The Construction Of CeRNA Network Mediated By CircRNAs And The Function Study Of CircSLC38A1 In Bladder Cancer

Bladder cancer（BC）ranks first among urinary system malignancies in China,and the cancer incidence and mortality rates continue to rise in recent decades.Because of lack of sensitive diagnostic approaches,nearly one-quarter of BC patients are already in the middle and advanced stages as soon as they are diagnosed,which are associated with a worse prognosis because patients develop distant metastases.Although clinical treatments,including surgery,radiation therapy,chemotherapy,and immunotherapy have improved over the past decades,the overall therapeutic effects of BC are limited and the five-year survival rate keeps at a low level.Therefore,finding promising biomarker,exploring the molecular mechanisms of pathogenicity and screening effective therapeutic targets have enormous potential significance for improving the clinical strategies and outcomes of BC.Circular RNAs（circRNAs）are a novel group of noncoding transcripts that present stably covalent closed loop where the 3’ and 5’RNA ends are joined together,which make them resist with RNA exonuclease and can stably exist in tissues,cells and various body fluids.It has reported that circRNAs play functional regulators and widely involved in the processes of cancers.However,so far,there are few studies on the functions,pathogenic mechanisms and prognostic efficacy of circRNAs in bladder cancer.The research content is consistent with the following two parts:PartⅠ:The construction of circRNA-miRNA-mRNA network and prognostic evaluation model in Bladder CancerObjective:To identify the dysregulated circRNAs in BC and construct a ceRNA regulatory network.Then,a prognostic model was established,and the efficacy of prognostic model was evaluated.Methods:Sequencing data of bladder cancer including circRNAs,mRNA and miRNAs were obtained from GEO and TCGA database.By using,"|log2FC|>1,P-value<0.05" as the standard,the sequencing data were analyzed and the RNA differential expression profiles were drawn.A ceRNA regulatory network mediated by circRNAs was constructed by integreting TCGA and multiple online databases.GO and KEGG were applied to predict the function of ceRNA network;Then the online database String was used to explore the interation relationship between molecules in ceRNA network;The BC prognostic related molecules were screened by the Cox regression and Kaplan-Meier analysis,then a prognostic model was constructed,and the effectiveness was further evaluated by drawing the ROC curve.Result:The differential analysis showed that 4 circRNAs were screened in 2 GEO datasets,among which hsa_circ₀103114,hsa_circ₀102402 and hsa_circ₀104387 were up-regulated,while hsa_circ₀102787 was down-regulated.By using multiple database,a ceRNA network was constructed,which mediated by 4 circRNAs and contains 23 miRNAs and 86 mRNAs.Functional analysis showed that the network has tumor regulation potential,and there were closely interation relationship between mRNAs in the network.Then,a prognostic risk assessment model was established,the risk score=（-0.3569）*ABRACL+（-0.2644）*MAP3K8.Kaplan Meier analysis showed patients with high risks had a poor prognosis（P<0.0001）.The AUC of the three and five-years overall survival rate were 0.656 and 0.744,respectively.Conclusion:4 circRNAs were screened in this study,then a ceRNAs network mediated by circRNAs was constructed.By further screened prognostic related molecules in this network,a prognostic model was established and evaluated,which provided theoretical support for the molecular mechanism research,diagnosis and prognosis evaluation of bladder cancer.PartⅡ:circSLC38A1 facilitates metastasis of bladder cancer by interacting with ILF3 and initiating TGFβ2 transcriptionObjective:To explore the biological function and pathogenic molecular mechanism of the differentially expressed circRNA,hsa_circ₀000396（circSLC38Al）in bladder cancer.Methods:We explored the expression profiles of circRNAs in five paired samples of BC and matched adjacent tissues by high-throughout sequencing,and determined circSLC38A1 as a new candidate according to function prediction and literature reports.The expression levels of circSLC38A1 in BC samples were detected by qRT-PCR.Function of circSLC38A1 was investigated by in-vitro and in-vivo assays.RNA pull down assay and RIP assay were applied to verify the interaction between circSLC38A1 and ILF3.CUT&Tag and RNA-seq were used to explore downstream targets of circSLC38A1-ILF3 complex.Serum exosomal RNA from BC patients and healthy controls were extracted,and the expression level of circSLC38A1 in exosomes was detected,ROC curve analysis was used to evaluate the diagnostic efficacy of exosomal circSLC38Al.Result:CircSLC38A1 is upregulated in bladder cancer tissues and cells.Fuctional assays showed that circSLC38A1 played an oncogenic role to promote invasion and metastasis in BC cells,and it also promoted lung metastasis of bladder cancer in mice model.Integrate CUT&Tag-seq and RNA-seq data,TGFβ2 was screened as the downstream target of circSLC38A1-ILF3 complex.CircSLC38A1 was highly expressed in exosomes from serum of bladder cancer patients compared with healthy control,exosomal circSLC38A1 could used as a potential diagnostic biomarker for BC,and its diagnostic efficiency could reach 87.8%.Conclusion:CircSLC38A1,which is highly expressed in BC,could act as an oncogene to promote the metastasis of BC by forming a complex with ILF3 and activating TGFβ2 transcription.In addition,surum exosomal circSLC38A1 can be used as a potential biomarker of BC.