Identification And Distribution Characterization Of Extrachromosomal Cyclic DNA In Peripheral Blood Mononuclear Cells Of Patients With Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus(SLE)is an autoimmune disease with multisystem involvement,the pathogenesis of which is still unclear.Increasingly,studies have demonstrated that extrachromosomal circular DNA(ecc DNA)molecules can induce innate immune responses and mediate inflammatory responses.ecc DNA is a circular DNA molecule that is isolated or shed from the normal genome and is free from the chromosomal genome.In this study,we intend to further explore the mechanism of SLE development by comparing the differences in ecc DNA between peripheral blood mononuclear cells(PBMC)of SLE patients and healthy controls.Methods:Sixteen patients with inactive SLE(Inactive-SLE group),sixteen patients with active SLE(Active-SLE group),and twelve healthy controls(NC group)were enrolled in this study.Ficoll density gradient centrifugation method was used to extract PBMC of the three groups of samples,respectively.Through high-throughput sequencing and bioinformatics analysis,the source and distribution characteristics of ecc DNA molecules identified in PBMC were compared,and GO enrichment analysis and KEGG enrichment analysis were performed for different ecc DNA target genes.Result:(1)Abundant ecc DNA fragments were detected in all three groups of samples.The length of ecc DNA in all three groups showed double peaks,but the ecc DNA fragment in patients with low-disease active SLE was generally smaller than that in patients with active SLE and healthy controls.The first peak value of ecc DNA fragment appeared at ～143bp in patients with either low disease activity or active SLE.(2)The ecc DNA fragments of all three sets of samples were widely distributed on the genome of human autosomes,especially on chromosome 19,which has a high gene density.(3)Cp G island sequence and r RNA sequence enrichment in repetitive elements was lower in patients with low disease activity and active SLE than in healthy controls.(4)Repeated trinucleotide motifs with high frequency were detected in all three groups of ecc DNA fragments,especially sequence I=sequence III=CAC and sequence II=sequence IV=ACA.(5)Compared with the inactive SLE group and the active SLE group,the main enrichment items and pathways of ecc DNA target genes in GO and KEGG enrichment analysis were roughly the same.Compared with the healthy control group,the up-regulated ecc DNA target genes in SLE patients were mainly enriched in biological processes related to cell growth and development in GO enrichment analysis.KEGG enrichment analysis showed that up-regulated ecc DNA target genes were significantly enriched in Focal adhesion and Rap1 signaling pathways.In addition,we found that up-regulated ecc DNA target genes enrichment pathways were mainly related to cell signal transduction mechanism.Conclusion:(1)This study proved for the first time that ecc DNA molecules are widely present in PBMC.(2)There were significant differences in ecc DNA molecules in SLE patients compared with healthy controls,including length distribution characteristics,chromosome distribution characteristics and repeat element distribution characteristics.(3)The formation of ecc DNA molecules may be related to the cyclization of DNA fragments mediated by homologous recombination.