| ObjectiveLung cancer(LC)is one of the malignant tumors with high morbidity and mortality worldwide.According to histological types,LC can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),of which the NSCLC accounts for about 85%.Although substantial progress has been made in the diagnosis and treatment of NSCLC(e.g.,surgical modalities,targeted therapies,and immunotherapy)in recent years,five-year overall survival of patients with NSCLC remains unsatisfactory.Exploring new molecular targets and their mechanisms in the occurrence and development of NSCLC is of profound significance for the search for new biomarkers and effective therapeutic targets of NSCLC.Circular RNA(circ RNA)is a novel type of endogenous non-coding RNA,which plays an important role in the epigenetic regulation and widely involved in the proliferation,metastasis,drug resistance and immune escape of tumor.However,the role and mechanism of circ RNA in NSCLC have not been fully elucidated.In this study,we found that hsa＿circ＿0000519 is highly expressed in lung adenocarcinoma(LUAD)via bioinformatics analysis,but its biological function and mechanism remains to be further investigated.Therefore,this study aims to confirm the expression pattern of hsa＿circ＿0000519 in LUAD,and further elucidate its function as well as mechanism in the tumorigenesis of LUAD.MethodsFirst,hsa＿circ＿0000519 was found to be significantly up-regulated in LUAD through searching the circ RNA microarray datasets of NSCLC in GEO database and conducting bioinformatics analysis.Subsequently,the expression pattern of hsa＿circ＿0000519 in LUAD tissues and cell lines was verified by RT-q PCR.RNase R digestion assay was used to verify the stability of hsa＿circ＿0000519,and FISH as well as nuclear plasma separation assay was used to confirm the subcellular localization of hsa＿circ＿0000519.Then,we established the stable overexpression or knockdown cell lines of hsa＿circ＿0000519,and determined the effect of hsa＿circ＿0000519 on the biological behavior of LUAD cells through a series of functional experiments in vitro and in vivo.To explore the downstream mechanism,we first used bioinformatics analysis to predict potential target miRNAs of hsa＿circ＿0000519,and investigated the interaction between hsa＿circ＿0000519 and its target miRNA by RNA immunoprecipitation and dual luciferase reporter assays.Next,we detected the expression of the target miRNA in LUAD tissues and cell lines by RT-q PCR,and further explored its biological function by functional experiments in vitro.After that,we used bioinformatics analysis again to predict the candidate target genes of miRNA,and verified the inhibitory effect of miRNA on the expression of its target genes by RTq PCR and western blot,and determined their direct interaction by dual luciferase reporter assay.Subsequently,we detected the expression of the target gene in LUAD tissues by RT-q PCR,and further analyzed the correlation among hsa＿circ＿0000519,miRNA and miRNA target gene expression.In addition,the effect of miRNA target gene on the biological behaviors of LUAD cell lines was further investigated by functional experiments in vitro.To elucidate that hsa＿circ＿0000519 modulate the tumorigenesis of LUAD via serving as a molecular sponge of miRNA to regulate the expression of miRNA target gene,we further constructed the cotransfected cell lines of hsa＿circ＿0000519 and miRNA to conduct molecular mechanism and functional recovery experiments.Finally,bioinformatics analysis was used to predict the possible signaling pathways involved in the hsa＿circ＿0000519/miRNA/m RNA axis,and western blot was performed to verify the pathway.ResultsIn this study,hsa＿circ＿0000519 was found to be highly expressed in LUAD tissues through differential expression analysis of 3 circ RNA microarray datasets,which was further verified in clinical LUAD tissues and cell lines.In addition,the expression of hsa＿circ＿0000519 was found to be positively correlated with the T grade and TNM stage of LUAD patients.We found that hsa＿circ＿0000519 was far more stable than its host gene,and was mainly located in the cytoplasm.Functional experiments in vitro and in vivo showed that overexpression of hsa＿circ＿0000519significantly promoted the proliferation,migration,invasion ability of LUAD cells and the subcutaneous tumor formation ability of nude mice,while knockdown hsa＿circ＿0000519 exhibited the opposite effect.Bioinformatics analysis showed that hsa-miR-1296-5p may be the miRNA target of hsa＿circ＿0000519,and their interaction was determined by Dual luciferase reporter assay and RNA immunoprecipitation.It was also proved that hsa＿circ＿0000519 negatively regulated the expression of hsa-miR-1296-5p We found that hsa-miR-1296-5p was down-regulated in LUAD tissues by RT-q PCR,and its expression was negatively correlated with that of hsa＿circ＿0000519.Functional experiments in vitro indicated that overexpression of hsa-miR-1296-5p could significantly inhibit the proliferation,migration and invasion ability of LUAD cells.Subsequently,bioinformatics analysis suggested that DARS,SF3B2 and TRIP12 may be potential downstream target genes of hsa-miR-1296-5p,while DARS was further identified as the direct target gene by RT-q PCR,western blot and dual luciferin reporter gene assay.Interestingly,DARS was found to be negatively regulated by hsa-miR-1296-5p but positively regulated by hsa＿circ＿0000519.Correlation analysis showed that the expression of DARS was significantly negatively correlated with that of hsa-miR-1296-5p,but positively correlated with that of hsa＿circ＿0000519 in LUAD patients.In addition,Functional experiments in vitro showed that overexpression of DARS could partially reverse the inhibitory effect of hsa＿circ＿0000519 on the proliferation,migration and invasion capability of LUAD cells,indicating the oncogenic role of DARS in the progress of LUAD.Trough molecular mechanism recovery experiments,we found that overexpression of hsa-miR-1296-5p could partially reverse the increased expression of DARS mediated by hsa＿circ＿0000519 up-regulation,while knockdown of hsa-miR-1296-5p could also partially recover the decreased expression of DARS mediated by hsa＿circ＿0000519 down-regulation.Through functional experiments of comeback,it’s found that overexpression of hsa-miR-1296-5p could reverse the enhanced proliferation,migration and invasion ability of LUAD cells induced by hsa＿circ＿0000519 up-regulation,while knockdown of hsa-miR-1296-5p could also reverse the decreased proliferation,migration and invasion ability of LUAD cells induced by hsa＿circ＿0000519 down-regulation.Finally,bioinformatics analysis suggested that hsa＿circ＿0000519/hsamiR-1296-5p/DARS axis might be involved in the PI3K/AKT/m TOR signaling pathway,which was further verified by western blot.Conclusion1.hsa＿circ＿0000519 was highly expressed in LUAD tissues and cell lines,and its high expression was correlated with the higher T grade and TNM stage of LUAD patients.2.hsa＿circ＿0000519 exerted an oncogene role in the tumorigenesis of LUAD.Down-regulation of hsa＿circ＿0000519 could significantly inhibit the proliferation,migration,invasion ability of LUAD cells and the subcutaneous tumor formation ability of nude mice.3.hsa＿circ＿0000519 promoted the progression of LUAD by serving as a miRNA sponge of hsa-miR-1296-5p to up-regulate DARS expression.4.hsa＿circ＿0000519 could activate the PI3K/AKT/m TOR signaling pathway via modulating the hsa-miR-1296-5p/DARS axis. |
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