LncRNAs Expression And Function Analysis In Prefrontal Cortex Of Alzheimer's Disease Patients And A Mouse Model

Background Alzheimer's disease(AD)is a common neurodegenerative disorder characterized by progressive cognitive dysfunction and behavioral impairment in the elderly.AD is characterized by the accumulation of amyloid-p(A?)plaques and neurofibrillary tangles,synaptic and neuronal loss in the hippocampus and cortex whose pathogenesis remains unknown.Long non-coding RNAs(IncRNAs)constitute a class of long(>200 nucleotides[nt])endogenous non-coding RNA molecules originally perceived to be the non-coding transcripts.LncRNAs can be involved in the regulation of gene expression in many ways.Recent studies have reported that the differential expression and function of lncRNAs in AD mainly focus on the hippocampus,but there is no systematic analysis of lncRNAs in AD prefrontal cortex(PFC).The neuroimaging study of 118 AD patients revealed that the delusions,apathy and depression which were the most prevalent neuropsychiatric symptoms,were related to the prefrontal cortex.We use RNA-seq to investigate the transcriptome changes of lncRNA expression in prefrontal cortex of AD patients and healthy individuals,as well as in AD mice and wild-type controls.to screen for AD-specific mRNA/IncRNAs and perform comparative analysis.Then we made further study on the discovery of high expressed IncRNAs associated with AD by using bioinformatics analysis.Combined with functional prediction and pre-experimental results,we selected the highly expressed IncRNA MEG3 to study its biological function and mechanism in AD with biological chemistry,molecular biology and cytology methods.Methods In this study,RNA-sequencing(RNA-seq)was performed to investigate the PFC expression patterns of dysregulated IncRNAs in AD patients and normal individuals,as well as in APP/PS1 mice(AD mice)and wild-type controls.Based on the bioinformatics methods of differentially expressed analysis and functional enrichment analysis,we carried out a comprehensive analysis.For the search of orthologous IncRNAs,human IncRNAs coordinates were converted to mouse coordinates using NCBI BLASTN.DAVID is used for target gene function annotation.Spearman correlation coefficient was used to analyze the expression patterns of IncRNAs in the prefrontal cortex of AD patients and AD mice.By constructing the eukaryotic overexpression vector of MEG3 in SH-SY5Y cells,we detected cell proliferation by cck-8 method and observed cell neurite structure and MEG3 location by RNAscope method.The methods of quantitative real-time polymerase chain reaction(qRT-PCR),western blot and immunofluorescence was used to detect the expression level of HDAC2 and synapse-associated proteins.Results The identified lncRNAs showed the same characteristics of shorter length,lower expression level,and less conserved than mRNAs in human and mouse,which have also been detected in other mammals.The expression pattern of orthologous mRNA and IncRNAs are highly conserved in human and mouse PFC.The dominating enriched BPs and KEGG pathways of the highly expressed genes in PFC were very similar across species.However,the number of functionally enriched BP terms of the highly expressed IncRNAs in human and mouse PFC were 52 and 40;the BP terms associated with neural activities were negative regulation of neuron apoptotic process,regulation of neuronal synaptic plasticity and nervous system development in human PFC.Compared with normal individuals,we found that 750 IncRNAs and 388 mRNAs were differentially expressed in AD patients.Differentially expressed IncRNAs are involved in biological processes such as apoptosis,p53 signaling pathways,cell biosynthesis,and vesicle-mediated translocation.Compared with wild-type mice,we found that 222 mRNAs and 227 IncRNAs were differentially expressed in AD mice which were mainly involved in biological process such as apoptosis,B cell receptor signaling pathway,establishment of synaptic vesicle localization and synaptic vesicle transport.In these mRNAs and IncRNAs of differentially expressed,we found that 17 conserved IncRNAs and 12 homogenous genes of these transcriptomes.At the signaling pathway level,genes involved in inflammatory reaction processes and the Toll-like receptor signaling pathway were most strongly conserved between mice and human AD.Among the correlated mRNAs targeted by IncRNAs,apoptosis,and the JAK-STAT signaling pathway were the most highly conserved between AD patients and AD mice.LncRNA MEG3 which is overexpressed in AD patients inhibits cell proliferation.Overexpression of MEG3 resulted in a decrease in SH-SY5Y cell growth rate according to the results of growth curve and the change of neurite structure.MEG3 is localized in the nucleus.The overexpression of MEG3 in SH-SY5Y cells can significantly promote the expression of HDAC2 and reduce the expression level of synapse-associated proteins such as NR2B,PSD-95,CamkII,synaptophysin,and tubulin.Conversely,after inhibiting the expression of MEG3,the expression of HDAC2 decreased,and the expression of synaptic-associated proteins NR2B,PSD-95,CamkII,synaptophysin and tubulin increased.Conclusion Our study shows that the biological functions of the highly expressed genes in PFC are similar between human and mouse;the expression pattern of PFC orthologous mRNA and IncRNAs are highly conserved across species,which can provide the genetic basis for understanding the high concordance of anatomical structure and cell type in human and mouse PFC.However,the number of functionally enriched BP terms of the highly expressed IncRNAs in human PFC was more than that of mouse PFC;the number of BP terms associated with neural activities was evidently more than that of mouse,including negative regulation of neuron apoptotic process,regulation of neuronal synaptic plasticity and nervous system development.Compared with controls,there were some aberrant lncRNAs in AD patients and AD mice,some of which are related to AD progress.However,the abundance and diversity of IncRNAs expression in PFC of AD patients is more than AD mice.GO and KEGG pathways analysis of differential expression IncRNAs which involved in apoptosis and JAK-STAT signaling pathway were similar in AD patients and AD mice.These results provided new ideas for the usage of double-transgenic mouse model to study AD-related pharmacological intervention studies,and provide a clue for biological evolution and comparative study.The IncRNA MEG3,by regulating the expression of HDAC2 and synapse-associated protein,probably inhibits the proliferation of neurons and the formation of neurite-like structure,and to participate in the cognitive impairment of AD.