Differential Expression And Function Of Circular RNA In Plasma Of Lung Adenocarcinoma Patients

Objective:Circular RNA?circRNA?is a new type of non-coding RNA molecules with a circular structure,which plays an essential role in cancer development.This study selected differentially expressed circRNAs related to lung adenocarcinoma?LUAD?by bioinformatics analysis and explored the differential expression of circRNA in the plasma of LUAD patients,in order to evaluate whether plasma circRNAs could be used as diagnostic biomarkers for LUAD.Moreover,in vitro experiment was performed to confirm the function of circRNA in LUAD and bioinformatics analysis was used to predict the possible mechanisms and functions of circRNAs in LUAD.Methods:1.Selection of candidate circRNAs:The GEO database was used to investigate differentially expressed circRNAs in paired LUAD tissues and adjacent nontumor tissues.The circBase and CSCD database were used to select LUAD-specific circRNAs.The expression levels of the host genes were analyzed in TCGA dataset,and the prognostic value was assessed using the Kaplan-Meier plotter.2.Validating the expression of circRNAs in plasma of LUAD:The expression of the circRNAs was verified in plasma samples of 153 primary LUAD patients and 54 normal controls using qRT-PCR.In addition,we examined the expression in plasma of 54 patients with LUAD before and after surgery.ROC curve analysis was used to investigate the diagnostic value of circRNA in distinguishing LUAD patients from normal controls.Clinical data of the subj ects were collected to assess the clinical significance of circRNAs.3.In vitro experiment:The siRNA targeting the back-splice junction of circRNA was synthesized to knockdown the expression of the circRNA in LUAD cell lines?A549 and H1299?.After transfection,the cells were processed to assess the knockdown activity by qRT-PCR.The CCK-8 assay was used to measure the function of circRNAs in cell proliferation.4.Prediction of the function and mechanism of circRNAs in LUAD:Potential interactions between the circRNAs and miRNAs were predicted using CircInteractome and circBank database.In addition,miRNA-target interactions were predicted with TargetScan and miRTarBase.The circRNA-miRNA-mRNA network was constructed and visualized with the Cytoscape software.To gain further insight into the functions,GO and KEGG pathway analyses were performed for the target genes using DAVID.Results:1.A total of 182 differentially expressed circRNAs?P<0.05 and |FC|>1.5?were obtained using the GE02R tool.Among the 182 deregulated circRNAs,154 were upregulated and 28 were downregulated.23 LUAD-specific circRNAs were detected using the CSCD database.Four circRNAs?hsa_circ₀005962,hsa_circ₀003958,hsa_circ₀086414,hsa_circ₀001936?were identified as candidate circRNAs,because their host genes showed differential expression in the LUAD and adjacent normal tissues.We found that these circRNAs had prognostic value.2.The expression of the four candidate circRNAs was verified in plasma samples of LUAD patients and normal controls using qRT-PCR.The results showed that hsa_circ₀005962 was highly upregulated in LUAD?P<0.0001?and hsa_circ₀086414 was downregulated?P<0.0001?.However,hsa circ 0003958 and hsa_circ₀001936 showed no significantly differential expression in the plasma between the LUAD patients and the normal controls?P = 0.63;P= 0.50?.3.ROC curve analysis confirmed that a signature comprising the two circRNAs?hsa_circ₀00596 and hsa_circ₀086414?had good diagnostic potential,with an AUC of 0.81?P<0.0001?.4.An statistical analysis was performed to assess correlations between the expression levels of the circRNAs and the clinicopathological features of the LUAD patients.we found that the expression of hsa_circ₀086414 was associated with gender?P = 0.046?and EGFR mutations?P =0.001?.5.Plasma hsa_circ₀005962 displayed significantly different expression before and after surgery in patients with LUAD?P<0.0001?.6.In vitro experiments indicated that knockdown of hsa_circ₀005962 greatly suppressed A549 and H1299 cell proliferation,suggesting that hsa circ 0005962 promoted LUAD cell proliferation.7.We had predicted the circRNA-miRNA-mRNA network for hsa circ 0005962.Bioinformatics analysis revealed that hsa circ 0005962 might be involved in LUAD development.Conclusion:1.The present study validated the significant upregulation of hsa circ 0005962 and downregulation of hsa circ 0086414 in LUAD plasma and cells,suggesting the potential for use of this two-circRNA signature as a novel noninvasive biomarker for LUAD diagnosis.2.We found that the hsa circ 0086414 levels were associated with EGFR mutations,suggesting that hsa circ 0086414 may be involved in the development of LUAD with EGFR mutation.3.Hsa circ 0005962 had different expression in the plasma of LUAD patients before and after surgery.In vitro experiments suggested that hsa circ 0005962 promoted LUAD cell proliferation.In silico analysis indicated that hsa circ 0005962 might be involved in the development of LUAD acting as ceRNAs.