RM.Part Ⅲ Lnc-HZ11 regulates BPDE-promoted human trophoblast cell cuproptosis and induces miscarriage 1.Cell cuproptosis significantly up-regulated in RM tissues and BPDE exposed trophoblast cells;2.Lnc-HZ11 activates cuproptosis by promoting the expression and membrane localization of SLC31A1 and up-regulation of intracellular copper ions;3.In the Bap-abortion model,AS-Hz11 intervention reduced the abortion rate of pregnant mice by inhibiting copper death.