Investigating The Regulation Mechanism Of Non-coding RNA In Peripheral Blood Of Schizophrenia Through Inflammation-related Genes

Objective:Schizophrenia,as one of the most common mental illness with a high disability rate,unknown etiology and pathogenesis,exerts serious and heavy burden to both patients' families and society.Furthermore,it has become a great challenge to public health of all human beings.Therefore,to ameliorate the therapeutic effect of schizophrenia,it is an urgent and significant task to explore the causes of schizophrenia,reveal its inherent genetic molecular mechanism,with the aim to promote the early prevention,diagnosis and ultimately the treatment.It has demonstrated that abnormal neuroinflammation may be closely related to the occurrence and development of schizophrenia,and inflammatory factors were found possibly play a crucial role in its pathogenesis.And more recently,non-coding RNA(ncRNA)showed to participate in the regulatory mechanism of schizophrenia.We found in previous study that inflammatory genes mRNA,including CXCL8,EGR1,EGR3,IL-1? and PTGS2,were abnormally high expression in the peripheral blood of patients with schizophrenia.Long non-coding RNA(LncRNA)can be used as a competitive endogenous RNA(ceRNA)for micro non-coding RNA(miRNA),and miRNA can degrade or inhibit target mRNA.Based on these,in the present study,by using use high-throughput transcriptomics,bioinformatics methods and traditional cell molecular biology techniques,we identify potential molecular diagnostic markers in peripheral blood of schizophrenia and further explore the detailed molecular mechanism of regulatory network among LncRNA-miRNA-mRNA ceRNA that significantly affect the onset of schizophrenia,which had been preliminarily verified by our previous study.Importantly,the role of non-coding RNA in regulating the mechanism of inflammation-related genes,which play a crucial role in the pathogenesis of schizophrenia will be intensively investigated.Out results will shed a new light on the development of novel and effective strategies for the diagnosis and therapy of schizophrenia.Methods:1.By using Illumina HiSeqTM 4000 sequencing technology,LncRNA high-throughput sequencing was performed in the peripheral blood leukocytes of 10 patients with schizophrenia and 10 healthy controls.The edge package of R software was employed to identify differentially expressed LncRNA in the peripheral blood,and simultaneously the LncRNA expression profile was analyzed.The data underwent weighted co-expression network analysis(WGCNA)to screen out characteristic modules that were related to schizophrenia.The LncRNA in the module and the differentially expressed LncRNA were collected for intersection genes.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to verify its actual expression in a larger peripheral blood sample(50:50),and finally the receiver characteristic operating curve(ROC)analysis was used to determine whether LncRNA would be taken as a potential molecular diagnostic marker for schizophrenia.Combining with the highly expressed mRNA identified in our previous study,the highly expressed LncRNA was selected and determined to be included in the subsequent construction of the ceRNA regulatory network.2.Meanwhile,Illumina HiSeqTM 2000 sequencing technology was used to perform miRNA high-throughput sequencing in the peripheral blood leukocytes of the similar 10 schizophrenia patients and 10 healthy control.The edge pac kage of R software was also employed to identify differentially expressed miR NA,and WGCNA was used in the analysis of miRNA expression profile data.From the characteristic modules related to schizophrenia,the differentially expressed miRNA and the miRNA targeted by the LncRNA-MEG3,which were iden tified in the first part,are taken to mix with the cross genes.Then,qRT-PCR method was used to verify its actual expression in a larger peripheral blood sam ple(50:50).To reveal the ceRNA regulatory network mechanism,low-expressed miRNAs were selected as the research object,GO analysis and KEGG pathway enrichment analysis of miRNA targeted mRNA were performed respectively to clarify the biology of the function of the target genes,construct protein inter action network and identify Hub gene.ROC analysis was used to ascertain whether miRNA could be taken as a potential molecular diagnostic marker for schizophrenia,with aim to preliminarily construct LncRNA-miRNA-mRNA ceRNA regulatory network.3.To verify the ceRNA regulatory network in vitro cell experiments:(1)qRT-PCR technology detects the relative expression of LncRNA,miRNA,and mRNA in cells,and preliminarily judges whether there was a regulatory relationship between LncRNA-miRNA and miRNA-mRNA according to the level of expression;(2)To perform bioinformatics methods to predict the interaction sites of LncRNA-miRNA and miRNA-mRNA;(3)To construct LncRNA and mRNA vector plasmids and employ dual luciferase reporter gene detection to verify whether LncRNA-miRNA and miRNA-mRNA can interact;(4)To perform rescue experiment for verifing the ceRNA regulatory network.Results:1.(1)A total of 413 differentially expressed LncRNA transcripts were identified in the peripheral blood leukocytes of patients with schizophrenia,of which 197 were highly expressed and 216 were lowly expressed;(2)WGCNA multi-dimensional analysis identified the module purple,containing 56 LncRNA transcripts,which were related to schizophrenia;(3)The LncRNA transcripts in the module and the differentially expressed LncRNA by sequencing were used to take the intersection genes.There were 7 LncRNA transcripts(5 low expression,2 high expression)of the intersection genes;(4)The qRT-PCR technology confirmed that 2 low expression LncRNA(MALAT1,XIST)and 1 highly expressed LncRNA-MEG3 were consistent with the expression of sequencing.Revealed by ROC curve analysis,LncRNA(MALAT1,MEG3)could be used as potential molecular diagnostic markers for schizophrenia.However,LncRNA-XIST had not enough diagnostic value;(5)Finally,the highly expressed LncRNA-MEG3 related to immune function was adopted in the research object for constructing ceRNA regulatory network.2.(1)A total of 46 differentially expressed miRNA were identified in the peripheral blood leukocytes of patients with schizophrenia,of which 36 were hi ghly expressed and 10 were lowly expressed;(2)miRNA sequencing data was analyzed by WGCNA and identified the schizophrenia-related module turquoise containing 298 miRNA,which were associated with schizophrenia;(3)The diff erentially expressed miRNA,the miRNA in the module turquoise,and the miR NA targeted by LncRNA-MEG3 were taken the intersection genes,3 key miRN A were identified and verified by qRT-PCR,confirming the low expression of both hsa-miR-6833-3p and hsa-miR-877-3p,while hsa-miR-125a-3p still exhibite d stably high expression in peripheral blood.Revealed by ROC curve analysis,3 miRNA could be used as a potential molecular diagnostic marker for schizop hrenia;(4)Furthermore,the mRNA genes which 2 downregulated miRNA(hsa-miR-6833-3p and hsa-miR-877-3p)targeted may participate in nervous system function and immune response due to their enrichment in important immune-rela ted pathways(such as Wnt signaling pathway);(5)Revealed by the bioinformatics methods,hsa-miR-6833-3p could target mRNA(EGR3 and PTGS2),and hsa-miR-877-3p could target mRNA(EGR1 and PTGS2);(6)In the constructed protein interaction network,the PTGS2 gene was the Hub gene targeting mRNA of hsa-miR-6833-3p;(7)Using high-throughput sequencing data and bioinforma tics analysis to construct 4 schizophrenia inflammations Related ceRNA molecular regulatory network:?LncRNA-MEG3/hsa-miR-6833-3p/EGR3;?LncRNA-MEG3/hsa-miR-6833-3p/PTGS2;?LncRNA-MEG3/hsa-miR-877-3p/EGRl;?LncRNA-MEG3/hsa-miR-877-3p/PTGS2.3.(1)qRT-PCR technology detected the relative expression of each molecule in cells,and found that LncRNA-MEG3 and hsa-miR-6833-3p,hsa-miR-6833-3p and mRNA(PTGS2 and EGR3)possessed a regulatory relationship;(2)Bioinformatics methods respectively predicted the interaction sites of LncRNA-MEG3 and hsa-miR-6833-3p,hsa-miR-6833-3p and mRNA(PTGS2 and EGR3);(3)After plasmid construction and transfection of cells,it was confirmed that LncRNA-MEG3 and hsa-miR-6833-3p,hsa-miR-6833-3p and mRNA(PTGS2 and EGR3)have an interaction relationship through dual luciferin reporter gene detection;(4)EGR3 could not be used as the core gene to construct the ceRNA regulatory network;(5)LncRNA-MEG3 could target hsa-miR-6833-3p to regulate the expression of inflammation-related mRNA(PTGS2)associated with schizophrenia.Conclusion(s):1.Differential expression of LncRNA and miRNA can be found in peripheral blood of schizophrenia,suggesting that the molecular regulation mechanism involved in non-coding RNA may be one of the reasons affecting the onset of schizophrenia.2.Low-expressed LncRNA-MALAT1,high-expressed LncRNA-MEG3,high-expressed hsa-miR-125a-3p,low-expressed 2 miRNA(hsa-miR-6833-3p,hsa-miR-877-3p)are related to schizophrenia in peripheral blood,and these non-coding RNA have the potential as molecular diagnostic markers for schizophrenia.3.LncRNA-MEG3 can target hsa-miR-6833-3p regulating the expression of inflammation-related gene PTGS2 associated with schizophrenia,suggesting that the regulatory network of LncRNA-MEG3/hsa-miR-6833-3p/PTGS2 may be involved in the pathogenesis of schizophrenia.