Effects And Mechanisms Of Hsa＿circ＿0001666 On Biological Behavior Of Non-small Cell Lung Cancer Through MiR-1184/miR-548I/AGO1 Axis

Objective: Lung cancer is one of the malignant tumors with the highest incidence and mortality in our country and even in the world.Non-small cell lung cancer(NSCLC)accounts for about 85% of the total incidence of lung cancers.The 5-year survival rate of early NSCLC after surgical treatment can reach 70% to 90%.However,about 75% of patients are in advanced or advanced stages at first diagnosis,and despite important advances in treatment for advanced NSCLC,such as targeted therapy and immunotherapy,the overall survival rate for NSCLC remains low.Meanwhile,the molecular pathogenesis of NSCLC is highly heterogeneous and complex.Therefore,further elucidated the pathogenesis of non-small cell lung cancer can provide important theoretical basis for improving the prognosis of NSCLC patients.Circ RNAs have been proved to play an important role in the occurrence and development of cancers.However,the dysfunction of circ RNAs in non-small cell lung cancer(NSCLC)has not been fully explored.This study aims to focus on an upregulated circ RNA,hsa＿circ＿0001666(circ0001666),and further explores its biological roles and mechanism in NSCLC progression and potential applications value clinically.Methods: In this study,we verified the existence and subcellular location of circ0001666 through Sanger sequencing and RNA FISH assay,and detected its expression in NSCLC tissues and cell lines by q RT-PCR assay.At the same time,the relationship between circ0001666 expression level and clinical data of patients was analyzed by combining clinicopathological data and follow-up data.Further,overexpression and knockdown vector of circ0001666 were constructed to explore its effect on the biological behavior of NSCLC through transwell,wound healing and animal experiments.Then,bioinformatics software(Target Scan,miRDB,Circ Interactome,Circbank,miRanda)was used to predict potential miRNAs adsorbed by circ0001666 and regulated downstream targets.The relationship and mechanism of circ0001666 sponging miR-1184/miR-548 I to regulate AGO1 was explored by pulldown assay,RNA-FISH experiment,Dual-luciferase assay,and western blot experiment.Wound healing and transwell assay were used to detect the co-regulation function of circ0001666,miR-1184/miR-548 I and AGO1 on the biological behavior of NSCLC cells.Finally,GSEA enrichment analysis and western blot assay were used to explore the effect of circ0001666 on PI3K/AKT/mTOR signaling pathway through targeting AGO1.Results: Circ0001666 was highly expressed in NSCLC tissues and cell lines through q RTPCR,and was positively correlated with pathological stage and lymph node metastasis in NSCLC patients.Kaplan-Meier survival analysis suggested that NSCLC patients with high circ0001666 expression had a lower overall survival rate.RNA FISH showed that circ0001666 was mainly located in the cytoplasm and promoted the invasion and migration of NSCLC in vitro and in vivo.Dual-luciferase,pulldown,FISH and western blot assay showed that circ0001666 could directly bind to miR-1184/miR-548 I,and then release the inhibition effect of miRNA on target gene AGO1 and consequently promote the protein expression of AGO1.Overexpression of circ0001666 can reverse the inhibition of miR-1184/miR-548 I on invasion and migration of NSCLC cells and the reduction of AGO1 protein expression by miR-1184/miR-548 I.Finally,GSEA enrichment analysis and western blot analysis showed that circ0001666 activated PI3K/AKT/mTOR signaling pathway by regulating AGO1 protein.Conclusion: This study demonstrated that circ0001666 was significantly up-regulated in NSCLC tissues and cell lines,and mainly localized in the cytoplasm.Circ0001666 was closely associated with tumor size,pathological stage,lymph node metastasis and poor prognosis.Mechanistically,circ0001666 could promote the expression of AGO1 by competitively binding miR-1184/miR-548 I and activate PI3K/AKT/mTOR signaling pathway,thus promoting the invasion and metastasis of NSCLC,which provided a new theoretical basis for the role of circ RNAs in the progression of NSCLC.