Differential Expression Profiles And Functional Prediction Of Circular RNAs In Peripheral Blood Leukocytes Of Children With Dilated Cardiomyopathy

BackgroundDilated Cardiomyopathy（DCM）in children is a kind of Cardiomyopathy mainly characterized by left ventricular or bicentricular dilation and left ventricular systolic insufficiency,which can often lead to progressive congestive heart failure,ventricular arrhythmia,and even sudden cardiac death.It is the leading cause of death and cardiogenic disability in children and adolescents,and the main indication for heart transplantation in children around the world.It is believed that the mechanism of DCM in children is caused by the combined action of genetic susceptibility and environmental damage.However,the specific occurrence and pathogenesis of the disease are still unclear.Compared with other adult DCM,DCM in children appears to have a wider range of etiologies,including idiopathic,genetic mutations,myocarditis,neuromuscular disorders,and congenital metabolic system disorders.Recently,gene modification and epigenetic regulation have become an important source of research on the pathogenesis of DCM in children.Circle RNAs（circRNAs）are a class of endogenous RNAs produced by reverse splicing of precursor mRNAs in eukaryotes.Structurally,circRNAs form unique covalent loops without a 5’cap or a 3’ polyadenosylated tail and exhibit greater stability and sequence conserved than common linear RNA molecules.In addition,circRNAs are often cell-and tissue-specific.It is well established that circRNAs are involved in regulatory processes,including transcriptional modulation,splicing interference,microRNA（miRNA）isolation,and translation.In recent years,more and more studies have shown that some circRNAs may act as "miRNA sponges",naturally isolating and competitively inhibiting miRNA activity,suggesting that circRNAs are highly likely to play an important role in post-transcriptional regulation.It hsa also been reported that circRNAs regulate gene expression by interacting with RNA-binding proteins and translation regulators or directly binding to mRNA.In addition,circRNAs can be translated in vivo and in vitro.Recent studies on circRNAs have improved our understanding of the mechanisms by which they play a role in cardiovascular disease.However,further studies on the role of circRNAs in the pathogenesis of DCM are needed to develop early diagnostic techniques and suggest new therapeutic targets.ObjectiveThis study was aimed to investigate the differential expression of circRNAs in peripheral blood leukocytes between children with DCM and healthy children,and to explore its possible regulatory mechanism according to the expression of circRNA-miRNA-mRNA network,in order to prepare ourselves for bigger tasks ahead for the study of the pathogenesis of DCM.Meanwhile,the differentially expressed circRNAs were verified to provide an important theoretical basis for the search of biomarkers for the treatment of dilated cardiomyopathy.MethodIn this study,peripheral blood samples from 25 children with DCM diagnosed in the pediatric cardiology ward of Shandong Provincial Hospital between February 1,2019 and June 31,2020 were collected as the experimental group.On the other hand,we collected peripheral 25 blood samples from healthy volunteers who underwent physical examination in the child health and development clinic of Shandong Provincial Hospital as the control group.The gene chip analysis was carried out by selecting the three cases of the experimental group and the three sex-and-age matched cases of control group,and the difference expression of the circRNAs and mRNAs was observed.We evaluated the biological functions of differentially expressed circRNAs,by using a circRNA-miRNA-mRNA interaction network constructed by Genome Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）to predict the potential functions of differentially expressed genes and DCM-related pathways.Finally,the accuracy of the chip data was further verified by quantitative real-time polymerase chain reaction（qRT-PCR）,and differentially expressed circRNAs were verified in 25 children with DCM and 25 healthy children,and the products were verified by Sanger sequencing.Results1.A total of 25 children with DCM and 25 healthy children met the inclusion criteria,and the two groups were basically matched in terms of gender and age.The data of left ventricular end-diastolic diameter Z fraction,left ventricular ejection fraction,brain natriuretic peptide precursor,hypersensitive troponin T,C-reactive protein and cardiothoracic ratio in echocardiography between the two groups were statistically analyzed,and the P value was<0.001,showing a statistically significant difference.In addition,15（60%）of the children with DCM had viral infection,21（84%）had ECG abnormality,and 16（64%）had genetic abnormality.None of these were found in the control group.2.By comparing peripheral blood leukocyte microarray data between children with DCM and healthy children,the trunking criteria of differentially expressed circRNAs were≥2 or≤0.5 and P<0.05.A total of 1156 circRNAs were differentially expressed in DCM.There were significantly more down-regulated circRNAs（899）than up-regulated circRNAs（257）.In addition,641 differentially expressed mRNAs were found,among which the up-regulated mR:NAs（482）were significantly more than the down-regulated mRNAs（159）.By preliminary analysis of the differentially expressed circRNAs,it was found that they were mainly derived from chromosome 2,chromosome 1 and chromosome 5.In addition,these circRNAs are typically less than 2000 nucleotides in length,with a concentration of less than 500 nucleotides.3.The three most enriched GO terms were ’response to wounding’,’inflammatory response’ and‘cytokine secretion’ in BP;the top three terms in CC were’endomembrane system’,’ruffle membrane’ and‘endosome membrane’.In MF,’enzyme binding’,’SMAD binding’,and ’peroxidase activity’ were the three most enriched GO terms.KEGG pathways in differentially expressed circRNAs were enriched in ’Toll-like receptor signaling’,and ’Fc gamma R-mediated phagocytosis’,processes commonly associated with DCM.4.We successfully designed and synthesized 4 circRNAs primer sequences,which were stably amplified by qRT-PCR,and the products were confirmed as target products by Sanger sequencing.5.The qRT-PCR demonstrated that,hsa_circ₀067735 was down-regulated（0.45 times,P<0.001）and hsa_circ₀070186 was up-regulated（2.82 times,P<0.001）in the DCM group,compared with the control group.Hsa_circ₀069972 was down-regulated（0.50 fold,P<0.05）,and the expression trends of the three were consistent with the chip data,which were statistically significant.Hsa_circ₀005495 was down-regulated by 0.69 times（P<0.05）,indicating that the expression trend of circRNAs was contrary to the results of microarray data.Functional analysis of these circRNAs showed that they were associated with hypertrophy,remodeling,fibrosis,and autoimmunity.6.The software was used to predict the top 10 miRNAs binding sites with the highest up-regulation and down-regulation ratios differentially expressing circRNAs,and to predict the miRNAs that might be adsorbed.In addition,the circRNA-miRNA-mRNA co-expression network of three circRNAs,hsa_circ₀067735,hsa_circ₀069972 and hsa_circ₀070186 were completed.Conclusions1.Through gene chip analysis,the expression of circRNAs and mRNAs in peripheral blood white blood cells of children with DCM was significantly different.2,Biological functions of differentially expressed circRNAs were evaluated by circRNA-miRNA-mRNA interaction network constructed by GO and KEGG analysis,and it was speculated that differentially expressed circRNAs might affect injury response and Toll-like receptor signaling pathway.Through the mechanism of ceRNA,it participates in the regulation of transcription level,affects the protein expression and the adsorption of miRNA,and thus participates in the mechanism of the occurrence and development of DCM.3.Three trends of the four circRNAs were consistent with the microarray results by qRT-PCR,which confirmed the reliability and reproducibility of the microarray results.Among them,the differential expression of hsa_circ₀067735,hsa_circ₀069972 and hsa_circ₀070186 were all statistically significant,which had the potential to be biomarkers for the non-invasive diagnosis of DCM in children.