Expression Profiles And Functional Analysis Of Non-coding RNAs In Children With Anti-NMDA Receptor Encephalitis

BackgroundAutoimmune encephalitis?AE?is a relatively new category of immune-mediated,induced by pathogenic auto-antibody autoimmune disease in which patients present with seizures,psychosis,amnesia and aberrant behavior.Brain Magnetic Resonance Imaging?MRI?and Electroencephalography?EEG?also typical lack specific and sensitive findings in children with autoimmune encephalitis.These are core symptom resembling infectious encephalitis,but AE patients generally do not have fevers or cerebrospinal fluid?CSF?pleocytosis.There are two kinds of pathogenic autoantibodies according to the location of antigen in neuronal cell,antibody against the neuronal cell surface proteins and antibody against intracellular proteins.Such identification and characterization of autoantibodies could create new categories of neurological diseases and promote the understanding of patho-physiologic roles of target proteins in human brain function.Anti-N-mythyl-D-aspartete?NMDA?receptor encephalitis is the most common form of autoimmune encephalitis associated with antibodies against the neuronal cell surface in children and also leads to severe disease with a certain mortality rate and relapsing rate.NMDA receptors are a type of ionotropic glutamate receptors and neurotransmitter receptors that are widely expressed in the central nerves system and they play a major role in excitatory transmission and synaptic plasticity.Most of NMDA receptors are heterotetrameres and mostly be composed of two NR1 and two NR2 subunits.Antibodies of NMDA receptors are against the extracellular part of NR1 subunit.When NMDA receptors bind with antibodies,the adjacent EphB2 receptor will be failed to stabilize NMDA receptor.The unstable NMDA receptor will be internalized by endocytosis and lysosome.Studies have shown that NMDA receptor encephalitis is associated with a tumor,typically ovarian teratomas in females.In pediatric and male patients,the type and frequency of tumors may be different.But,in addition to tumors,brain viral infection could trigger autoimmunity to neuronal surface proteins,as suggested by observations in patients with herpes simplex virus?HSV?infection.In particular,children who had had herpes simplex virus encephalitis with recurrence of neurological symptoms showed negative CSF HSV PCR and positive NMDAR antibodies,they manifested movement and behavioral disorders with decreased levels of consciousness as main symptoms.The clinical manifestations of anit-NMDA receptor encephalitis in children are also different from adults.In children the disease usually starts with neurological symptoms,a very peculiar speech disturbance is more common and dysautonomic manifestations,central hypoventilation particularly is less frequent.Base on the above,there is an urgent need to study the gene molecular mechanisms of pediatric anti-NMDA receptor encephalitis.Non-coding ribonucleic acid?ncRNA?including microRNA?miRNA?,long non-coding RNA?lncRNA?,and circular RNA?circRNA?are defined as RNA that is not translated into protein.Recent sequencing results revealed that the quantity of ncRNA is large.They participate in many physiological functions including regulation of translation,RNA modification and degradation and they are also be proven to participate in many diseases.LncRNAs are defined as transcripts of more than 200 nucleotides that are not or less translated into proteins.LncRNA participates in various developmental processes in transcriptional,post-transcriptional,protein translational,protein post-translational,or epigenetic regulation leading to gene expression alteration or serving as competing endogenous RNA?ceRNA?,they also involved in RNA degradation.And also,lncRNA is even more remarkable in central nervous system specificity.Notably,40%of 10000-50000 lncRNA in the human genome are expressed specifically in the brain,which indicated IncRNAs may play a crucial role in the physiological or pathological condition of the CNS.Compared to protein-coding genes,lncRNA provides more information about cell type identity during mammalian cortical development and some IncRNAs also participate in controlling neuronal plasticity.CircRNAs are a large class of noncoding RNAs and with the characteristic of tissue-,cell type-and stage-specific expression patterns.They are single stranded with highly stable closed loop structure.CircRNA ring structure is thought to be more efficient in suppressing miRNA activity versus linear transcripts.Because of their stability,circRNA can accumulate in quiescent and post-mitotic cells,such as neurons.Prior studies have identified an enrichment of circRNAs in the brain,relative to other tissues.An increasing number of ncRNAs in human body were proven to be involved in many neurological disorders including Parkinson's disease,Alzheimer's disease,acute stroke,glioma,multiple sclerosis and major depressive disorder.They can be used as diagnostic biomarker,to predict outcomes of disease or as drug targets.However,data related to anti-NMDA receptor encephalitis and ncRNA are scarceObjectiveTo explore the differential expression profiles of lncRNA,circRNA and mRNA in blood leukocytes from pediatric ani-NMDA receptor encephalitis patients and healthy controls,and then uncover the potential roles of lncRNA and circRNA related to anti-NMDA receptor encephalitis in order to provide a new clue for further researchMethodIn this study,we recruited seven children with typical anti-NMDA receptor encephalitis according to the Graus and Dalmauand criteria?2016?and seven healthy pediatric volunteers and collected their peripheral blood samples.Then we chose six samples?three patients and three volunteers?for microarray analysis.We use bioinformatics analysis including differential expression analysis of lncRNA,circRNA and mRNA,cis/trans IncRNA target prediction,GO and KEGG pathway analysis,lncRNA/circRNA-miRNA interaction network,and lncRNA/circRNA-mRNA co-expression network analysis.Next,seven pairs were used for further validation of microarray assays with qRT-PCR and additional analysis.Results1.Microarray analysis is an efficient approach for investigating the biological function of IncRNAs and circRNAs.The microarray data showed that the expressions of lncRNAs,circRNAs and mRNAs in anti-NMDA receptor encephalitis were different from those in controls.There were 894 significantly dysregulated IncRNAs including 469 up-regulated and 425 down-regulated in the pediatric anti-NMDA receptor encephalitis group in comparison with the healthy control group?fold change?2 or?0.5,p<0.05?.1195 circRNAs were significantly dysregulated.Of these.501 were up regulated and 694 were down regulated.Additionally,502 up regulated and 217 down regulated mRNAs were detected in the anti-NMDA receptor encephalitis group compared with the control group?fold change?2 or ?0.5,p<0.05?.2.Cis/trans lncRNA target prediction was performed for differentially expressed lncRNAs to find the potential target mRNAs.The results showed that 57 differential regulated IncRNAs target 82 mRNAs which were also significantly dysregulated in NOD-like receptor signaling pathway and Fc gamma R-mediated phagocytosis pathway.3.The most prominent GO terms classification of IncRNAs and circRNAs were all enriched in biological regulation,cellular process and single-organism process in biological process;cell,cell part and organelle in cellular component;binding,catalytic activity and transport activity in molecular function.The GO enrichment analysis of significant dysregulated IncRNAs showed that leukocyte differentiation,lymphocyte differentiation and T cell activation were enriched and these biological functions were related with immunity.Lysosome organization in biological process,calcium-dependent protein binding in molecular function and azurophil granule membrane in cellular components which were all involved in receptor internalization,were significantly enriched in GO enrichment of circRNAs4.The notable pathways in KEGG enrichment of significantly dysregulated lncRNAs include autophagy,apoptosis and HIF-1 signaling pathway.KEGG classification of IncRNAs were nervous system and immune system in organismal systems;neurodegenerative diseases,infectious diseases and immune diseases in human diseases;signal transduction and signaling molecules and interaction in environmental information processing.The two most KEGG pathway enrichment of host gene of significantly dysregulated circRNAs were endocytosis and lysosome pathways.5.LncRNA/circRNA-mRNA co-expression network in NOD-like receptor signaling pathway and Fc gamma R-mediated phagocytosis pathway was constructed to predict gene function.In the lncRNA-mRNA co-expression network,it was shown that lncRNAs may have positive or negative correlation with these mRNAs which may play essential roles in pediatric anti-NMDA receptor encephalitis.ARPC2,GABARAPL1,FCGR2B,PYDC1,FCGR2A,MYD88,CXCL2,MFN2,PRKCA,TXN,MAVS,TRIP6,CARD 17,CXCL1,PIP5K1B and LA2G4A were shown in the network.Of the top 20 dysregulated circRNAs?10 up-and 10 downregulated?,17 circRNAs correlated with the two pathways,while 12 mRNAs of PYDC1,PPAP2B,PIP5K1B,PLA2G4A,TRIP6,PRKCA,DNM1,CXCL2,FCGR2B,FCGR2A,HSP90B1,CXCL1 were also part of this network.In the network,dysregulated lncRNAs\circRNAs could correlate with one or more up-or down-regulated mRNAs,the correlation may be positive or negative.6.lncRNA/circRNA-microRNA interaction network predicted that mRNAs playing important role in the NOD-like receptor signaling pathway and Fc gamma R-mediated phagocytosis pathway were released through the competitive endogenous mechanisms.The released mRNAs were MAVS,PRKCA,CXCL2,TRIP6,FCGR2B,FCGR2A,TXN,MFN2,PIP5K1B,CXCL1,ARPC2,PYDC1,GABARAPL1,GABARAPL1 and PLA2G4A.LncRNAs may play biological function through those released mRNAs.On the other hand,of the top 20 dysregulated circRNAs?10 up-and 10 downregulated?,three circRNAs of hsa_circ₀074239,hsa_circ₀075949 and hsa_circ₀027275 can'sponge' miRNAs to further regulate mRNAs of DNM1?PRKCA?RIP5K1B?FCGR2B?FCGR2A?CXCL1?TRIP6?PYDC1?ARPC2?HSP90B1.7.qRT-PCR analysis validate that the results of hsa_circ₀070185,hsa_circ₀070976,hsa_circ₀042407,hsa_circ₀081953 and hsa_circ₀039997 were consistent with the microarray data,and hsa_circ₀042407 was statistically significant.Conclusions1.Long non-coding RNAs and circular RNAs were abundant in blood leukocytes from ani-NMDA receptor encephalitis patients and healthy children,and a part of lncRNAs and circRNAs were significantly dysregulated which may play essential roles in the occurrence and development of pediatric anti-NMDA receptor encephalitis2.Biological analysis suggested that these significant dysregulated lncRNAs and circRNAs may involve in pathogenesis of anti-NMDA receptor encephalitis through kinds of biological process and pathway.