The Molecular Characterization Of Extrachromosomal Circular DNA In Plasma Of Lung Adenocarcinoma

Backgrounds:Lung cancer is one of the highest morbidity and mortality of cancer in the world,among which lung adenocarcinoma accounts for the most common pathological type.The application of targeted therapy and immunotherapy in patients has greatly improved the survival rate.Subsequently,drug resistance and oncogene mutations have brought new challenges to the clinic.Finding the mechanism that affects oncogene mutations has become the top priority.Due to its unique function and structure,extrachromosomal circular DNA(eccDNA)has been focused recently.It is independent of the chromosomal genome without being regulated;Its ring structure is more stable and not easily degraded;its fragments can carry a certain number of genes.All of these may play an important role in the formation of tumors.Therefore,based on next-generation sequencing(NGS),this study profile the cell-free eccDNAs in plasma and described the molecular characteristics,potential functional roles of the cell-free eccDNAs in lung adenocarcinoma.Methods:After review and approval by the ethics review committee,peripheral blood was collected from 12 lung adenocarcinoma patients and 6 healthy people from the First Affiliated Hospital of Guangzhou Medical University,and then circulating free DNA was extracted.In addition,chromosomal DNA of lung adenocarcinoma cell lines H3122 and H1975 were extracted as reference.To enrich the eccDNA,the exonuclease V was used to digest and remove linear DNA,after which was verified by q PCR.And then using a rolling-circle amplification method amplified the eccDNA by Phi29 DNA polymerase and random primers.The eccDNA library was constructed,prepared and sequenced Based on the MGISeq-2000 RS sequencing method of BGI.The Circle-Map algorithm is used to find the circular fragments in the Reads of the aligned reference genome,and then the eccDNA will be reconstructed.Finally,the structure characteristic of eccDNA fragment was explored through the biological databases such as UCSC and Repeat Masker,and used R Studio to perform statistical analysis and graph the results.Results:(1)Both lung adenocarcinoma patients and healthy people contain trace amounts of eccDNA in their plasma.(2)The length of eccDNA fragments of lung adenocarcinoma patients are shorter than those of healthy people(median values are 340 bp and 361 bp,respectively).However,it is found that the proportion of long fragment eccDNA in lung adenocarcinoma patients is significantly higher than the healthy people when analyzed the proportion of eccDNA with a fragment length of more than 3000 bp.(3)Among the eccDNA fragments larger than 3000 bp,the proportion of 5’-UTR and 3’-UTR regions in the long-fragment eccDNA of lung adenocarcinoma patients is higher than the healthy people.Further analysis about the intersection between the functional regulatory elements that can be modified by H3K4Me1,H3K4Me3,and H3K27 Ac histones and eccDNA have found that compared with healthy people,the level of enrichment of regulatory elements in lung adenocarcinoma patients is higher.(4)The number of repetitive elements in the eccDNA fragment of lung adenocarcinoma patients is higher than the healthy people,and the proportion of repetitive elements in LINE and LTR is higher than that too.Conclusion:In this study,it was found that the eccDNA in plasma of lung adenocarcinoma has significant differences in length,genomic elements,regulatory elements,and repetitive elements compared with healthy people.Further characterization of the extracellular eccDNAs in peripheral blood will facilitate understanding of their molecular mechanism and potential clinical utilities of lung adenocarcinoma.