| Background:Major Depressive Disorder(MDD)is one of the most common mental disorders in human beings,and its pathological mechanism is complex.miRNA(microRNA),lncRNA(long non-coding RNA)and circRNA(circular RNA)have been proved to be related to the occurrence and development of depression.However,a single index is difficult to fully evaluate the pathological process of depression,so the discovery of depression-related networks has more potential to explore its pathological mechanism.LncRNA and circRNA can competitively combine with miRNA and then regulate the expression of mRNA,which together form a competitive endogenous RNA(Competitive Endogenous RNA,ceRNA regulatory network and participate in the process of many diseases.WKY rats,bred by Wistar rats,have behavioral and physiological abnormal similar to those of human depression patients,which are often used in the genetic study of depression.Therefore,Wistar-Kyoto(WKY)was used in this study,and Wistar rats as control group.Objective:1.To screen and validate the specific expression of circRNAs,lncRNAs,miRNAs and mRNAs in the hippocampus of depression rats.2.To construct the depression-related specific circRNA / lncRNA-miRNA-mRNA regulatory networks.Methods:1.Experimental animal grouping and behavioral test:(1)Experimental animal grouping: WKY depressed rats were divided into two groups: depression group(n = 10)were used saline intragastrical administered for 21 day,treatment group(n = 10)weretreated with citalopram 5mg/kg for 21 days.Wistar rats(n=7)were act as control group,and normal saline was intragastrical administered for 21 days.(2)Behavioral test: after the intervention,the open field test(OFT),forced swimming test and Sucrose preference test(SPT)were performed to detect whether the rats had depression-like behavior.2.RNA sequencing and functional annotation:(1)isolated rat hippocampal tissue,extracted total RNA to construct cDNA library,and sequenced the whole genome with Illumina HiSeq 4000.Then,screened the depression-related differentially expressed circRNAs,lncRNAs,miRNAs and mRNAs.(2)functional annotation of differentially expressed circRNAs,lncRNAs,miRNAs and mRNAs were performed by GO enrichment analysis and KEGG pathway analysis.3.Construction of depression-related specific ceRNA regulatory networks:(1)candidate circRNAs and lncRNAs were obtained according to the results of difference analysis.(2)depression-related specific circRNAs regulated ceRNA networks and lncRNAs regulated ceRNA networks were constructed by bioinformatics techniques respectively.(3)GO enrichment analysis and KEGG pathway analysis were used to annotate the functions of the specific ceRNA regulatory networks.Results:1.The results of behavioral test:1.1OFT: the total movement time in the treatment group and the control group was significantly longer than that in the depression group(P=0.007,P=0.031),and the central stay time in the depression group was significantly lower than that in the treatment group(P=0.013)and the control group(P<0.001).1.2 forced swimming test: the immobility time of rats in the depression group was significantly longer than that in the treatment group(P<0.001)and the control group(P<0.001).1.3 SPT: the sucrose preference coefficient of depression group was significantly lower than treatment group(P=0.032)and control group(P<0.001).2.RNA-Seq results and differential gene analysis:2.1 A total of 4312 circRNAs were detected.There were 78 differentially expressedcircRNAs in depression group vs control group,46 up-regulated and 32 down-regulated,and their functions were mainly concentrated in immune response and metabolic pathway,while in treatment group vs depression group,there were 30 differentially expressed circRNAs,15 up-regulated and 15 down-regulated,which were mainly related to immune response and virus defense response.2.2 A total of 523 lncRNAs were detected.A total of 31 lncRNAs were differentially expressed between the depression group and the control group,20 were up-regulated and 11 were down-regulated.The analysis of its function was mainly related to nerve cells,immune response and virus defense response.A total of 10 lncRNAs were differentially expressed between the depression group and the treatment group,including 3 up-regulated and 7 down-regulated,and their functions were mainly concentrated in immune response and viral defense response.2.3 A total of 559 miRNAs were detected.A total of 48 miRNAs were differentially expressed between the depression group and the control group,of which 18 were up-regulated and 30 down-regulated,which were mainly related to protein binding,system development and metabolic pathway.A total of 10 miRNAs were differentially expressed between the depression group and the treatment group,including 2up-regulated and 8 down-regulated,which were mainly related to protein binding,system development and autophagy / phagocytosis.2.4 A total of 14974 mRNAs were measured.A total of 352 mRNAs were differentially expressed between the depression group and the control group,which were176 up-regulated and 176 down-regulated,and their functions were mainly concentrated in nerve signal transduction,Ras signal pathway and alcoholism.There were 124 differentially expressed mRNAs between depression group and treatment group,of which 60 were up-regulated and 64 down-regulated,which were mainly related to virus infection,virus response and virus defense response.3.Construction of depression-related specific ceRNA regulatory network:3.1 There were 3 circRNAs(circRims2,circFhod3,circPsmb1)and 2 lncRNAs(ENSRNOT00000087486,ENSRNOT00000085401)differentially expressed indepression group and control group,depression group and treatment group at the same time,taking them as candidate circRNAs and lncRNAs.3.2 The depression-related specific circRNA-miRNA-mRNA regulatory network was constructed,including 3 circRNAs,24 miRNAs,939 mRNAs,956 edges and 991 nodes,mainly enriched in hormone receptor binding,phylogeny,GABA-ergic synapses and so on.3.3 The depression-related specific lncRNA-miRNA-mRNA regulatory network was constructed,including 2 circRNAs,46 miRNAs,1446 mRNAs,10940 edges and1493 nodes,mainly enriched in circadian rhythm,HIF-1 signal pathway,Cushing syndrome and so on.Conclusion:In this study,we found that there were specific circRNAs,lncRNAs,miRNAs,mRNAs and abnormally regulated ceRNA networks in the hippocampus of depressed rats,and their functions were annotated.Our study not only provides novel insight for the pathogenesis of depression,but also provides a reference basis for the later study of the pathological mechanism of depression.In the future,we need to further screen depression-related regulatory pathways in the ceRNA regulatory network and expand the sample verification. |
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