Mechanism Study Of CircRPPH1 Regulating The Biological Behavior Of Bladder Cancer Cells Through The MiR-296-5p/STAT3 Pathway

Objective: Bladder cancer is a prevalent malignant tumor in the urinary system,seriously affecting human health.As a novel type of non-coding RNA,circ RNA has been demonstrated to have significant regulatory functions in the pathogenesis and progression of diverse diseases.However,the role of circ RNA in bladder cancer remains to be further explored.The aim of this study is to investigate the functional effects and molecular mechanisms of circ RPPH1 in regulating the malignant biological behavior of bladder cancer cells.Methods: 1.The transcriptome data set of circ RNA for bladder cancer in GEO database was used for differential expression analysis,and then molecular biology experiments were used to explore the expression of circ RPPH1 in bladder cancer cell lines and its influence on biological behavior of bladder cancer cells.2.Bioinformatics methods were used to predict the miRNAs bound to circ RPPH1.In vitro experiments,the regulatory relationship among circ RPPH1,miR-296-5p and STAT3 was verified by RIP assay,dual luciferase reporter assay,fluorescence in situ hybridization assay,real-time fluorescence quantitative PCR(RT-q PCR)and western blotting assay.Then,the expression levels of circ RPPH1 and miR-296-5p in bladder cancer cell lines were specifically regulated,and functional rescue experiments were conducted to observe changes in the malignant biological characteristics of bladder cancer cells.3.Bioinformatics methods were used to predict the RNA binding protein that was targeted to circ RPPH1 or its precursor RNA,and then the expression of this protein in bladder cancer cell lines was explored by RT-q PCR and western blotting assay.Based on the TCGA database and functional experiments,the relationship between this protein and the clinicopathologic features of bladder cancer and its effect on the biological behavior of bladder cancer cells were further studied.The regulatory relationship between RNA binding protein and circ RPPH1 was investigated by RIP assay,RT-q PCR,western blotting assay and functional rescue assays.Results: 1.Bladder cancer circ RNA transcriptome data set(GSE92675 and GSE97239)was download from the GEO database,including 7 bladder cancer tissue samples and 7 normal bladder tissue samples.The two data sets had 575 and 753 DEcirc RNAs,respectively.The intersection of DEcirc RNAs in the two data sets showed that only one circ RNA was highly expressed in bladder cancer,namely circ RPPH1(hsa＿circ＿0000512).Meanwhile,the results of RT-q PCR confirmed that circ RPPH1 expression level was increased in bladder cancer.By constructing interfering and overexpressing plasmids,we specifically regulated the expression level of circ RPPH1 in bladder cancer,and found that it could promote the proliferation,invasion and metastasis of bladder cancer cells in vitro and in vivo.2.The Circ Interactome and CIRCBANK databases predicted miR-296-5p as the targeted miRNAs.Fluorescence in situ hybridization assay showed that circ RPPH1 co-localized with miR-296-5p in the cytoplasm of bladder cancer cells.Compared with the anti-Ig G group,circ RPPH1 and miR-296-5p were significantly enriched in the anti-Ag O2 group.Luciferase reporter gene assay further confirmed the targeting relationship between circ RPPH1 and miR-296-5p.Besides,miR-296-5p was predicted to bind the 3’UTR of STAT3 m RNA with a high score based on the prediction of Target Scan database.Luciferase reporter gene assay also verified the targeting relationship between miR-296-5p and STAT3.Furthermore,RT-q PCR and western blotting confirmed that miR-296-5p could inhibit STAT3 expression at the post-transcriptional level.The regulatory relationship among circ RPPH1,miR-296-5p and STAT3 were then verified by several functional rescue experiments.3.The Circinteractome database predicted that EIF4A3 could bind to the flanking region of circ RPPH1 precursor RNA,with two binding sites.RT-q PCR and western blotting results revealed that EIF4A3 expression was significantly increased in bladder cancer cell lines compared to the normal bladder epithelial cell line.TCGA database analysis showed that the expression level of EIF4A3 was closely correlated with the pathological subtypes,pathological stages,histological grades,overall survival and disease-specific survival of bladder cancer(P < 0.05).RIP experiment results indicated that EIF4A3 could bind to the flanking region of circ RPPH1 precursor RNA.More importantly,results of RT-q PCR and western blotting experiments showed that EIF4A3 could promote the generation of circ RPPH1 in bladder cancer cells.In addition,functional rescue experiment showed that the proliferation,invasion and migration ability of bladder cancer cells in the sh-EIF4A3 and OE-circ RPPH1 co-transfected group were stronger than that in the sh-EIF4A3 group alone.Conclusions: 1.Bioinformatics analysis and molecular biology experiments showed that circ RPPH1 expression was increased in bladder cancer cells compared with normal bladder epithelial cells.Besides,circ RPPH1 can promote the proliferation,invasion and metastasis of bladder cancer cells.2.Circ RPPH1 can regulate the expression of STAT3 by competitively binding to miR-296-5p,thus promoting the proliferation,migration and invasion of bladder cancer cells.3.The expression of EIF4A3 is significantly elevated in bladder cancer and positively associated with the malignant pathological characteristics of the disease.In addition,EIF4A3 can bind to the flanking region of circ RPPH1 precursor RNA,thereby promoting the generation of circ RPPH1 and facilitating bladder cancer cell proliferation,invasion,and migration.