Screening And Validation Of LncRNA Of Differential Expression Of Peripheral Blood In Patients With Schizophrenia

Objective: Using long noncoding RNA?lncRNA?microarray chip to select the lncRNA differences expression profiles of schizophrenia in the peripheral blo od,verifying the screening results,and constructing of the lncRNA Artificial Neural Network?ANN?forecasting model,to find a convenient,good specificity,high sensitivity and widely applicable scope of the biological molecular diagnostic marker of schizophrenia.Methods: 1.Collecting samples: A case-control study,in strict accordance with inclusion and exclusion criteria for collecting samples of schizophrenia?case group?and healthy subjects?control group?by EDTA anticoagulated peripheral blood of 55 cases each,a total of 110 cases of peripheral blood extracted total RNA.2.Chip screening: Randomly selecting 5 samples in case group and control group separately and using Beijing Boao company's GeeDom? LncRNA+mRNA Human Gene Expression Microarray V4.0?containing 40916 lncRNA probes and 34235 mRNA probe?to detect different expreesion profiles of lncRNA and mRNA in peripheral blood,with Fold Change?FC?of the absolute value is greater than or equal to 1.5,and the P value is less than or equal to 0.05 as the standard of screening the differential expression profiles of lncRNA and mRNA spectrum of schizophrenia and the differences in the expression of lncRNA and mRNA cluster analysis to compare the differential expression of lncRNA and mRNA between the two groups.3.qRT-PCR validation: Selecting ENST00000454043.1,TCONS₀0029776,TCONS₀0014810,ENST00000539163.1,uc001 aiu.1,TCONS₀0007244,ENST00000431705.1 seven differentially expressed lncRNA and ?-actin as the internal gene,using quantitative Real-time PCR?qRT-PCR?to verify,the case group and the control group 50 samples separately.Then applying SPSS20.0 to analy the result of PCR.4.Artificial neural network predictive model construction: Using Back-Propagation Algorithm Error?BP?to construct the prediction model of peripheral blood lncRNA artificial neural network in schizophrenia.5.ROC curve: The artificial neural network simulation results and 7 kinds of lncRNA to carry out the ROC curve detection together,in order to find the specific high sensitivity,good sensitivity of the biological markers of schizophrenia.6.lncRNA-mRNA co expression network construction: On the base of the chip data,calculating the correlation between the expression of the lncRNA and mRNA and according to the threshold setting that Pearson correlation coefficient?correlation?> 0.95 or < 0.95 and P < 0.05 screening criteria to screen significantly expressed lncRNA and mRNA and construct differentially expressed lncRNA-mRNA coexpression network using Cytoscape generate whole network diagram.7.Target gene prediction,pathways and functional analysis: On the base of the lncRNA-mRNA coexpression network,by looking for the genomic location in less than 10 kb lncRNA-mRNA relationship,and for lncrna?3 'UTR?sequences alignment using blat,was screened for sequence similarity lncRNA-mRNA relationship of lncRNA to predict differentially expressed target genes.At the same time,functional annotation analysis,GO analysis and pathway of target gene and target gene enrichment analysis for prediction.8.Transcription factor prediction: Through the analysis of each lncRNA transcription start site upstream 2000 bp and downstream 500 bp region,using Match-1.0Public transcription factor prediction tools to predict the the regional lncRA transcription factor binding sites.Results: 1.Chip screening results: The chip raw data were normalized,selected |FC| ? 1.5,and P < 0.05 lncRNA/mRNA for differentially expressed lncRNA/mRNA.Experimental results show that the differential expression of lncRNA were 1200,723 high and 477 low expression;differential expression of mRNA 1364,384 high and 980 low expression.2.Verification results: Through the qRT-PCR verification of schizophrenia peripheral blood differentially expressed 7 lncRNA: ENST00000454043.1,TCONS₀0029776,TCONS₀0014810,ENST00000539163.1,uc001 aiu.1,TCONS₀0007244,ENST00000431705.1,after SPSS20.0 software analysis found that ENST00000454043.1,TCONS₀0029776,TCONS₀0014810,ENST00000539163.1 and uc001 aiu.1 were up-regulated,and P < 0.05,the difference is statistically significant;TCONS₀0007244 and ENST00000431705.1 were down-regulated,and p < 0.05,the difference is statistically significant.At the same time,through the qRT-PCR results comparing with the chip results,7 kinds of lncRNA qRT-PCR detection results are generally consistent with the chip,the changing trends of credibility chip results are proved.3.Construct the prediction model and the ROC curve detection: Using of the seven kinds of lncRNA constructed schizophrenia peripheral blood lncRNA ANN prediction model.By the ROC curve analysis,the results show that,the simulation results of the calculation of the area under the ROC curve was 0.915,specificity was 77.4%,the sensitivity was 90.3%.4.The co-expression network constructed results: Successfully built schizophrenia peripheral whole blood lncRNA-mRNA differential expression of co-expression network,lncRNA most differentially expressed from positive regulatory role.5.Prediction of the target genes,pathways and functional results: On the base of lncRNA-mRNA co-expression network,predicted its target genes which was a relationship of lncRNA-mRNA which is p15557-A₂1_P0014496,both in detection It was tested in the down state,and plays a sense regulatory role in cis-regulation.Through GO analysis found that lncRNAs mainly involved in regulating apoptosis signaling pathways,mitochondrial membrane organelles membrane of regulation,the extracellular protein kinase 1/2 silk crack the original activated protein kinase signaling pathways,FAS signaling pathways,Hippo signaling pathways activities and so on;pathway analysis found that lncRNA can also participate in GABA synaptic activity,decorated mRNA and RNA polymerase II pre-transcriptional activity by means of regulation;and diseases analysis found that the differences in expression of genes may be linked to schizophrenia,split affective disorder or bipolar disorder,cognitive ability,behavior disorders,meningioma and immunodeficiency syndrome and other diseases.6.Transcription factors predicting results: Through the transcription factor prediction software,this study found 48 transcription factors of p3151,p6190,p8919,p15557,p21917,p35367_v4 and p37754_v4 7 lncRNA probes.Conclusion: 1.This study found that there was a differential expression profile of lncRNA in peripheral blood of patients with schizophrenia.2.After on selecting ENST00000454043.1,TCONS₀0029776,TCONS₀0014810,ENST00000539163.1,uc001 aiu.1,TCONS₀0007244,ENST00000431705.1 seven lncRNA for qRT-PCR,the result is statistically significant difference.These genes may as schizophrenia diagnosis or prognosis biomarkers.3.ANN model of lncRNA in peripheral blood was established,AUC was 0.915,sensitivity was 90.3%,specificity was 77.4%,which could be the objective prediction index of schizophrenia.