CircRNA Of Gene Expression Profile Analysis In Hippocampal CA1 Regions In CMS-induced Depressed Mice

Objectives:Depression is an affective mental illness that seriously jeopardizes the physical and mental health of humans.Elucidating the pathogenesis of depression and establishing an effective treatment strategy are always an important part of the study of depression,and are also hot and difficult in the field of neuroscience.The etiology of depression is complex,and environmental factors and gene interactions influence the occurrence and development of depression.Stress,especially chronic negative stress,is an important environmental factor in the pathological process of depression.Increasing evidence indicates that epigenetic regulation,mainly including DNA methylation,histone modification,and noncoding RNA(ncRNA)exerts important roles in the development of depression and are critical for the antidepressant effects of drugs and therapeutic strategies via regulating the expression of specific target genes.As a novel ncRNA,circRNA(circular RNA)is abundantly expressed in the central nervous system with cell type-and developmental phase-dependent characters.Series of evidence reported that circRNAs,acting as micro-RNA(miRNA)sponge,binding molecules of RNA-binding proteins,exert apparent regulatory modifications and participate in normal physiological processes and in pathological processes of many kinds of diseases such as cancer,and aging.Most recently,Data from clinical study indicated that circRNAs aredifferently expressed in blood sample of depressed patients and could be reversed by antidepressants treatment,suggesting that there may be a correlation between circRNA and the development of depression.At present,no evidence was provided that whether the circRNAs were also differently expressed in the depression-related brain regions,such as hippocampal CA1 region,and what's the potential roles of these differentially expressed circRNAs during the pathological process of depression.In the present study,for the first time,at least to our knowledge using chronic mild stress(CMS)-induced depression mouse model,combining with animal behavioral tests,transcriptomics,bioinformatics,and molecular biological methods,we aimed to investigate the expression patterns of mRNA and circRNAs in the hippocampal CA1 region of CMS-induced depression mice,analyze the biological information of differentially expressed mRNA and circRNAs,and construct a circRNA-miRNA-mRNA interaction networks.We tried to obtain the expression pattern of circRNAs in hippocampal CA1 region of CMS-induced depressed mice and provide directly experimental evidence for investigating the potential roles of circRNAs in depression.Methods:1 Establishment of depression mouse model induced by chronic mild stress.Fifty ICR mice were habituated for 5 days and divided into na?ve group and stress group.Mice in na?ve group were normally reared without any disturbance.Mice in stress group were exposed to a 28-day stress treatment according to an CMS procedure.The day after the last day ofstress exposure,sucrose preference test(SPT),forced swimming test(FST),open field test(OFT)and novelty suppression feeding(NSF)were carried out to evaluate the depressive-and anxiety-like behaviors of mice.Mice were decapitated and the tissue sample of hippocampal CA1 area was isolated and stored for transcriptional analysis or quantitative real-time polymerase chain reaction(qRT-PCR).2 Expression analysis of circRNAs and mRNAs Using RNA chip.Tissue samples(n=3 in each group)were selected,total RNA was extracted and quantified,and circRNA and mRNA expression analysis was performed by using Arraystar expression microarray.The differential expression was evaluated as fold change?2 or?-2,P<0.05.3 Revaluation of differently expressed circRNAs and mRNAs.To verify the reliability of data from chip,four differently expressed circRNAs and five differently expressed mRNAs were selected and primers were designed.qRT-PCR was used to detect the expression of the transcripts in the hippocampal CA1 region of mice.The 2~-??Ct??Ct method calculates the difference multiple.4 Bioinformatics analysis of differently expressed mRNA and circRNA.Gene onlogy analysis and pathway analysis of differently expressed mRNAsandcircRNAsmaternalgeneswereperformed;circRNA-mi RNA-mRNA interaction network were constructed.Results:1.Behavioral results showed that mice with a 28-d CMS treatment exerted significant depressive-like behaviors,reflected by significant decrease of sucrose preference in the SPT test(P<0.01),increased floating time in the FST(P<0.05);CMS-treated mice also exerted significant anxiety-like behaviors,assessed by OFT and NSF,reflected by decreased spending time in central zone(P<0.01)and reduced and the spontaneous activity in the OFT(P<0.05),and prolonged latency time to feeding in the NSF(P<0.05).2.The results from qRT-PCR validation indicated that the expression microarray data was reliable,and the expression microarray data suggested that there was differential expression of circRNA and mRNA in the hippocampal CA1 region of CMS-induced depressed mice.Compared with that of mice in na?ve group,448 mRNAs and 3215 circRNAs were differently expressed in the hippocampal CA1 region of CMS mice.GO analysis suggested that,in addition to synapses,neuronal components,receptors,and other related functional/genetic differences,which have been reported,there are differences in the relevant mRNA and circRNA parential genes genes,such as neural projections,activin receptor pathways,and lymphocyte migration.Pathway analysis of the results showed that the steroid biosynthesis,Hippo signaling,TGF-beta pathway and other related pathway mRNA and circRNA maternal genes are different,suggesting that the mentioned pathways may be involved in the pathological process of depression,and its specific function remains to be verified.Based on the basic bioinformatic analyses,circRNA-miRNA-mRNA interaction networks were constructed for two differently expressed circRNAs(circ-SYNDIG1.1 and circDTNB.377).Conclusion:1.Our present data showed that circRNAs were differently expressed in the hippocampal CA1 region of CMS-induced depression mice.1845circRNAs were up-regulated and 1370 circRNAs were down-regulated after CMS treatment.Beside this,CMS treatment induced differential expression of mRNAs in the hippocampal CA1 region of mice.2.Bioinformatic analyses indicated several newly downstream target/signaling pathways that may be involved in CMS-induced depression.our present results provides a preliminary experimental evidence for further investigating the functions and even the underlying molecular mechanism of candidate circRNA in stress-induced depression.