| Objective: Preeclampsia(PE)is a disorder characterized by hypertension in pregnancy.Neonatal hypoxic-ischemic encephalopathy(HIE)is one of the common clinical diseases in the offspring of PE patients,leading to neurodegenerative changes,cognitive changes,severe mental retardation,learning and behavior problems,motor disorders,and so on.Neonatal hypoxic-ischemic encephalopathy involves reduced oxygen supply to the blood and reduced cerebral blood flow.Both processes lead to immediate neuronal death(necrosis)in the embryo or delayed neuronal cell death(apoptosis),which causes permanent brain damage to the offspring.DNA methylation plays an important role in the development and neurodegeneration of the central nervous system.During ischemia and hypoxia,the DNA methylation level of embryonic brain tissue changes.In Down Syndrome,three copies of DNMT3 L could be responsible for extensive genome methylation,and the expression of some genes related to neural development was altered.At the same time,high DNA methylation levels increased neuronal apoptosis,but these mechanisms were not clear.Therefore,on the basis of existing studies on neural development in preeclampsia fetuses,it is important to further explore the molecular mechanism of whether DNMT3 L induces neuronal apoptosis through DNA methylation for our understanding of neural development and the underlying neuropathology of degenerative diseases.Methods:Section 1:Effects of preeclampsia on rat embryonic neuronsThe SD female rats in the experiment were divided into Sham and PE groups.The abdominal aorta and uterine artery were coarcted in female rats in the PE group at 13.5 days of pregnancy.At 12.5d and 19.5d of pregnancy,the pregnant female rats were placed in metabolic cages,and urine was collected for 24 hours to detect the total protein content.At 20.5 d of pregnancy,the carotid artery blood pressure of the pregnant rats was measured;HE staining was performed on the kidneys of pregnant rats.At 20.5 days of pregnancy,the uterus was separated,and the fetal rats were taken out to measure the body weight and brain tissue weight.The prefrontal cortex of fetal rat was taken for:(1)TUNEL staining on frozen sections was used to detect the number of apoptotic cells;(2)neuronal changes were observed under a transmission electron microscope;(3)m RNA and genomic DNA were extracted for q PCR detection;(4)total protein was extracted and the protein level was detected by Western blot;(5)genomic DNA was used to detect the overall methylation changes of genomic DNA by dot blot hybridization;(6)after sulfite conversion of genomic DNA,MSP was used to detect changes in Lmna promoter methylation.Section 2:The regulation mechanism of hypoxia-inducible factor 1A(HIF1A)on methyltransferase 3L(DNMT3L)Rat hypoxia-inducible factor 1 subunit alpha(Hif1a)overexpression plasmid,luciferin plasmid containing DNA methyltransferase 3 like(Dnmt3l)or DNA methyltransferase 3 alpha(Dnmt3a)promoter were constructed;these plasmids were then used for double luciferase assay.The binding site of HIF1 A to Dnmt3 l promoter was determined by site-directed mutation assay and chromatin immunoprecipitation.After overexpression of Hif1 a in PC12 cells,the regulation of HIF1 A on DNMT3 L and DNMT3 A was verified by q PCR and Western blot.Section 3:Mechanism of HIF1A-DNMT3L-LMNA axis on apoptosisPackaged rat Dnmt3 l overexpression virus,infected with primary neurons in rats,m RNA extracted to detect Lmna changes.In PC12 cells:(1)q PCR and Western blot detected changes in Lmna m RNA and protein levels after overexpression of Hif1 a and Dnmt3l;(2)flow cytometry detected apoptosis after overexpression of Dnmt3 l or knocking down Lmna;(3)double luciferase assay was used to detect the luciferase activity of Lmna promoter after overexpression of DNMT3 L and DNMT3 A,and q PCR was used to detect the expression of Lmna in cells treated with different concentrations of decitabine after overexpression of Dnmt3a;(4)overexpression of Hif1 a while knocking down Dnmt3 l,Western blot was used to detect changes in LMNA.In Dnmt3 l knock-in mice,Western blot and q PCR detected embryo 20.5 d(E20.5 d)LMNA protein and Lmna m RNA changes in the prefrontal cortex,and used transmitted electron microscopy to observe the nuclear membrane changes of prefrontal neurons;immunoprecipitation of prefrontal protein was performed to verify the protein interaction between DNMT3 L and LMNA.In in vitro GST pulldown experiments,the results confirmed that there was a direct relationship between DNMT3L and LMNA.Results:Section 1:Effects of preeclampsia on rat embryonic neurons1.The PE animal model was successfully constructed.At 20.5 days of gestation,the female rats in the PE group had increased proteinuria,increased arterial blood pressure,and manifested renal hypertension and nephropathy.2.The fetal body weight and brain tissue weight of the PE group decreased.3.The prefrontal cortex of fetal rats:(1)In TUNEL staining of the frozen section,the number of apoptotic cells increased in the PE group;(2)under an electron microscope,abnormal nucleus,incomplete nuclear membrane,and cell membrane were observed in PE group,and damaged mitochondria were observed;(3)q PCR and Western blot methods showed that the expression levels of HIF1 A,DNMT3A and DNMT3 L increased in the prefrontal cortex of PE group,and the apoptosis-related protein level increased,and the nuclear membrane related protein LMNA decreased;(4)the global methylation changes in PE group increased;(5)using MSP to detect methylation sites,Lmna promoter methylation level was significantly increase in the PE model.Section 2: The regulation mechanism of HIF1 A on DNMT3L4.HIF1 A had transcriptional activation effect on Dnmt3 l and Dnmt3 a promoters,and the transcriptional activation sites of HIF1 A and Dnmt3 l promoters were confirmed by a mutation kit..5.The chromatin immunoprecipitation experiment verified the regulatory site of HIF1 A on the Dnmt3 l promoter.6.In PC12 cells,it was verified that overexpression of Hif1 a could increase the expression levels of Dnmt3 l and Dnmt3 a m RNA and protein.Section 3: Mechanism of HIF1A-DNMT3L-LMNA axis on apoptosis7.Lmna m RNA expression was decreased in rat neuronal neurons after infection with lentivirus overexpressing Dnmt3 l.8.In PC12 cells,after overexpression of Hif1 a and Dnmt3 l,the expressions of Lmna and LMNA detected by q PCR and Western blot decreased;DNMT3L had a transcriptional inhibition effect on the promoter of Lmna,and this effect was independent of DNMT3 A to regulate Lmna through the non-methylated pathway.9.After Dnmt3 l overexpression or Lmna knockdown,the amount of apoptosis of PC12 cells increased.Hif1 a was overexpressed,while Dnmt3 l was knocked down,and the reduced level of LMNA recovered.10.In Dnmt3 l knock-in mice,expression of LMNA protein and Lmna m RNA in the prefrontal cortex of E20.5 d fetuses were decreased;nuclear membrane of prefrontal neurons was incomplete by transmission electron microscope;by immunoprecipitation,the protein interaction between DNMT3 L and LMNA was verified.11.GST pulldown experiment verified that DNMT3 L and LMNA had a direct interaction relationship,and DNMT3 L bound to the(nuclear localization sequence)NLS region of LMNA.Conclusion:1.In the preeclampsia model of fetal rat(E20.5 d),The expressions of HIF1 A,DNMT3A and DNMT3 L were increased,and the overall methylation level was increased.The nuclear membrane of neurons in the prefrontal cortex was incomplete.The expression of LMNA decreased,and the apoptosis-related protein increased,resulting in the phenomenon of apoptosis of prefrontal neurons.2.It was found that HIF1 A could act as a Dnmt3 l transcription factor to activate its transcription,which may also be one of the important mechanisms affecting neural development in the development of HIE.3.Increased Dnmt3 l expression or decreased Lmna expression led to apoptosis.DNMT3 L regulated Lmna promoter through non-methylation pathway independently of DNMT3 A.HIF1A could regulate LMNA through DNMT3 L,leading to apoptosis. |
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