Characterization Of Plasma Extrachromosomal Circular DNA In Cerebral Infarction

Research background and purpose:Stroke is the leading cause of death and disability in Chinese residents,among which ischemic stroke accounts for about 70% of all stroke.Ischemic stroke has a high disability rate and recurrence rate,causing a huge burden to patients,their families,and the whole society.With the application and maturity of intravenous thrombolysis,interventional thrombectomy technology,surgery and other treatments,the survival rate and prognosis of stroke patients have been significantly improved,but there are also problems such as time window limitation and ischemia-reperfusion injury.The occurrence and development of acute ischemic stroke(AIS)is closely related to inflammatory response and oxidative stress.Extrachromosomal circular DNA(eccDNA)has functions such as signal regulation,intercellular communication,and immune response,and can participate in the inflammatory response and oxidative stress process of AIS.However,no study has reported the relationship between eccDNA and AIS.Therefore,we explored the differences of plasma eccDNA between AIS patients and healthy controls by collecting plasma samples from clinical AIS patients and healthy controls,to provide new research ideas and targets for the diagnosis and treatment of AIS.Materials and Methods:From June 2021 to August 2022,5 AIS patients and 5 healthy controls were enrolled in the First Affiliated Hospital of Chengdu Medical College,and plasma samples were collected.Plasma eccDNA was obtained using QIAamp Circulating Nucleic Acid Kit.NEBNext Ultra DNA Library Prep Kit for Illumina was used to construct the library.High-throughput sequencing and Circle-Seq were used to analyze plasma eccDNA.The length characteristics,chromosome distribution characteristics and gene annotation results of the two groups of eccDNAs were described.The molecular characteristics of AIS eccDNAs were described and analyzed from multiple directions,such as the motif sequences on both sides of the eccDNA junction and the enrichment analysis of eccDNA target gene bases.Result:1.A total of 247521 and 208978 eccDNAs were identified from AIS and healthy controls,respectively.The proportion of eccDNAs less than 1kb was 98.95% and98.93%,respectively.The average length of eccDNA was 3.96 kb in AIS and 3.04 kb in healthy controls.There were 171 and 123 eccDNAs greater than 100 kb in length in AIS and controls,respectively.2.eccDNA was derived from each chromosome,and the lowest density of eccDNA was found on X and Y chromosomes.The top three eccDNA density in both AIS and control groups were chromosome 17,19 and 20.3.The results of eccDNA gene annotation and classification showed that the AIS group and the control group had the highest proportion of m RNA and lnc RNA,and the two groups had the highest proportion of three types of repeat sequences,LINE,SINE and LTR.4.The transcription factors corresponding to the AIS motif sequences were At5g58900,ERF5 and SHN3,respectively.The transcription factors corresponding to the control group motif sequences were AT3G52440,Jun and E2 F,respectively.5.The differentially expressed eccDNA target gene enrichment results showed that eccDNA genes in AIS were involved in the regulation of inflammatory response and neurological function after cerebral infarction,and the main pathways included platelet activation,glutamatergic synapses and MAPK signaling pathway.6.mir378 C may be a potential protective factor of AIS and improve the prognosis of AIS patients by preventing atherosclerosis,and its corresponding eccDNA may become a biomarker for the prognosis of AIS.Conclusions:There are a variety of eccDNA in the plasma of AIS patients and healthy controls,and there are differences in the length distribution,chromosome distribution and gene function enrichment between the two groups.The differentially expressed eccDNA annotated genes in AIS were involved in the inflammatory response,oxidative stress,neuroprotection and other biological processes and pathways in AIS.eccDNA genes may affect the occurrence and development of AIS by participating in platelet activation,glutamatergic synapses,MAPK signaling pathways.mir378 C may be a potential protective factor in AIS,and its corresponding eccDNA may become a biomarker for the prognosis of AIS.