Exploring The Mechanism Of Circular RNA CircLMBR1 Inhibiting The Progression Of Bladder Epithelial Malignant Transformation Cells

Background:Bladder cancer is one of the most common malignant tumors in the urinary system,with high mortality,recurrence rates and treatment costs.The etiology of bladder cancer is complicated,and chemical carcinogens play an important role.Circular RNA(circ RNA)has been identified to play an indispensable role in the occurrence and development of bladder cancer.The high conservation of its sequence and the stability of its structure make it a research hotspot.Recent studies have shown that circ RNA is involved in the regulation of chemical carcinogenesis.However,the mechanism of circ RNA in the chemical carcinogenesis of bladder cancer is not clear and needs to be further explored.Objective:To explore the interaction mode between circLMBR1 and protein in the occurrence and development of malignant transformed cells of bladder epithelium,and to obtain the signal pathways that may be involved in,so as to provide a new potential target for early diagnosis and treatment of bladder cancer.Methods:1.3-methylcholine induced malignant transformation of human bladder epithelial immortalized cells(MC-SV-HUC-1)and cadmium chloride induced malignant transformed human bladder epithelial immortalized cells(Cd Cl2-SV-HUC-1)were used for overexpression of circLMBR1.RNA pulldown assay was carried out with a specific probe,and the eluate was detected by Coomassie brilliant blue staining and sent to the company for protein sequencing.2.The differentially expressed proteins were analyzed by GO annotation,KEGG pathway analysis and protein interaction analysis.3.The four proteins with the most binding amount,ACTA2,ALDH1A3,AKR1B1 and LAMB3,were selected from the differentially expressed proteins for analysis and verification.4.Western blot assay was carried out on the eluate after RNA pulldown assay to verify the reliability of the sequencing results of RNA binding protein.5.RNA Binding Protein Immunoprecipitation(RIP)assay and qPCR assay were used to verify that ACTA2 and ALDH1A3 were RNA binding proteins of circLMBR1.6.The probe of specific circLMBR1 with fluorescence labeling was designed,and the co-localization of circLMBR1 and proteins were confirmed by fluorescence in situ hybridization assay(FISH).7.Cd Cl2-SV-HUC-1 cells were treated with overexpression and knockdown of circLMBR1,respectively.The effect of circLMBR1 expression on ALDH1A3 was verified by western blot assay.8.Three pairs of sg RNAs of ALDH1A3 were designed by using CRISPR/Cas9 system,and the cell line stably knockout ALDH1A3 was constructed by means of lentivirus packaging plasmids.Finally,the effect of knockout ALDH1A3 was verified by western blot assay.9.Knockout ALDH1A3,Transwell,MTS proliferation,colony formation and wound healing assays to detect the changes of cell invasion,proliferation and migration in stable cell lines overexpressing circLMBR1.10.The subcutaneous tumor model of nude mice was established,and the effect of knockout ALDH1A3 after overexpression of circLMBR1 on the growth of bladder cancer cells in nude mice was analyzed.Results:1.Circ LMBR1 specific probe successfully pulled down proteins.The number of up-regulated and down-regulated proteins in Cd Cl2-SV-HUC-1 cell probe group was59 and 54,while that in MCA-SV-HUC-1 cell probe group was 95 and 123,respectively.2.GO analysis of functional classification of differential proteins showed that the first three enrichment items of biological processes were cellular processes,biological regulation and metabolic processes,KEGG pathway analysis showed that the first three enrichment pathways in Cd Cl2-SV-HUC-1 cells were homologous recombination,non-homologous terminal junction and base excision repair,while in MCA-SV-HUC-1 cells were citrate cycle,homologous recombination and amino acid biosynthesis.3.Western blot assay showed that proteins ACTA2,ALDH1A3 and AKR1B1 could be successfully pulled down by circLMBR1 specific probe.4.RIP assay and qPCR assay reverse verify that ACTA2 and ALDH1A3 are RNA binding proteins of circLMBR1.5.FISH assay verified the co-localization of ACTA2,ALDH1A3 and circLMBR1 in the cytoplasm.6.Western blot assay confirmed that the expression of protein ALDH1A3 was regulated by circLMBR1.7.Compared with the control group,the invasion,proliferation and migration ability of Cd Cl2-SV-HUC-1 and MCA-SV-HUC-1 cells overexpressing circLMBR1 were restored after knockout of ALDH1A3.8.In the subcutaneous tumor model of nude mice,compared with the control group,the size and body weight of subcutaneous tumors of T24 cells in which ALDH1A3 expression was knocked out after overexpression of circLMBR1 were significantly increased(P < 0.05).Conclusion:1.Circ LMBR1 specific probe successfully pulled down RNA binding proteins.The amount of up regulation and down regulation of RNA binding protein in malignant transformed bladder epithelial cells induced by different chemicals was different.2.GO analysis and KEGG pathway analysis by bioinformatics analysis revealed the possible biological processes and signal pathways involved in differential proteins.Protein interaction network map shows the interaction between proteins and proteins.3.RNA pulldown,Western blot,RIP,qPCR and FISH assays verified the co-localization of proteins ACTA2,ALDH1A3 and circLMBR1 in the cytoplasm.4.The expression of protein ALDH1A3 is regulated by circLMBR1.5.After overexpression of circLMBR1,the invasion,proliferation and migration ability of Cd Cl2-SV-HUC-1 cells and MCA-SV-HUC-1 cells knocked out of ALDH1A3 expression were restored,while the size and body weight of nude mouse subcutaneous tumor model with T24 cells increased significantly.