The Study Of The Relationship Between RNA N6-methyladenineand Alzheimer’s Disease

Background:Alzheimer’s disease(AD)is the most common form of dementia among the elderly and is characterized by impairment of cognitive and memory functions.At present,about 50 million people worldwide suffer from AD and related diseases With the extension of life expectancy,it is estimated that this number will increase to 80 million by 2030 and 160 million by 2050.With the aggravation of population aging in China,the prevalence rate of AD is increasing year by year.According to statistics,the number of AD patients among the elderly population in China has exceeded 6 million,and it is estimated that the number of AD patients will exceed 20 million by 2050.It is the region with the largest population and the fastest growth rate of AD patients in the world,bringing a heavy burden to patients,families,society and medical treatment.AD has complex etiology and pathogenesis,which is not fully understood at present.In the treatment of AD,there is a lack of effective radical means,mainly symptomatic treatment.Therefore,it is an urgent problem to study the etiology and pathogenesis of AD and seek effective prevention and treatment measures accordinglyThe deposition and excessive aggregation of amyloid-β peptide(Aβ)in the brain tissue are the major etiology and pathologic changes of AD.However,the specific mechanism of AD is still unclear.Currently,the widely recognized mechanisms include:A kind cascade reaction theory,Tau protein theory,synaptic dysfunction,au tophagy dysfunction and mitochondrial dysfunction,including mitochondrial dysfunction,mitochondrial autophagy disorder and mitochondrial dynamics disorder.Both genetics and epigenetics are involved in the pathogenesis of AD.m6A is the most abundant modification in eukaryotic RNAs,accounting for more than 80%of RNA base methylation,and exists in a variety of species.With advances in technology,methyltransferase complexes including METTL3,METTL14,Wilms tumorel binding protein(WTAP)have been identified.The demethylase mainly consists of:fat mass and obesity-associated protein(FTO),which is the first protein found to catalyze the demethylation of m6A,and its homologue 5(A1kB Homologue 5).In addition,YT521-B homology(YTH)family structure including YTHDF1,YTHDF2,YTHDF3,YTHDC1 and heterogeneous nuclear ribonucleoprotein A2B1(HNRNPA2B1)are proved to be m6A methylation binding protein that play a regulatory role by affecting mRNA stability,output,translation,splicing,and decay.ALKBH5,on the other hand,is a demethylase similar to FTORecent studies have shown that dysregulation of RNA methylation is associated with many biological processes,including neurodevelopmental and neurodegenerative diseases.However,is m6A RNA methylation involved in the pathogenesis of AD,and how is it regulated by what mechanism?Does it affect synaptic function and thus regulate the pathogenesis of AD?Or by affecting the mitochondrial autophagy pathway?Therefore,in this study,we explored the differential expression of RNA m6A methylation in AD model and control group,and explored the expression of methylase in AD model.At the same time.lentivirus-mediated RNA interference transfection was used to observe the changes in the expression levels of related genes,autophagy related proteins,and the changes in the ultras tructure of mitochondria under projection electron microscopy after METTL3 RNA interference in AD cell model,and to speculate the possible mechanism of RNA m6A methylation in AD.At the same time,we preliminarily observed the possible role of circRNAs in the pathogenesis of AD.Part I mRNA N6-methyladenine is involved in the pathogenesis of Alzheimer’s disease by regulating synaptic functionObjectivesCombined with the above background,this research uses the classic APP/PS1 double transgenic mice as the AD animal models,with Aβ25-35 deal with 24 hours of SH-SY5Y cells as the AD cell model,the main content of the research include:1.through high-throughput sequencing in the AD animal models and the control group were observed mRNA m6A methylation expression profiles.2.To observe the expression of methylated modifying enzyme in different brain tissues of the two groups of mice.3.Observe the occurrence mRNA m6A methylation differentially expressed genes,gene expression related to synaptic function quantity.The purpose is to determine whether there is involvement of mRNA m6A methylation in the pathogenesis of AD,and to explore the possible mechanism of mRNA m6A methylation in the pathogenesis of AD,so as to provide a new idea and direction for the search for therapeutic targets of AD.Methods1.Animal tissue:Male APP/PS1 mice aged 9 months were used as the animal model of AD in the experimental group and male C57BL/6 mice aged the same month were used as the control group.Each group had 15 mice2.The cell model,SH-SY5Y as control group,Aβ25-35 processing as AD SY5Y cells model of 24 hours,at 37 ℃,5%CO2 conditions,regular training in DMEM medium,containing 10%fetal bovine serum3.mRNA m6A methylation sequencing.High-throughput sequencing of mRNA m6A methylation was performed on the hippocampus of 9-month-old APP/PS1 mice and C57BL/6 control mice,with 3 mice in each group4.Quantitative detection of mRNA m6A methylation level.This part use animals to test:the AD model group and control group in mice brain tissue in mice,divided into cortex,hippocampus and cerebellum 3 part of the organization,each part of the extracted RNA,respectively,using mRNA m6A methylation quantitative kit is complete.5.The expressions of METTL3 and FTO were detected in AD animal model group and control group.Two groups of mice were constructed,cortex,hippocampus and cerebellum of histones,using Western Blot method for validation.6.According to the result of high-throughput sequencing,methylation of the differentially expressed 3 genes related to synaptic function,there are two gene mRNA in the AD animal models m6A methylation level is raised,the other genes in the AD model mRNA m6A methylation level is lower,the qRT-PCR methods to verify the amount of gene expression.Results1.High-throughput sequencing results showed that there were a large number of genes differentially expressed in mRNA m6A methylation in the hippocampal tissues of mice in AD model group compared with those in control group,many of which were involved in synaptic function.2.Quantitative results of mRNA m6A methylation level:Compared with the control group,the quantitative levels of mRNA m6A methylation in the cortex and hippocampus of the AD animal model group were increased,and there was no statistical difference in the levels of mRNA m6A methylation in the cerebellar area.3.Expression of methylase in the brain of the two groups:the protein expression of methyltransferase METTL3 in the cortex and hippocampus was increased in the AD model group compared with the control group,and there was no statistical difference in the cerebellum;Compared with the control mice,the protein expression level of demethylase FTO in the hippocampus of AD model was decreased,while there was no statistical difference in the protein expression level in the cortex and cerebellum.4.Gene ontology(GO)analysis and KEGG pathway analysis showed that mRNAs differentially expressed in mRNA m6A methylation are related to dendritic development,transport,positive regulation of cell components and processes,tissue or biogenesis of cell components,nervous system development,as well as the binding and catalytic activity of specific substances(such as proteins,enzymes,metal ions,cations and heterocyclic compounds.KEGG pathway analysis predicted the pathways affected by changes in methyation of mRNAs m6A in AD brain tissue,including those related to synapses,glutamate synapses,axonal guidance,long-term enhancement and calcium signaling pathways.5.Three genes with differentially expressed methylation related to synapse function were screened out,and their gene expression levels were statistically different between the two groups.Among them,the mRNA m6A methylation levels of AMPA and NMDA were down-regulated in the AD animal model group,while their gene expression levels were increased in the AD animal model group,while the SEMA gene showed opposite trend to the two genes mentioned above.Conclusion1.Compared with the control group,AD model mice had a large number of genes differentially expressed in mRNA m6A methylation,so we speculated that mRNA m6A methylation might play an important role in the pathogenesis of AD.2.We speculate that mRNA m6A methylation,as an important epigenetic regulation mode,may be involved in the pathogenesis of AD by regulating synaptic function,but this needs to be confirmed by further studies.Part Ⅱ mRNA N6-methyladenine is involved in the pathogenesis of Alzheimer’s disease by influencing autophagyObjectivesCombined with the above research background,the changes of gene expression levels of differentiated mRNA m6A after METTL3 RNA interference were studied,and the changes of autophagy-related proteins and mitochondrial ultrastructure in AD cell model and control group were also detected after METTL3 RNA interference.To further explore the possible mechanism of mRNA m6A methylation involvcd in ADMethods1.Cell culture:same as part 1.2,Quantitative mRNA m6A methylation kit was used to detect the inethylation level of mRNA in6A in AD cell model and control group3.Western Blot was used to detect the protein expression levels of METTL3 and FTO in the two groups of cells.4.Construction of lentivirus-mediated METTL3 RNA interference vector Shanghai Jikai Company assisted in the construction and packaging of lentivirus vector.A total of 3 primer targets were provided.5.Three lentivirus vectors were transfected into SH-SY5Y cell lines and a blank control group was set up.After 72 hours,the transfection rate of lentivirus was observed by fluorescence microscope.qRT-PCR was also used to further verify the transfection efficiency of lentiviral vectors.The target with high transfection efficiency was selected for reserve.6.Cell group:select stable transfection bead,the cells are divided into SH-SY5Y,slow virus empty vector group and METTL3 RNA interference group,and then use A β25-35 respectively dealing with the above cells for 24 hours,the cells were divided into 4 groups,SH-SY5Y group,A β-SY5Y group,Aβ empty carrier group,Aβ-METTL3-SY5Y group(METTL3 AD cell model of RNA interference)7.Verify the expression levels of genes related to autophagy in mRNA m6A methylated differential expression.qRT-PCR was used to detect the gene expression of autophagy related genes.A total of 4 genes(MAPK,Mtor,PI3K,CDC42)were screened for detection8.Detection of expression levels of mitochondrial autophagy-related proteins:Proteins from the cells were extracted and the expression of mitochondrial autophagy-related protein Parkin was detected by Western Blot9.The ultrastructure changes of mitochondria were observed by transmission electron microscope.The ultrastructure of mitochondria was observed by electron microscopyResults1.The quantitative detection results of mRNA m6A methylation level showed that the methylation level of mRNA m6A in the AD cell model group was higher than that in the control group.2.Western Blot analysis of methylase protein expression showed that the protein expression level of METTL3 in AD cell model group was higher than that in the control group,while the protein expression level of FTO in the two groups showed no statistical difference.3.After the use of lentivirus vector mediated METTL3 RNA interference,the expression level of METTL3 gene was significantly decreased and the transfection efficiency was high.One target reduced METTL3 gene expression by up to 90%and was used in subsequent experiments.4.It was verified at the cell level that there were statistically significant differences in the gene expression levels of m6A methylation differential expression associated with autophagy.Among them,the expression level of MAPK gene in AD cell model was increased compared with that in the control group,while the expression level of MAPK gene was decreased in AD cell model after METTL3 RNA interference.Mtor and PI3K showed opposite trend to MAPK.The expression level of CDC42 gene in AD cell model was decreased compared with that in the control group,while there was no statistical difference in the expression level of CDC42 gene in AD cell model and AD cell model with METTL3 RNA interference.5.The expression of Parkin,a protein related to mitochondrial autophagy,showed that the protein expression in AD cell model was increased compared with that in the control group,while the protein expression in AD cell model after METTL3 RNA interference was decreased.6.The ultrastructure of mitochondria is different under transmission electron microscope.The mitochondrial structure of SH-SY5Y cells was clear,the membrane structure was complete,and the cristae were clearly visible.The empty vector virus transfected cells showed no significant difference from SH-SY5Y cells in the mitochondrial structure,while the mitochondria in the AD cell model showed large volume,swelling,incomplete structure,unclear cristae,and many damaged,degenerate and fragmented mitochondria.Most of the AD model cells after METTL3 RNA interference had clear and complete:mitochondrial structuresConclusion1.Compared with the control group,there were differences in the methylation level of mRNA m6A in the AD cell model,the protein expression level of methyltransferase METTL3 in the two groups was different,and the expression levels of some genes related to autophagy were different in each group.The mitochondrial structure of the AD cell model was changed after the interference of METTL3 RNA,suggesting that mRNA m6A methylation may be involved in the pathogenesis of AD.2.mRNA m6A methylation may be involved in the pathogenesis of AD by affecting the autophagy function.Part Ⅲ circRNAs N6-methyladenine is involved in the pathogenesis of Alzheimer’s diseaseObjectives1.The AD animal model group and control group in circRNAs m6A methylation spectrum,from methylation screening differentially expressed genes in which gene detects the change of gene expression to explore circRNAs m6A whether methylation may be involved in the pathogenesis of AD2.To explore the mechanism by which circRNAs may participate in the pathogenesis of AD.Methods1.Animal tissue:Male APP/PS1 mice aged 9 months were used as the AD animal model,and male C57BL/6 mice aged the same month were used as the control group.There were 10 mice in each group.2.CircRNAs m6A methylation sequencing.High-throughput circRNAs m6A methylation sequencing was performed on the hippocampus of 9-month-old APP/PS1 mice and C57BL/6 control mice,with 3 mice in each group.3.According to high-throughput sequencing results,5 genes with differential methylated expression related to autophagy were screened out,and their gene expression levels were verified by qRT-PCR.Results1.Compared with the hippocampal area of the control group,the hippocampal area of the AD model group showed a large number of differentially expressed circRNAs,and many genes were involved in autophagy.2.The distribution of circRNAs m6A methylated genes in each chromosome was different between the two groups.In AD animal model mice,circRNAs m6A methylated genes were distributed on all chromosomes,and the first five chromosomes with more up-regulated genes included chromosome 1,2,5,7 and X,while the first five chromosomes with more down-regulated genes included chromosome 1,2,5,6 and 9.3.Among the 5 genes screened out for differential expression of methylation related to autophagy function,the expression levels of some genes were statistically different in the two groups:the gene expression levels of PAK3 in the AD animal model group were decreased compared with the control group,while the gene expression levels of Erbb4 showed an opposite trend;in addition,the gene expression levels of Axin2,ache and USP7 in the mice of the two groups were not statistically differentConclusion1.There are a large number of circRNAs m6A methylated differentially expressed genes in AD model mice,and some genes have different expression levels,suggesting that circRNAs m6A methylation may be involved in the pathogenesis of AD.2.Methylation may involve in the pathogenesis of AD through affecting autophagy.