Preliminary Study On PTPRT Methylation Modification And Learning And Memory Ability Of Offspring In Hippocampus Of Streptozotocin-Induced Gestational Diabetes Rats

Part 1:Effect of Gestational Diabetes on Learning and Memory Ability of OffspringObjective:To establish a model of GDM offspring and explore the influence on the learning and memory ability of the GDM offspring.Method:Pregnancy SD rats were randomly divided into control group and GDM group.The GDM group was injected with 25 mg/kg streptozotocin(STZ)solution on days 5.The control group received the same amount of citric acid-sodium citrate buffer in the tail vein for 5 days.All pregnant rats were weighed on days 13,16 and 19,respectively.Blood glucose and blood insulin levels were measured in pregnant mice based on ELISA on days 0,7 and 14 of pregnancy.The erythropoietin(EPO)in amniotic fluid at birth was also measured.The offspring of the two groups were tested with Morris water maze(MWM)at the age of 8 weeks as well as 1 year.The effect of GDM on the spatial learning and memory ability of the offspring was tested with MWM.Results:1.Compared with the control group?the body weight,fasting glucose,fasting blood glucose(FBG),Fasting insulin(FINS)were significantly elevated in GDM group.What's more,the litter size,birth weight were also significantly reduced in GDM offsprings than that in control group.The EPO content in amniotic fluid in GDM group was significantly higher than that in control group(P<0.01).2.When the two groups was in 8 weeks old the overall performance of GDM offspring in MWM test was significantly weaker than that in the control group.However when the offspring were 1 year old,the experiment was carried out again.At that time no significant difference in MWM was found between the two groups.Conclusion:This experiment successfully established the model of GDM offspring.Through the MWM,it was found that the GDM can affect the learning and memory ability in the early life of its offspring(8 weeks old)(p<0.05).GDM offspring performed significantly worse than the control group in terms of spatial learning and memory ability in 8 weeks old but this difference was improved when the offspring were 1 year oldPart 2:Exploring the Key genes of GDM Intrauterine Environment Affecting Learning and Memory Ability of OffspringObjective:To screen and verify the key genes of intrauterine environment of GDM affecting the learning ability of offspringMethods:According to the scheme in the first part of the study,GDM and control group offspring rat models were established,and the whole genome DNA of GDM and normal pregnant offspring rat hippocampal tissue was extracted to prepare MeDIP-seq(methylated DNA immunoprecipitation sequence).After obtaining the original data,high-quality data were screened out.KOBAS was used to localize the differential sequences to the gene elements.BOWTIE was used to compare the obtained sequences with the reference genome.MACS was used to identify the MeDIP peaks of a single sample for the analysis of differentially methylated genes.The candidate of methylation specific key genes was screened out,and the PCR primer design of candidate methylation specific genes was carried out,and the real-time quantitative PCR reaction amplification was conducted.At the same time,primary hippocampal neurons were isolated and cultured,and 5-azo deoxycytidine was used to inhibit DNA methylation,and then protein extraction was used to detect the protein expression level of key genes by Western Blot,so as to explore the inhibitory effect of methylation on the expression of key genes.Results:1.Compared with the control group,specific-difference methylation genes(DMGs)in the GDM group were 256.Among them,a total of 180 hypomethylated genes and 76 hypermethylated genes.Five genes,PTPRT,PP2BA,ESR2,SLC2A1 and PPP1CA,were selected as candidate key genes for DMGs in GDM progeny brain tissues.2.Results of methylation specific PCR showed that non-methylation bands were amplified in all the 5 samples of the control group,while methylation bands were amplified in the GDM group,and the methylation level of PTPRT gene was the most significant among the five key genes.3.The relative mRNA expression levels of the five candidate key genes PTPRT,PP2BA,SLC2A1,PPP1CA and ESR2 in the GDM group were significantly lower than those in the control group(P<0.01;P<0.01;P<0.01;P<0.01;P<0.01),But the reduction in PTPRT was the most significant.4.In the protein expression levels of five candidate key genes PTPRT,PP2BA,SLC2A1,PPP1CA and ESR2 in GDM group,the expression level of PTPRT was significantly lower than that of the control group.5.The expression of PTPRT was inhibited in primary cells of GDM group.The expression level of PTPRT in the primary cells of GDM group was partially recovered by inhibiting DNA methylation with 5-azo deoxycytidine.Conclusion:There were specific difference methylation genes in the brain tissue of GDM offspring.The PTPRT gene had a good correlation with the methylation of GDM group.Whether the PTPRT gene is related to the learning and memory ability of the offspring of GDM rats needs to be confirmed by further experiments.Part 3:Study on the Effect and Molecular Mechanism of PTPRT Gene on the Cell Cycle and Apoptosis of Hippocampal Neurons in RatsObjective:To investigate the effect of PTPRT gene on cell cycle and apoptosis in rat hippocampal neurons,and to explore its molecular mechanism.Method:The PTPRT overexpression plasmid vector and the interference plasmid vector were separately constructed and transfected into rat hippocampal neurons.The subjects were divided into 4 groups:(1)Overexpression control group(Vector group);(2)Overexpressing PTPRT group(EGFP-PTPRT group);(3)Knockdown control group(siNC);(4)Knockdown of the PTPRT group(PTPRT-siRNA group).The relative expression of mRNA was detected by mRNA qRT-PCR.The protein expression level was detected by Western Blot.The cell cycle and apoptosis were detected by flow cytometry.The influence of apoptosis and molecular mechanism,and the relationship between PTPRT and synaptic vesicle fusion-related protein Syntaxin and its binding protein STXBP1(Syntaxin-binding protein 1)were detected by immunoprecipitation,and the mechanism of PTPRT-indueed learning and memory dysfunction was preliminarily explored.Results:1.The content of Cyclin A,the S phase marker protein of cell division in the knockout PTPRT group was significantly lower than that in the siNC group,and the content of Cyclin A in the overexpression PTPRT group was slightly higher than that in the Vector group.2.Flow cytometer showed that the number of cells in S phase in the PTPRT overexpression group was significantly higher than that in the Vector group,the proportion of cells in the S phase in the Vector group and the siNC.group was similar,and the number of cells in the S phase in the PTPRT-siRNA group was significantly lower than that in the siNC group.3.The mRNA qRT-PCR results showed that the relative mRNA expression of the apoptotic antagonist protein Bcl-2 in the hippocampal neurons of the EGFP-PTPRT group was significantly higher than that of the Vector group(P<0.01),while the relative mRNA expression of the apoptosis-related protein BAX was significantly lower than that of the Vector group(P<0.01).The relative mRNA expression of Bcl-2 in hippocampal neurons of PTPRT-siRNA group was significantly lower than that of siNC group(P<0.01),and the relative mRNA expression of BAX was significantly higher than that of siNC group(P<0.01).4.Western Blot analysis of apoptosis-related protein expression level showed that the protein expression level of Bcl-2 gene in the PTPRT-siRNA group was significantly lower than that of the other three groups including the siNC control group,while the BAX,Cleaved caspase-3 and Cleaved caspase-9 protein expression level was higher than that of the other three groups.The PTPRT overexpression group is exactly the opposite.5.By Co-immunoprecipitation,PTPRT overexpression(EGFP-PTPRT group)promoted the binding of STXBP1 and Syntaxinl and increased the expression of STXBP1 and Syntaxinl.Knockdown of PTPRT(PTPRT-siRNA group)inhibited the binding of PTPRT to STXBP1 and Syntaxin1,and decreased the expression of STXBP1 and Syntaxinl.Conclusion:Overexpression of PTPRT gene can increase the proportion of cells in the hippocampal neurons of S-phase cells,antagonize the apoptosis of rat neuronal cells and increase the expression of STXBP1 and Syntaxinl.In contrast,knockdown of PTPRT gene reduced the proportion of cells in the hippocampal neurons of S-phase cells,promoted neuronal apoptosis and decreased the expression of STXBP1 and Syntaxinl.Part 4:Preliminary Study on the Mechanism of PTPRT Gene Affecting Learning and Memory Ability of GDM Offspring RatsOBJECTIVE:To explore the effect of PTPRT gene on spatial learning and memory in GDM offspring rats.Method:A viral plasmid vector linked to the PTPRT gene was constructed.Lateral ventricle cannulation was performed,and followed by the MWM.The offspring were divided into 4 groups for lateral ventricle injection before the MWM.The names of the 4 groups and the injected drugs were respectively:(1)control group:normal offspring rats,injected artificial cerebrospinal fluid(2)GDM group:GDM Offspring rats,injected with artificial cerebrospinal fluid(3)Lentivirus control group:GDM offspring,injected with empty plasmid;(4)Lentivirus overexpression PTPRT group:GDM offspring,injected with PTPRT lentiviral plasmid.The corresponding indicators of the water maze test were recorded to explore the effect of PTPRT gene on the learning ability of GDM offspring rats.Results:1.The escape latency of the GDM group was significantly prolonged in the D1,D2,D3,and D4 days of the directional navigation test compared with the control group(P<0.0001,P<0.0001,P<0.0001,P<0.001).The over-expression group(GDM+PTRRT)was significantly smaller in the D1,D2,and D3 days than in the GDM+Vector group,but at the D4 day,it was comparable to the GDM+Vector group(P<0.001,P<0.001,P<0.001,P>0.05).Compared with the control group,the the swimming distances in GDM group on D1,D2,D3 and D4 days were significantly prolonged(P<0.01,P<0.0001,P<0.0001,P<0.01).In the overexpression group(GDM+PTRRT)group,the swimming distance in the D1,D2,and D3 days directional navigation test was significantly smaller than that in the GDM?Vector group,but there was no significant difference in swimming distance between the D4 and GDM+Vector groups(P<0.05,P).<0.01,P<0.05,P>0.05).2.In the space exploration experiment of Morris water maze,the number of GDM progeny rats entering the target area was significantly lower than that of the control group(P<0.0001);while the number of GDM+PTPRT overexpression groups entering the target area was significantly higher than that of the overexpression control group.The group(P<0.05);the GDM progeny group had a shorter target time in the target quadrant than the control group(P<0.0001);the GDM+PTPRT overexpression group stayed in the target quadrant significantly longer than the GDM group.(P<0,05).3.The expression level of PTPRT in each group is consistent with its typical Western Blot performance.The expression levels of PTPRT,Syntaxin 1 and STXBP1 in the GDM group were lower than those in the control group.In the overexpressed PTPRT vector group,the content of Syntaxin 1 and STXBP1 was slightly higher than that in the GDM group.Conclusion:Methylation of PTPRT gene is associated with decreased spatial learning and memory in GDM progeny rats.Overexpression of PTPRT gene can partially improve spatial learning and memory ability in GDM offspring rats.Summary:This study reveals the relationship between the methylation of PTPRT gene and the decline of spatial learning ability in hippocampal neurons of GDM offspring rats from levels of gene,transcription,protein and behavioral experiments,and preliminary proved that the overexpression of PTPRT gene can partially improve the spatial learning and memory ability of GDM offspring rats.