Regulation Of LncRNA NEAT1 On NLRP3 Inflammasome And Autophagy In Preeclampsia

BackgroundPreeclampsia(PE)is a unique syndrome that occurs after 20 weeks of gestation,usually with hypertension and proteinuria as the first clinical manifestations.As the disease progresses,severe cases may be accompanied by multiple organ dysfunction,placental abruption,and intrauterine death,resulting in adverse outcomes.It is one of the main reasons for the maternal and perinatal mortality.Due to the lack of effective methods for cure,many cases have to be terminated early,in order to prevent serious complications,resulting in extremely premature birth or even miscarriage.Therefore,in-depth research on the pathogenesis of PE,seeking effective treatment,is an urgent problem to be solved at present.The pathogenesis of PE is complex,which is the result of multiple factors such as the mother,fetus and placenta.And it is influenced by genetic,epigenetic,environmental and physiological factors,etc.It is believed that the pathogenesis includes chronic ischemia and hypoxia of placenta,immune disorders,genetic imprinting,trophoblast dysfunction,autophagy disorder and excessive inflammatory responses.Long noncoding RNA(lncRNA)refer to RNAs with a length of more than 200 nucleotides.They are generally not considered to be involved in the coding of protein,but as the potential key regulators,they are involved in the development of various diseases.A variety of lncRNA can regulate the functions of trophoblast and participate in the development of PE by changing gene expression and interfering signaling pathways.Nuclear Enriched Abundant Transcript 1(NEAT1)is an important component of paraspeckles,which can regulate the cell function and chemical resistance of tumor cells,and participate in the occurrence of tumor.Due to the similarity between trophoblast cells and tumor cells in biological functions,the role of lncRNA NEAT1 in PE has been concerned,but the mechanism remains unclear.Inflammatory response is a protective immune response,and inflammasome is the important factor.The most characteristic inflammasome is NLRP3.Excessive activation of NLRP3 inflammasome leads to various pregnancy-related diseases,such as recurrent spontaneous abortion,chorioamnitis,preterm birth,PE,and gestational diabetes mellitus(GDM),etc.Autophagy is a protective mechanism for cells to self-digest and decompose some proteins in cells under adverse living conditions,thereby ensuring cell survival.Autophagy disorders can lead to a variety of pregnancy-related diseases,such as PE,Intrauterine growth restriction and GDM,etc.Hypoxia,oxidative stress and inflammatory responses in PE are closely related to autophagy.The researches show that lncRNA NEAT1 plays an important role in tumors,neurodegenerative diseases,cardiovascular diseases and other diseases by regulating NLRP3 and autophagy.And there is also a complex interaction between inflammasome and autophagy.Therefore,we speculate that lncRNA NEAT1 may regulate NLRP3 or autophagy in PE.This study aims to investigate the regulation of lncRNA NEAT1 on NLRP3 and autophagy,and their effect on the cell function and animal phenotype through clinical specimen detection,cell experiments and animal experiments.So we can clarify the roles of lncRNA NEAT1,NLPR3 and autophagy in PE,and provide new ideas and theoretical basis for the study of the pathogenesis and clinical treatment of PE.Part Ⅰ Expressions of lncRNA NEAT1,NLRP3 inflammasome and atuophagy in placenta of women with preeclampsiaObjectiveTo investigate the expressions of lncRNA NEAT1,NLRP3 inflammasome and autophagy in placenta of normal pregnancy and PE.MethodsWomen who underwent cesarean section from January 2018 to December 2018 were selected.Among them,30 women were the PE group;30 women with normal pregnancy who delivered during the same period were the normal group.The expression of lncRNA NEAT1 in placenta was detected by real-time quantitative PCR(qRT-PCR).The localization and protein expressions of NLRP3 inflammasome and autophagy were detected by immunohistochemistry(IHC)and Western blot.Bioinformatics analysis of lncRNA NEAT1 was conducted to predict its target genes,and enrichment analysis were used to explore its possible mechanism.Results1.qRT-PCR showed that the expression of lncRNA NEAT1 in the placenta of the PE group was significantly increased than that of the normal group.And the expression of lncRNA NEAT1 was positively correlated with the blood pressure.2.IHC showed that NLRP3,Caspase-1,LC3,and P62 were mainly expressed in the cytoplasm of trophoblast cells.The expressions of NLRP3,Caspase-1 and LC3 in the PE group were significantly stronger than the normal group,while the expression of P62 was significantly weaker.3.Western blot showed that the expressions of NLRP3,Caspase-1,Beclin-1,LC3Ⅱ and LC3Ⅱ/Ⅰ in the PE group were significantly increased than the normal group,while the expression of P62 was significantly decreased.4.The CeRNA and PPI protein interaction network showed that NLRP3 was the one of the target genes of lncRNA NEAT1,and the correlation between them was good.The GO and KEGG/PANTHER enrichment analysis showed that some biological processes and pathways of target genes were closely related to the PE,such as hypoxia,inflammation,autophagy and oxidative stress,etc.Conclusion1.The expression of lncRNA NEAT1 was increased,the NLRP3 inflammasome was activated and the autophagy was enhanced in the placenta of PE.2.Biological processes and signaling pathways enriched by the target genes of lncRNA NEAT1 are closely related to PE,and NLRP3 is one of the target genes.3.The up-regulation of lncRNA NEAT1,and activation of NLRP3 inflammasome and autophagy in placenta were associated with the pathogenesis of PE.Part Ⅱ Expressions of lncRNA NEAT1,NLRP3 inflammasome and autophagy in trophoblast cells under hypoxia and the effect on cell functionsObjectiveTo investigate the expressions of lncRNA NEAT 1,NLRP3 inflammasome and autophagy in JEG-3 cells cultured under hypoxia for different times,as well as the effect on the cell functions.Methods:Hypoxia played an important role in the development of PE.JEG-3 cells were cultured under hypoxia condition(2%O2,93%N2,5%CO2)to establish the hypoxia models(namely hypoxia groups).The hypoxia groups were further divided into 12h,24h and 48h groups according to the different hypoxia time.The cells cultured under normoxia condition(20%O2,75%N2,5%CO2)were the normoxia group.The mRNA expressions of lncRNA NEAT1 and NLRP3 in each group were detected by qRT-PCR.The protein expressions of NLRP3 inflammasome,inflammatory factors and autophagy were detected by Western blot.Flow cytometry(AnnexinV-FITC/PI apoptosis assay),MTT and Transwell assay were used to evaluate the apoptosis,proliferation and invasion of the trophoblast cells in each group.Results1.Compared with the normoxia group,the expressions of lncRNA NEAT1 and NLRP3 mRNA were significantly increased in the hypoxia groups,and the expressions gradually increased with the duration time of hypoxia.2.Compared with the normoxia group,the expressions of NLRP3,Caspase-1,IL-18,IL-1β,Beclin-1 and LC3II were significantly increased in the hypoxia groups,and the expressions gradually increased with the duration time of hypoxia.P62 was significantly decreased,and the expression gradually decreased with the duration time of hypoxia.3.Compared with the normoxia group,the apoptosis of JEG-3 cells were significantly increased in the 24h and 48h hypoxia groups,and the apoptosis was the highest in the 48h hypoxia group.The apoptosis in the 12h hypoxia group was decreased than that in the normoxia group.4.Compared with the normoxia group,the proliferation of JEG-3 cells were significantly decreased in the 24h and 48h hypoxia groups,especially in the 48h hypoxia group.However,there was no significant difference between the 12h hypoxia group and normoxia group.5.Compared with the normoxia group,the invasion of JEG-3 cells were significantly decreased in the 24h and 48h hypoxia groups,and the invasion in the 48h hypoxia group was decreased more obvious.However,the invasion in the 12h hypoxia group was increased than that in the normoxia group.Conclusion1.The expressions of lncRNA NEAT1,NLRP3 inflammasome and autophagy were significantly increased in the trophoblast cells cultured under hypoxia,which were positively correlated with the duration time of hypoxia.2.Hypoxia for short--term could promote the invasion of trophoblast cells and inhibit cell apoptosis,while hypoxia for long-term could inhibit the proliferation and invasion of trophoblast cells,and promote cell apoptosis,resulting in the damage to cell functions.3.Long-term hypoxia may affect the functions of trophoblast cells and participate in the occurrence of PE by regulating the expressions of lncRNA NEAT1,NLRP3 inflammasome and autophagy.Part Ⅲ Regulation of lncRNA NEAT1 on NLRP3 inflammasome and autophagy in trophoblast cells under hypoxia and the effect on cell functionsObjectiveTo investigate the effect on the expressions of NLRP3 inflammasome and autophagy as well as the cell functions after interfering the expression of lncRNA NEAT1 in JEG-3 cell under hypoxia condition,and to further investigate the effect on the expressions of related factors and the cell functions after inhibiting NLRP3 and autophagy,so as to clarify the regulatory mechanism between lncRNA NEAT1,NLRP3 inflammasome and autophagy and the possible role in the pathogenesis of PE.MethodsJEG-3 cells were treated with hypoxia for 24h to establish the model of PE in vitro.Under hypoxia condition,JEG-3 cells were transfected with small interfering RNA(si-NEAT1)to interfere the expression of lncRNA NEAT1,and transfected with NLRP3 over-expression plasmid(pcDNA-NLRP3)to over-express NLRP3.Their negative controls(si-NC and pcDNA-NC)were also transfected.The transfection efficiency was verified by qRT-PCR.The mRNA expressions of NLRP3 inflammasome related factors in each group(normoxia,hypoxia,hypoxia+si-NC,hypoxia+si-NEAT1,hypoxia+si-NEAT1+pcDNA-NC and hypoxia+si-NEAT1+pcDNA-NLRP3)were detected by qRT-PCR.The protein expressions of NLRP3 inflammasome,inflammatory factors and autophagy were detected by Western Blot.The expression of LC3 was detected by immunofluorescence(IF).The apoptosis and invasion of cells were detected by Flow cytometry and Transwell assay.Furthermore,JEG-3 cells were treated with autophagy inhibitor 3-MA and NLRP3 inhibitor MCC950 to inhibit autophagy and NLRP3 under hypoxia condition.The expressions of the related factors,the apoptosis and invasion of cells in each group(normoxia,hypoxia,hypoxia+3-MA,hypoxia+MCC950 and hypoxia+3-MA+MCC950)were detected by the above methods.Results1.Under hypoxia condition,the expressions of NLRP3,Caspase-1,IL-18,IL-1β,Beclin-1 and LC3II were significantly decreased and P62 was significantly increased after interfering the expression of NEAT1 by si-NEAT1.Further transfected with pcDNA-NLRP3,the expressions of NLRP3,Caspase-1,IL-18,IL-1β,Beclin-1 and LC3Ⅱ were significantly increased,and P62 was significantly decreased.2.Under hypoxia condition,the apoptosis of the JEG-3 cells was significantly decreased and the invasion was significantly increased after interfering the expression of NEAT 1 by si-NEAT1.Further transfected with pcDNA-NLRP3,the apoptosis of the cells was significantly increased and the invasion was significantly decreased.3.Under the hypoxia condition,the expressions of NLRP3,Caspase-1,IL-18,IL-1β,Beclin-1 and LC3Ⅱ were significantly decreased,while P62 was significantly increased,after inhibited the NLRP3 by MCC950.And inhbiting the autophagy by 3-MA,the expressions of the Beclin-1 and LC3Ⅱ were significantly decreased,P62 was significantly increased,but the expressions of NLRP3,Caspase-1,IL-18 and IL-1β were further increased.4.Under hypoxia condition,the apoptosis of the JEG-3 cells was significantly decreased and the invasion was significantly increased after inhibited the NLRP3 by MCC950.However,the apoptosis of the cells was further increased and the invasion was further decreased after inhibited autophagy by 3-MA.Conclusions1.Interfering the expression of lncRNA NEAT1 can inhibit the activation of NLRP3 inflammasome and autophagy and reverse the increase of apoptosis and the decrease of invasion induced by hypoxia.And these changes can be reversed again by over-expression of NLRP3.2.Autophagy was enhanced with the activation of NLRP3 inflammasome,and decreased with the inhibition of NLRP3 inflammasome.Inhibition of autophagy can aggravate the activation of NLRP3 inflammasome induced by hypoxia.3.Activation of NLRP3 inflammasome can lead to the damage of the cell functions,while inhibition of autophagy may aggravate the damage of cell functions caused by hypoxia by aggravating the activation of NLRP3 inflammasome.4.LncRNA NEAT1 can mediate the damage of trophoblast cell functions by regulating NLRP3 inflammasome and autophagy,and NLRP3 inflammasome and autophagy also have mutual regulation between each other,participated in the occurrence of PE.Part Ⅳ Regulation of lncRNA NEAT1 on NLRP3 inflammasome and autophagy in preeclampsia mice and the effect on the phenotypeObjectiveThe mouse model of PE was established to explore the regulation of lncRNA NEAT1 on NLRP3 inflammasome and autophagy,as well as the effects on the phenotype and pregnancy outcomes of mice,and then to clarify the mechanism of IncRNA NEAT1 in PE.MethodsPE mice model were established by intraperitoneal injection of lipopolysaccharide(LPS),and normal pregnant mice were used as the control.The lentivirus sh-NEAT1 was transfected into PE mice to interfere the expression of lncRNA NEAT 1,and sh-NC was transfected as the control group.The blood pressure of mice in normal,PE,PE+sh-NC and PE+sh-NEAT1 groups was monitored,and their placenta,fetus and kidney were collected.The mRNA expressions of lncRNA NEAT1 and NLRP3 inflammasome related factors in placenta were detected by qRT-PCR.The protein expressions of NLRP3 inflammasome,inflammatory factors and autophagy in placenta were detected by Western Blot.The expression of LC3 in placenta was detected by IF.HE staining was used to detect renal pathology.The number of fetuses and embryo absorption were recorded,and the weight of fetuses and placenta were weighed respectively.Results1.The expression of lncRNA NEAT1 in placenta of PE group was significantly higher than the normal group,while the expression of NEAT1 in PE+sh-NEAT1 group was significantly lower than the PE+sh-NC group.2.Before injection of LPS,there were no significant difference in blood pressures among the four groups.After injection of LPS,the blood pressures of PE and PE+sh-NC groups increased gradually,and were significantly higher than the normal group.And after interference with sh-NEAT1,the blood pressures of PE+sh-NEAT1 group decreased significantly compared with PE+sh-NC group.3.The kidney of PE group showed glomerular swelling,endothelial cell proliferation,capillary stenosis and renal capsule occlusion.And after interference with sh-NEAT1,the pathological changes of kidney in PE+sh-NEAT1 group were improved.4.The embryo absorption rate of PE group was significantly higher than the normal group,but the fetal and placental weights were significantly lower.After interference with sh-NEAT1,the embryo absorption rate of PE+sh-NEAT1 group was significantly lower than the PE+sh-NC group,and the fetal and placental weights were significantly higher.5.The expressions of NLRP3,Caspase-1,IL-18 and IL-1 β in the placenta of PE group were significantly increased,while those were significantly decreased after interference with sh-NEAT1.6.The expressions of Beclin-1 and LC3Ⅱ in the placenta of PE group were significantly increased,and the expression of P62 was significantly decreased.After interference with sh-NEAT1,the expressions of Beclin-1 and LC3Ⅱ were significantly decreased,while the expression of P62 was significantly increased.Conclusions1.The expression of lncRNA NEAT1 was up-regulated in the placenta of PE mice,while the NLRP3 inflammasome was activated and the autophagy was enhanced.2.Interference with lncRNA NEAT1 can inhibit the activation of NLRP3 inflammasome and autophagy in placenta of PE mice,and improve hypertension,kidney injury and pregnancy outcomes of the PE mice.3.The abnormal expression of lncRNA NEAT1 is involved in the occurrence and development of PE,and interference with NEAT1 can improve the phenotype and outcomes of PE mice by regulating NLRP3 inflammasome and autophagy.LncRNANEAT1 is expected to be a potential target for the treatment of PE.