Occurrence And Molecular Mechanism Of Stress Granules Formation In Severe Preeclampsia

Preeclampsia(PE)is a kind of hypertensive disorder complicating pregnancy which is caused by multiple factors,mechanisms and pathways,such as gene,inheritance,epigenetics and immunity.So far,its etiology and pathogenesis remain to be fully clarified.It poses a serious threat to maternal and infant health,with an incidence of 3-5%worldwide.As one of the main causes of maternal mortality and perinatal morbidity and mortality,PE is a kind of hypertensive disorder complicating pregnancy(HDP).With the continuous development of medicine,the classification of HDP is becoming more and more detailed.PE is a unique existence of HDP,which belongs to idiopathic diseases during pregnancy.In recent years,the studies on PE have been deepened and the relevant guidelines have been updated.No matter in the field of basic research or clinical research,the studies related to PE has made some progress.Traditionally,PE is defined as emerging hypertension complicated with proteinuria after pregnancy for 20 weeks.The new definition also includes maternal organ dysfunction,such as renal insufficiency,liver involvement,neurological or blood system complications,uterine and placental dysfunction and fetal growth restriction.Currently,the understanding of the etiology and pathogenesis of this disease is based on the two-stage theory of PE theory.The first stage is preclinical phase.During early embryogenesis,there are inadequate preparation of endometrium and superficial tunica muscularis(junction area),trophoblast ischemia and hypoxia,impaired trophoblast invasion function in muscular layer,spiral artery maladjustment,ischemia-reperfusion injury,placental oxidative stress and endoplasmic reticulum stress,release of a variety of cytokines(TNF-α,IL-2,etc.),anti-angiogenic factors(soluble Fms such as tyrosine kinase-1,sEng,etc.)or alarmins(uric acid,acellular fetal DNA,etc.)released into the maternal blood circulation system,thus causing the excessive activation of maternal systemic inflammatory reaction,extensive vascular endothelial damage,activation of white blood cells,complement and coagulation,thrombosis and activation of renin-angiotensin-aldosterone system,decrease of maternal blood volume and enhancement of vascular response.Eventually,it leads to diversified clinical manifestations of PE,and enter the second stage-clinical manifestation stage(elevated blood pressure,edema,hypoproteinemia,increased urinary protein,pulmonary edema,etc.).PE is a dynamic disease,which can progress continuously.In the absence of effective treatment,the treatment of PE is mainly to choose the best time to terminate pregnancy after stabilizing the condition of mother and fetus.This will inevitably bring an inevitable consequence-premature birth and its related complications,especially early-onset PE,which will have a more serious impact on the fetus.Simple preventive measures,such as diet and lifestyle intervention,calcium supplementation and aspirin,are effective,but it wasn’t significant.At present,there are many studies on the prediction of PE,but they have not been verified and cannot be widely used in clinical practice.There is no downward trend in the incidence of PE.Therefore,it is imperative to develop new intervention targets and treatment strategies.Stress granules(SGs)are dense granular substances formed in the cytoplasm of eukaryotic cells,which are mainly composed of various proteins and nucleic acids.SGs are closely related to the remodeling of messenger ribonucleoprotein(mRNP)under the stimulation of external environment.Under the stimulation of external environment,protein translation of eukaryotic cells is stopped,and untranslated mRNA and some proteins with mRNA binding ability gather together to form SGs in cytoplasm.SGs are formed when the initiation of translation is inhibited,so mRNAs that stop translation are recruited in these particles.The SGs are mainly composed of poly(A)+mRNAs containing 48 S initiation complex,small ribosome subunits,mRNA attenuation factor tristetraprolin,translation initiation factors such as eukaryotic translation initiation factor 4E(eIF4E),eIF4G,eIF4A and eIF4B,and many RNA binding proteins regulating mRNA structure and function,including HuR,Staufen,Smaug,TTP,fragile X mental retardation protein,G3BP,CPEB and SMN.SGs also contain some scaffold proteins,such as Fas-activated serine/threonine phosphoprotein.The aggregation of SGs is a variable and dynamic process,and its formation depends on the type of mRNPs in particles.Due to the different external stimulation conditions,the protein components of SGs are also different.The assembly of SGs is considered to be a protective mechanism of cells against various stress stimuli such as ischemia,hypoxia,oxidative stress,heat shock or viral infection.When the external environment stimulates,the process of protein translation is stopped.MRNAs are released from ribosomes and carry envelopes of specific RNA-binding proteins into SGs.Once the external stimulus is released,mRNAs leave the SGs structure and are then transported to cytoplasm for protein translation.SGs are highly conserved RNP cytoplasmic granules,which play an important role in translation regulation,inflammatory response,immune response,apoptosis,virus infection and cell damage caused by protein misfolding.Overactivation of inflammatory response is recognized as a factor related to PE.SGs play an important role in regulating inflammation and immune response.However,whether SGs are involved in the pathogenesis of PE is still unclear,and there are few related reports at present.In this study,tissue specimens(placenta of late pregnancy,including PE group and NC group)were used to detect the formation and localization of SGs by quantitative PCR,Western-blot and immunofluorescence,which confirmed the expression of landmark components of SGs in PE,and found that SGs in placenta of PE patients had obvious aggregation and formation phenomenon.It is also found that the serum of PE patients can induce the aggregation and formation of SGs in HTR-8/Svneo trophoblast line.CoCl2(a hypoxia inducer)was used to stimulate the hypoxia model of human extracorporeal trophoblast line HTR-8/Svneo,and study the formation of SGs under hypoxia.Furthermore,by knocking down the expression of kinase protein related to stress granule formation,the changes of stress granule formation under hypoxia were detected,so as to explore the related molecular mechanism of stress granule formation in PE.Chapter 1 Research on the Aggregation and Formation of Stress granules in Placenta of PEObjective:There are many studies on the pathogenesis of PE,and the over-activation of inflammatory immune response plays an important role in the pathogenesis of PE.Stress granules(SGs)play an important role in translation regulation,inflammatory response,apoptosis,virus infection and cell damage caused by protein misfolding.SGs and their related proteins are closely related to inflammatory immune response,and play a role in regulating inflammation and immune response.However,there is no relevant report on whether SGs are involved in the occurrence and development of PE.Here,we collect clinical samples of PE and test whether there is abnormal expression of SGs in placenta of PE through related experiments.Research methods:1.The clinical information of severe PE patients and matched pregnant women was collected,and them were divided into PE group(PE group)and normal control group(NC group).PE group was further divided into eo-PE group(early-onset PE group)and lo-PE group(late-onset PE group).The related clinical data of PE group and NC group were statistically analyzed by SPSS software.2.Hematoxylin and eosin(HE)staining were used to observe the pathological features of placenta in PE group and NC group under microscope.3.Real-time quantitative PCR was used to detect the difference of mRNA expression of stress granule related genes in placenta between PE group and NC group.4.Western-blot technique was used to detect the difference of HuR and G3BP expression in placenta of PE group and NC group.5.Immunofluorescence staining was used to detect the aggregation and formation of SGs in placenta of PE group and NC group.6.Immunofluorescence staining was used to detect the aggregation of SGs in HTR-8/Svneo cells treated with serum from PE group and NC group.Research results:1.SPSS analysis showed that there was no difference in age,height and hemoglobin between PE group and NC group,but there were significant differences in body mass index,systolic blood pressure,diastolic blood pressure,mean arterial pressure,platelets,alanine aminotransferase,aspartate aminotransferase,uric acid,lactate dehydrogenase,newborn birth weight and Apgar score.There was no significant difference in body mass index,platelets,alanine aminotransferase,aspartate aminotransferase,uric acid and lactate dehydrogenase between eo-PE group and lo-PE group.2.HE staining was used to analyze the pathological features of placenta,and non-specific pathological features of placenta in PE patients were found,including villus ischemic infarction,villus interstitial vasodilation and congestion,and fibrin deposition around villi.3.Real-time fluorescence quantitative PCR technique showed that the expression of SGs related genes elavll,lsm2,lsm4 and agolmRNA in placenta of PE group was significantly higher than that of NC group.There was no significant difference in mRNA expression of elavl1,1sm2,lsm4 and ago1 between eo-PE group and lo-PE group.4.Western blot was used to detect 2 classic SGs marker proteins HuR and G3BP by.The results showed that compared with NC group,the expression of HuR and G3BP protein in placenta of PE group increased significantly.Further grouping for Western-blot detection compared with real-time fluorescence quantitative PCR results were similar between eo-PE group and lo-PE group HuR expression in placenta was not statistically significant difference.5.By immunofluorescence experiment,we stained HuR and G3BP as marker proteins of SGs.The results showed that HuR green labeled fluorescent particles and G3BP red labeled fluorescent particles were obvious in placenta of PE group,while almost no labeled fluorescent particles were found in control group.6.HTR-8/Svneo cells were exposed to 10%serum of PE patients or matched serum of pregnant women for 24h.By immunofluorescence staining technique,it was found that the serum of PE patients could induce the aggregation of HuR or G3BP positive SGs in HTR-8/Svneo cells.Research Summary:The aggregation of HuR/G3BP positive SGs was observed in placenta of PE patients,and the aggregation of HuR/G3BP positive SGs in human villous trophoblast HTR-8/Svneo was induced by serum of PE patients.It is suggested that SGs are involved in the occurrence and development of PE.Chapter 2 Hypoxia-induced stress granule aggregation in HTR-8/Svneo cellsObjective:Under many external stimuli,such as sodium malonate,sodium arsenite,heat shock at 45℃,cold shock and virus infection,the cells will produce stress response and form stress granules(SGs).CoCl2 is a hypoxia inducer,which can be used to induce hypoxia environment in vitro.It is commonly used in the study of cell proliferation,differentiation and stress response.The two-stage theory of PE is based on the premise that hypoxia and insufficient blood supply of trophoblast result in trophoblast injury.The recasting disorder of uterine spiral artery leads to shallow implantation of placenta,continuous ischemia and hypoxia of placenta,continuous oxidative stress and inflammatory reaction,and finally leads to clinical onset.Here,hypoxia model was constructed by CoCl2 in vitro,so as to detect whether there is aggregation of SGs in HTR-8/Svneo cells under hypoxia.Research methods:1.The hypoxia model of trophoblast was constructed by CoCl2.The proliferation of trophoblast cells was detected by CCK-8 cell proliferation assay after CoCl2 stimulation for 0h,24h and 48h;cell scratch test was used to detect the cell migration of trophoblast cells after CoCl2 stimulation for 0h,24h and 48h.Western blot assay was used to detect the expression of HIF-1 a protein in trophoblast cells stimulated by CoCl2.2.Real-time quantitative PCR was used to detect the mRNA expression of stress granule-related genes in trophoblast cells after CoCl2 stimulation for 24h.3.Western blot was used to detect the expression of HuR,P-EIF2 α and 4EBP1 in trophoblast cells after CoCl2 stimulation for 24h.4.Immunofluorescence staining was used to detect the aggregation of SGs in trophoblast cells after CoCl2 stimulation for 24h.Research results:1.After treatment with CoCl2,the proliferation and migration of trophoblast cells were significantly inhibited,and the level of HIF1α protein was significantly increased.2.The expression levels of Elavl1,Ago1,Lsm2,Lsm4,Dcp1a,Ddx6 and Fubp1mRNA associated with trophoblastic SGs were significantly increased after treatment with CoCl2.3.After treatment with CoCl2,the expression levels of HuR and P-eIF2 α in trophoblast SGs increased significantly,while P-4EBP1 decreased significantly.4.After CoCl2 treatment,HuR/G3BP positive stress granule structure was observed by immunofluorescence staining,but hardly observed in the control group.Research Summary:The hypoxic model of HTR-8/Svneo cells constructed by CoCl2 could inhibit the proliferation and migration of HTR-8/Svneo cells and induce the aggregation of HuR and G3BP positive SGs in HTR-8/Svneo cells.SGs may play an important role in the occurrence and development of PE.Chapter 3 Research on molecular mechanism of hypoxia-induced stress granule formation in HTR-8/Svneo cellsObjective:According to the aforementioned research results,it was found that there is a phenomenon of stress granule aggregation in the pathogenesis of preeclampsia(PE),and the serum of PE patients can induce the aggregation of stress granules(SGs)in HTR-8/Svneo cells.The hypoxia model of HTR-8/Svneo cells was successfully established by using CoCl2,and the aggregation of SGs in the cytoplasm of HTR-8/Svneo cells induced by CoCl2 was successfully detected by real-time quantitative PCR,western-blot and immunofluorescence staining.EIF2α phosphorylation pathway and 4EBP1 phosphorylation pathway are two classical pathways involved in the formation of SGs.In order to clarify the molecular mechanism of stress granule formation in PE,we explored the pathway of stress granule formation in HTR-8/Svneo trophoblast by small RNA interference technique.Research methods:1.4 kinases(HRI,PKR,PERK and GCN2)and 4EBP1 in eIF2α pathway were knocked down by RNA interference,and the effect of gene silencing was detected by real-time quantitative fluorescence PCR.2.4 kinases(HRI,PKR,PERK and GCN2)and 4EBP1 in eIF2α pathway were knocked down by RNA interference.After treatment with CoCl2,the expression of P-eIF2α and 4EBP1 was detected by western blot.3.RNA interference and immunofluorescence staining were used to detect the changes of SGs formation after CoCl2 treatment under different interference pathways.Research results:1.4 kinases and 4EBP1 in eIF2α phosphorylation pathway were knocked down by small RNA interference technique,and the silencing effect after knockdown was detected by real-time quantitative PCR technique.We found that the expression of four kinases in HTR-8/Svneo cells was inhibited after gene knockdown.The expression of 4EBP1 was also down-regulated.2.4 kinases affecting the formation of SGs were knocked down by RNA interference technique,and then HTR-8/Svneo cells were cultured under hypoxia induced by CoCl2 for 24h.It was found that HRI siRNA could significantly weaken eIF2α phosphorylation induced by CoCl2 and inhibit the formation of SGs induced by CoCl2.3.RNA interference technique was used to knock down 4EBP1,and then cultured HTR-8/Svneo cells under hypoxia induced by CoCl2 for 24h.It was found that 4EBP1 siRNA also significantly inhibited the formation of SGs induced by CoCl2.Research Summary:RNA interference with HRI pathway weakened CoCl2-induced eIF2α phosphorylation,while interference with other three kinase pathways had no effect.After RNA interference with 4EBP1,the expression of 4EBP1 was decreased,and the formation of SGs induced by CoCl2 was reduced.HRI-mediated eIF2α phosphorylation pathway and 4EBP1 pathway are involved in CoCl2 induced SGs formation in villous trophoblast cells.Research Conclusion:1.The aggregation of HuR/G3BP positive SGs was observed in placenta of PE patients.It is suggested that SGs may be involved in the occurrence of PE.2.The hypoxic model of HTR-8/Svneo cells constructed by CoCl2 could inhibit the proliferation and migration of HTR-8/Svneo cells and induce the aggregation of HuR and G3BP positive SGs in HTR-8/Svneo cells.It is suggested that the formation of SGs may be related to the severity of PE.3.RNA interference with HRI pathway weakened CoCl2-induced eIF2α phosphorylation,while interference with other three kinase pathways had no effect.After RNA interference with 4EBP1,the expression of 4EBP1 was decreased,and the formation of SGs induced by CoCl2 was reduced.HRI-mediated eIF2α phosphorylation pathway and 4EBP1 pathway are involved in CoCl2 induced SGs formation in villous trophoblast cells.It is suggested that SGs may become an effective monitoring index for early diagnosis,condition monitoring and prognosis follow-up of PE in the future.