Plasma Exosomal Circular RNAs Are Potential Early Markers Of Lung Adenocarcinoma And Preliminary Functional Study Of Hsa＿circ＿0001492/0000690

Objective: Lung cancer has the highest death rate of any cancer in the world,and most of the patients were in the middle and late stages at the time of diagnosis.Therefore,it is particularly important to find effective early diagnosis and prognostic markers for the treatment of lung cancer.Various studies have found that circular RNA(circRNA)carried by exosomes can be used as noninvasive screening markers.Lung adenocarcinoma(LUAD)is one of the most important histological types of lung cancer.This study aimed to identify differential circRNA in the plasma exosomes of patients with LUAD using high-throughput sequencing and confirm them by fluorescence quantitative real-time polymerase chain reaction(qRT-PCR),RNase R treatment and Sanger sequencing,proposing to find potential markers for the early-stage LUAD patients,and explore its preliminary effect on the proliferation and metastasis of LUAD cell.Methods: First,exosomes were isolated using an exosome isolation kit and confirmed by Western blotting,transmission electron microscopy(TEM),and Nano Sight Assay(NTA).Subsequently,high-throughput sequencing of plasma exosomes for circRNA was performed to construct differential expression profiles of plasma exosomes for lung adenocarcinoma.Four differentially expressed circRNAs were verified by qRT-PCR,RNase rase tolerance and Sanger sequencing in 15 pairs of clinical specimens.Finally,the effects of hsa＿circ＿0001492/0000690 on the proliferation,metastasis and invasion of lung cancer cells were investigated by CCK8,scratch,invasion and migration experiments in A549 cell line.Results: First,Western blot results showed that exosomes extracted from the plasma of lung adenocarcinoma patients and healthy people expressed exosome molecular markers CD9 and CD63.TEM and NTA showed that the size and morphology were consistent with exosomes.Then the result of high-throughput sequencing data documented that 182 differentially expressed exosomal circRNAs in all were screened,which included 105 that were upregulated and 78 that were downregulated in LUAD patient plasma compared with controls.The four upregulated circRNAs including circ＿0001492,circ＿0001346,circ＿0000690,and circ＿0001439 were identical to the sequencing data by qRT‐PCR,and had reverse splicing-site,which could not be degraded by RNase R.Finally,the cell function tests in A549 cells showed that hsa＿circ＿0001492/0000690 could significantly promote the metastasis and invasion of lung cancer cells.Conclusion: The expression levels of hsa＿circ＿0001492,hsa＿circ＿0001346,hsa＿circ＿0000690 and hsa＿circ＿0001439 in plasma exosomes of lung adenocarcinoma patients were significantly increased.Hsa＿circ＿0001492 can promote the proliferation,metastasis and invasion of A549 cells.Hsa＿circ＿0000690 can promote the metastasis and invasion of A549 cells.Plasma derived exosomes hsa＿circ＿0001492 and hsa＿circ＿0000690 are expected to be early molecular markers of lung adenocarcinoma and play an important role in the development of lung cancer.