Mechanism Of Exosomal CircPPP2R2D In Lung Adenocarcinoma Progression

BACKGROUND:Lung adenocarcinoma(LUAD)is the most prevalent pathological type of non-small cell lung cancer(NSCLC),accounting for approximately 50%of all lung cancer types.LUAD is insidious in its onset with no symptoms,and can only be detected by imaging and blood tumour markers,and there is still a lack of means for early diagnosis and treatment.In recent years,circular RNAs(circRNAs)have become a hotspot for researchers,especially in the field of oncology,and their mechanism of action mainly serves as a combination of competing endogenous RNAs and micro RNAs(mi RNAs),which in turn regulates the expression of downstream m RNAs.Exosomes can exist in various body fluids and tumour microenvironments,serving as a bridge of information exchange between tumour cells and a potential stable tumour biomarker in the future.Several studies have shown an association between LUAD and differential circRNAs in exosomes,but the relationship between circPPP2R2D and LUAD in plasma has not yet been reported.Therefore,in this research,we screened differential circRNAs by RNA sequencing,and then elaborated in the pathogenesis of LUAD by cellular phenotyping experiments and in vitro experiments,and provided a new basis for the diagnosis and treatment of LUAD.Objectives:1.To investigate the expression level and prognosis of circPPP2R2D in LUAD.2.To investigate the effect of abnormal expression of circPPP2R2D on the proliferation and metastasis of LUAD cell lines.3.To explore the involvement of circPPP2R2D in the progression of LUAD through mediating EP300 expression and the specific molecular regulatory mechanism.4.To explore the mechanism by which EIF4A3 promotes circPPP2R2D cyclisation and nuclear export.5.To explore the mechanism of HUR-mediated m~6A modification through IGF2BP3 to enhance the stability of circPPP2R2D.6.To explore the specific mechanism of SYNCRIP-mediated packaging of circPPP2R2D into exosomes.7.To explore the mechanism by which exosomal circPPP2R2D accelerates the exhaustion of anti-tumour CD8+T cells.Methods:1.High-throughput sequencing was used to search for differential exosomal circRNAs in the plasma of lung adenocarcinoma and normal control,and the exosomes were verified and identified by transmission electron microscopy and Western blot experiments;circular identification experiments were done on circRNAs,and validation was given to the sequencing results by RT-q PCR,and functional studies were carried out on the screened circRNAs.2.To find the parental genes,sequences,chromosomal localisation and mi RNAs that can be adsorbed for circPPP2R2D via circBase database(http://www.circbase.org)and circbank database(http://www.circbank.cn/).3.The information of patients included in the subgroups was counted,and the correlation between the expression level of circPPP2R2D in plasma exosomes and clinicopathological features of lung adenocarcinoma patients was assessed by statistical analysis.4.Overexpression and knockdown lentiviral vectors were constructed for circPPP2R2D gene,and lung adenocarcinoma cell lines were infected to establish stable transfections.The proliferation of lung adenocarcinoma cell lines was detected by CCK8;the migration and invasion ability of lung adenocarcinoma cells were detected by cell scratch assay and Transwell assay,respectively.5.The target mi RNA of circPPP2R2D and its downstream target genes were predicted by bioinformatics analysis;the binding targets and interactions between the two were verified by dual luciferase reporter assay.6.After overexpression or inhibition of EP300,EIF4A3,HUR and SYNCRIP genes,the stability,cyclisation and nuclear export of circPPP2R2D,and changes in circPPP2R2D content in exosomes were observed.7.CircPPP2R2D expression was detected by collecting exosomes secreted by LUAD cell lines and T cells in co-culture.The output of exosomes was inhibited,and intra-and extracellular circPPP2R2D expression was again detected.8.Tumour-forming models were constructed for NSG immunodeficient mice to determine the effect of circPPP2R2D on tumour formation in vivo,as well as to explore the effect of exosomal circPPP2R2D on tumours in vivo through human intervention.Results:1.RNA sequencing results showed that circPPP2R2D showed a trend of significantly high expression in the plasma of lung adenocarcinoma patients compared with that of healthy human plasma.2.The expression level of circPPP2R2D in plasma exosomes of lung adenocarcinoma patients was positively correlated with tumour size and smoking.3.Overexpression or knockdown of circPPP2R2D in can promote/inhibit the proliferation of lung adenocarcinoma cell lines and tumour growth in nude mice in vivo.4.Overexpression or knockdown of circPPP2R2D showed promotional and inhibitory functions on the number and size of metastatic foci in lung tissues.5.EP300-mediated activation of histone acetylation increased circPPP2R2D expression.6.The RNA-binding protein EIF4A3 could bind to circPPP2R2D in LUAD cells,and the cytoplasmic circPPP2R2D level was significantly reduced when EIF4A3 was inhibited.7.The HUR-m~6A-IGF2BP3 axis is dysregulated in circPPP2R2D in LUAD and enhances circPPP2R2D stability.8.CircPPP2R2D expression is enhanced in SYNCRIP-deficient LUAD cells,and circPPP2R2D expression is reduced in exosomes secreted by SYNCRIP-deficient LUAD cells.9.Co-culture of exosomes circPPP2R2D produced by lung adenocarcinoma cell lines and T cells promotes proliferation,migration,invasion and other functions.CONCLUSION:In this study,we systematically elucidated the increased expression of circPPP2R2D in lung adenocarcinoma after EP300-mediated binding to RNA-binding protein EIF4A3 in the cytoplasm,which promotes the output of circPPP2R2D,at the three levels of cells,tissues and nude mices.The stability of circPPP2R2D was greatly increased after regulation by the HUR-m~6A-IGF2BP3 axis.CircPPP2R2D content in exosomes was promoted by SYNCRIP.Functional studies showed that circPPP2R2D could promote proliferation,migration invasion and metastasis,and could enter exosomes to affect co-cultured lung adenocarcinoma cell lines and promote lung adenocarcinoma development.