The CircINTS4/miR-146b/CARMA3 Axis Promotes Tumorigenesis In Bladder Cancer

Introduction:Bladder cancer(BC)is one of common malignant tumor in the urinary system and is the ninth most common cancer worldwide.Much progress has been achieved in understanding the pathogenic mechanisms of BC.However,the main treatments for BC are still limited to surgery and chemotherapy,and due to its recurrence and metastasis,there are few effective therapeutic measures.Emerging evidence demonstrates that the CARMA3 plays a significant role in inflammation and carcinogenesis.Accumulating evidence demonstrates that CARMA3 is overexpressed in all types of cancers,such as breast,lung,renal,and colorectal cancers.In addition,CARMA3,considered to be an oncogene,regulates the proliferation,invasion,migration and apoptosis of tumour cells in association with activation of GPCR and PKC.Circular RNAs(circRNAs)act as competing endogenous RNA(ceRNA)to modulate gene expression and promote the occurrence and development of diseases and the role of circRNAs in carcinogenesis has recently been implicated in many cancers.However,the role of circRNAs are rarely reported in BC.Therefore,we explore the pathway of specific circINTS4/miR-146b/CARMA3 in bladder cancer,and to find the medical molecular markers,so as to provide an effective diagnosis and treatment of bladder cancer.Methods:1.Screening and detection of CARMA3 upstream microRNAThe public online database of Targetscan,miRWalk,miRBase and Starbase were searched to identify the potential role of specific miRNAs that have a consensus binding site in the 3'-untranslated region(3'-UTR)of CARMA3.Cases diagnosed of bladder cancer were randomly selected out to compare the expression of CARMA3 and miR-146 b between tumor and adjacent tissues.In addition,the expression of bladder cancer cell lines and SV-HUC-1 immortalized epithelial cell line was also examined for the same purpose.2.Regulation mechanism of miRNA on CARMA3 and its function in bladder cancer cell lineTo further confirm interaction between miR-146 b and CARMA3,we transfected 293 T cells with a wild-type(WT)or mutant(Mut)CARMA3 3'-UTR plasmid and measured luciferase activity.With exogenous regulation of target miRNA or CARMA3-overexpress plasmid in bladder cancer cell lines,and the changes of cell proliferation,migration,invasion,cell cycle and apoptosis abilities were measured.3.Screening and binding verification of miRNA upstream circRNAThe public online database of RNAhybrid was searched to identify the potential role of specific circRNA that have a consensus binding site of miR-146 b.To further confirm interaction between miR-146 b and circRNA.Luciferase activity reporter,RNA pulldown and RIP and FISH assays were performed in bladder cancer cells.4.The function role of upstream circRNA in bladder cancerCases diagnosed of bladder cancer were randomly selected out to compare the expression of circ INTS4 between tumor and adjacent tissues.In addition,the expression of bladder cancer cell lines and SV-HUC-1 immortalized epithelial cell line was also examined for the same purpose.With exogenous regulation of target circINTS4 in bladder cancer cell lines,and the changes of cell proliferation,migration,invasion,cell cycle and apoptosis abilities were measured.Tumour-formation experiments were also performed for cell function.5.Regulation mechanism of circINTS4 on miRNA in bladder cancer cellDeep investigate the regulatory relationship and regulation mechanism of upstream circINTS4 and downstream miRNA and target proteins6.Validation of circRNA for pathway activation/inhibitionWestern blot assay was performed to detect the effect of endogenous overexpression/silencing circRNA on the changes of corresponding NF-?B pathway and P38 MAPK pathway.Results:1.CARMA3 expression is upregulated and miR-146 b participates in the regulation of CARMA3 in bladder cancer.2.miR-146 b acts as a tumor suppresser by targeting CARMA3 in bladder cancer.3.circINTS4 functions as an efficient miR-146 b sponge in bladder cancer4.circINTS4 plays an oncogenic role of bladder cancer in vitro and vivo.5.circINTS4 affects the cell function and upregulates expression of the miR-146 b target gene CARMA3 by sequestering miR-146 b.6.circINTS4 activates the NF-?B signalling pathway and suppresses the P38 MAPK signalling pathway in a CARMA3-mediated manner in BC cells.Conclusions:circINTS4 could serve as a sponge for miR-146 b and regulate the expression of CARMA3 in a miRNA-mediated manner in BC.circINTS4 is upregulated in BC tissues and affects the proliferation,migration,cell cycle and apoptosis,and circINTS4 may increase tumorigenicity in bladder cancer.Additionally,the NF-?B and P38 MAPK signalling pathways are involved in the circINTS4/CARMA3 regulatory axis.All these findings may contribute to our knowledge regarding BC progression and to the development of a novel therapy target for BC.