Screening And Bioinformatics Analysis Of Differentially Expressed LncRNA In Peripheral Blood Leukocytes Of Schizophrenia

Schizophrenia（SZ）is one of the most serious psychiatric diseases with a unknown etiology incidence,the morbidity,recurrence rate and disability rate were higher,the lifetime prevalence rate in the world is about 1%.Multiple studies have shown that SZ is caused by a combination of genetic and environmeiital factors.Long non-coding RNA（LncRNA）is an RNA fragment that is transcribed in the RNA domain and greater than 200 nucleotide residues in length.At first,IncRNA is considered to be a non-functional gene fraglent left over from evolution.However,in recent years,studies have found that IncRNA plays an important role in many vital activities such as Dosage compensation effect,epigenetic regulation,cell cycle regulation and cell differentiation regulation,and participates in X chromosome silencing,genomic imprinting and chromatin modification,Various important regulatory processes such as transcriptional activation,transcriptional interference,and intranuclear transport.LncRNA is mostly located in the nucleus and is highly expressed in mammalian brain and genome.The gene expression of brain tissue and peripheral blood leukocytes has a common regulatory pathway.Many cytokines and neurotransmitters secreted in brain tissue are also exists in PBL,so many IncRNAs that are high expressed in brain tissue also clearly expressed in peripheral blood,The detection of IncRNA expression levels by PBL has clear feasibility and reliability.In summary,we carried out the following experimental protocol:1.By collecting peripheral blood leukocytes（PBL）for whole transcriptome sequencing,verifying the differential expressed IncRNA of sequencing results by qPCR（Real Time PCR）experiments,screening for differentially expressed lncRNA in PBL of schizophrenia.2.Processing functional annotation and analysis of the genes encoding which is co-expressed with mRNA,using bioinformatics GO（Gene Ontology）functional annotation analysis and KEGG（Kyoto Encyclopedia of Genes and Genomes）signal pathway enrichment analysis to construct IncRNA-target Gene-transcription factor network map to explore the biological functions and lncRNA-target gene-transcription factor pathways that involved in the development of schizophrenia,to find theoretical basis and support for cell-level mechanism research,and to provide relevant research clues.Objective（s）:1.Processing the whole transcriptome sequencing by collecting peripheral blood leukocytes（PBL）to find potential biomarkers for clinical diagnosis of schizophrenia.2.Through the bioinformatics GO functional annotation analysis,functional annotations were made for the target genes of IncRNAs with abnormal expression in SZ patients,analysis of which target genes may be associated with differential expressed IncRNAs in the GO classification entry;annotation of aberrantly expressed IncRNAs-related target gene signaling pathways by enrichment analysis of KEGG signaling pathway,investigating the role of this signaling pathway,to explore the genetic and molecular biological signaling pathway mechanisms which associated with the target genes.Method（s）:1.Whole transcriptome sequencing（Illumina HiSeqTM 4000）was performed by collecting peripheral blood leukocytes（PBL）.The expression of IncRNA was analyzed by using edgeR software.The differential transcripts were screened by FDR and log2FC.The screening conditions were FDR<0.05 and|log2FC|>1;qPCR experiments were performed on the differentially expressed IncRNAs screened by sequencing,and the data were analyzed by two independent samples to analyze whether there was a difference between the T group and the CK group,thereby verifying the accuracy of the sequencing results;screening of differentially expressed IncRNA in schizophrenic PBL;2.Using CPC,CNCI and other software to predict the encoding ability of new transcripts,and obtaining new predicted IncRNA.Then,the expression levels of mRNA and IncRNA in the sample were analyzed,and finally the correlation analysis of lncRNA-mRNA was performed.Functional annotation and analysis of the genes encoding the co-expressed nRNA were performed by using the Functional Annotation function in the DAVID database V.6.8（http://david.abcc.ncifcrf.gov/）,and processing GO（Gene Ontology）functional annotation analysis and KEGG（Kyoto Encyclopedia of Genes and Genomes）signal pathway enrichment analysis,construct lncRNA-target gene-transcription factor network map,explore the enriched biological functions related to the development of schizophrenia and IncRNA-target gene-Transcription factor pathway.Result（s）:1.Through experiments,20 IncRNAs with significant differential expression in peripheral blood of patients with schizophrenia were found,which were TCONS₀0055999,TCONS₀0134168,TCONS 00011466,ENST00000623640,TCObjS₀0112279,etc,and the differential expression levels between groups were significant（FDR<0.05）.qPCR experiments were performed on the differentially expressed IncRNA-TCONS₀138311 and TCONS 00134168.The TCONS₀0138311 was significantly down-regulated in the T group（P<0.05）,and the TCONS₀0134168 was significantly up-regulated in the T group（P<0.05）,verifying the accuracy of the sequencing results;indicating that IncRNA may be associated with the pathogenesis of schizophrenia,and can be further used as a biomarker for SZ patients.Bioinformatics analysis of the above 20 IncRNAs revealed that some of the items related to neuropsychiatric diseases were enriched.Through the prediction of Trans target genes,70 target genes co-expressed with differential IncRNA were obtained,which were ZNF2,TSEN34,TREM1,etc.through GO biological process enrichment analysis and target gene-related transcription factor network analysis,40 target genes with transcription factor regulation were obtained,which were IL5RA,BTG2,THBD,EREG,GYPA,ZEB2,PTGDR2,CLK3.The mTOR biological process（GO:0032008）discovered by enrichment analysis is associated with schizophrenia,Enriched into 13 KEGG signaling pathways related to schizophrenia,significant related pathways include:GABA synaptic energy（hsa04727）,RaP1（hsa04015）,5-HT（hsa00380）,NF-κB（hsa04064）.Transcription factors such as JUN,KLF4,TCF,and SP participate in key processes such as cell proliferation,differentiation,and apoptosis,or regulate cellular inflammatoryness.Factor gene expression leads to the persistence and development of inflammation,which mediates the formation,proliferation,migration and synapse formation of the nervous system.Conclusion（s）:20 IncRNAs with significant expression up-regulation in peripheral blood of patients with schizophrenia were TCONS—00055999,TCONS 00134168,TCONS₀0011466,ENST00000623640,TCONS₀0112279,etc.,and the differences in expression levels between the groups were significant（FDR<0.05）;The differential expression of lncRNA-TCONS₀0138311 and TCONS₀0134168 for qPCR experiments proved that the sequencing results were accurate,indicating that IncRNA may be associated with the pathogenesis of schizophrenia,and can be further used as a biomarker for SZ patients.Through the prediction of Trans target gene,70 target genes co-expressed with differential IncRNA were obtained,which were ZNF2,TSEN34,TREM1,etc.,and were enriched by GO biological process analysis and target genes,and the mTOR biological process was found by enrichment analysis.（GO:0032008）is associated with schizophrenia,Enriched into 13 KEGG signaling pathways related to schizophrenia,significant related pathways include:GABA synaptic energy（hsa04727）,Rap1（hsa04015）,5-HT（hsa00380）,NF-κB（hsa04064）.Related target gene-transcription factor network analysis,obtained 8 target genes with transcription factor regulation,which were IL5RA,BTG2,THBD,EREG,GYPA,ZEB2,PTGDR2,CLK3.The transcription factors JUN,KLF4,TCF,SP,etc.participate in key processes such as cell proliferation,differentiation and apoptosis,or regulate the expression of cellular inflammatory factor genes,leading to the persistence and development of inflammation,mediating the formation,proliferation,migration and Synapse formation.