Study On Differential Expression Of CircRNAs In PBMCs From Individuals With Schizophrenia

BackgroundSchizophrenia?SZ?is a severe neuropsychiatric disorder.The main clinical features include positive symptoms?eg.hallucinations,delusions,and psychomotor excitement?and/or negative symptoms?eg.emotional indifference,emotional dullness,social withdrawal,and hypopsychosis?.The prevalence of schizophrenia at time point was about 1%,and the lifetime prevalence was slightly highe.Individuals living with SZ have an average life expectancy that is 10–20 years shorter than that of the general population.The inability to maintain employment?80–90% unemployment?with SZ create a severe social burden.Despite advances in new technologies that have improved our understanding of schizophrenia,the mechanism of schizophrenia remains unknown.At present,the diagnosis of schizophrenia mainly depends on the spiritual interview and examination,but no effective objective diagnostic indicators have been found.Such syndromes diagnosed according to phenomenology may have great differences in etiology,pathological mechanism and biological characteristics of treatment response.Therefore,the exploration for sensitive and specific biomarkers to improve the biological homogeneity of SZ diagnosis has become a hot spot of current research.A growing evidence have shown that ncRNA plays an important role in physiological and pathological regulation by epigenetic modification.Circular RNA?circRNA?is a eukaryotic RNA species and its native form is circular,which makes it resistant to RNase R degradation.CircRNA has some characteristics as a ‘perfect' biomarker: stable property,not easyly degraded by RNase R,and convenient collection and measurement.Furthermore,circRNAs may potentially serve as minimally invasive biomarkers for disorders of central nervous system?CNS?,since CNS diseases often present a certain degree of damage to the blood-brain barrier,leading to small molecules entering the blood and cerebrospinal fluid.Recent studie suggests that the circRNA Cdr1 as genome knockout mice displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with human neuropsychiatric disorders.The above evidence provides indirect evidence that circRNAs may be involved in the pathogenesis of schizophrenia.Considering a strong deficit in prepulse inhibition of individuals diagnosed with SZ,we supposed that some circRNAs might participate in the pathogenesis and development of schizophrenia.Despite the growing evidence that circRNAs might participate in the mechanisms underlying SZ pathophysiology,no data have been published regarding the direct relationship between circRNA and schizophrenia.Considering the stability of exon circRNAs in cells and their instability in serum,since they are easily affected by other RNAs,PBMCs were extracted from individuals.AIM To investigate whether circRNAs are differentially expressed in PBMCs from individuals with SZ.To predicte target genes of miRNA,which were to correspond to the miRNA binding sites of differentially expressed circRNAs,and explore their biological functions via the GO and Pathway significance enrichment analysis.To investigate the differential diagnosis value of five differentially expressed circRNAs in schizophrenia of schizophrenia and depression,obsessive-compulsive disorder,bipolar disorder.To explore the effect of olanzapine treatment on the expression levels of differentially expressed circRNAs.METHOD First of all,using sex-and age-stratified sampling,nine individuals with SZ and nine age-and sex-matched healthy controls were collected.Differentially expressed circRNAs between the two groups was screened via microarray and then quantified using real-time quantitative reverse-transcription PCR?qRT-PCR?in SZ?n=102?and HC?n=103?groups.ROC curve analysis was used to evaluate the diagnostic value of differentially expressed circRNA for schizophrenia.Secondly,.the target genes of the five binding points?hsa-mir-659-3p,hsa-mir-548d-5p,hsa-mir-651-3p,hsa-mir-548c-5p,and hsa-mir-548a-5p?of hsa_circRNA₁04597 were predicted by miRBase,TargetScan and miranda,and then FunNet was executed to analysize the GO enrichment terms and KEGG pathways enrichment of target genes Next,40 individuals with schizophrenia,40 individuals with depression,40 individuals with obsessive-compulsive disorder and 40 individuals with bipolar disorder were selected by convenience method.Qrt-pcr was used to test the expression levels of 5 differentially expressed circRNAs in schizophrenia in the other three disorders.Finally,using the random number table method,30 individuals with schizophrenia,who met the inclusion criteria,were selected from the total SZ enrollment and received olanzapine monotherapy for 8 weeks.Then five circRNAs expression in these 30 individuals was assessed by qRT-PCR.RESULTS1.Nine circRNAs were differentially expressed according to a gene microarray.Of which,six were up-regulated and three were down-regulated.And then five differentially expressed circRNAs were identified between SZ and NC group via qRTPCR on large samples.Of the five remaining circRNAs,the expression levels of hsa_circRNA₁02101,hsa_circRNA₁01836,and hsa_circRNA₁04597 in the SZ group were down-regulated?P<0.001?.Conversely,the expression levels of hsa_circRNA₁03102 and hsa_circRNA₁03704 were significantly up-regulated?P<0.001?.No differences in the expression levels of the five circRNAs were observed between the two age subgroups,or sex?P>0.05?.The area under the curve?AUC?values of hsa_circRNA₁02101,hsa_circRNA₁04597,and hsa_circRNA₁01836 were 0.6376,0.8848,and 0.6397,respectively.When all three circRNAs were used to create,the AUC of a combined ROC curve was 0.8967.2.Cross-over analysis revealed that 223 target genes of 5 miRNAs,including hsamiR-659-3p,hsa-miR-548d-5p,hsa-miR-651-3p,hsa-miR-548c-5p and hsa-miR-548a-5p,were got from the intersection of 3 databases.By miRNA target gene prediction and functional annotation analysis,there were significant enrichments in several gene ontology?GO?biological process associated with nervous system and brain functions.Kyoto encyclopedia of genes and genomes?KEGG?pathway analysis indicated significant enrichments in several pathways related to the pathophysiology of SZ.3.Compared with SZ group,the expression levels of hsa_circRNA₁02101,hsa_circRNA₁01836 and hsa_circRNA₁04597 in MDD group were significantly up-regulated,all of which had the certain value in differential diagnosis of SZ and MDD;the expression levels of hsa_circRNA₁02101 and hsa_circRNA₁04597 in the OCD group were significantly up-regulated,and had the differential diagnosis value between SZ and OCD;the expression levels of hsa_circRNA₁02101 and hsa_circRNA₁04597 in BP group were significantly up-regulated and the expression levels of hsa_circRNA₁03704 and hsa_circRNA₁03102 were significantly downregulated,except for hsa_circRNA₁03704,the other three circRNAs had certain value in differential diagnosis of SZ and BP.4.Compared with those scores before treatment,the total score,subscales and factor scores of PANSS significantly decreased after 4 weeks treatment?P<0.01?and after 8 weeks treatment?P<0.001?.Repeated measures one-way ANOVA showed significant differences among three time points in hsa_circRNA₁03704,hsa_circRNA₁01836,and hsa_circRNA₁04597.Bonferroni's post-hoc test for multiple comparisons identified only hsa_circRNA₁04597 expression was significantly up-regulated between the 0-and 8-week time points?P<0.001?.The ??Ct value of hsa_circRNA₁04597 was significantly positively related to the change of the psychotic positive symptoms,activating factor,paranoid factor or aggressive factor?P<0.05?.CONCLUSION Hsa_circRNA₁04597 may serve as a novel potential diagnostic and therapeutic biomarker for schizophrenia and involve in the pathogenesis of schizophrenia.Hsa_circRNA₁02101 and hsa_circRNA₁04597,especially hsa_circRNA₁02101,may play a key role in the differential diagnosis between schizophrenia and depression,obsessive-compulsive disorder and bipolar disorder.