Role And Mechanism Of Circular RNA CircEPB41L2 In Regulating Bladder Cancer Development

Objectives: Bladder cancer(BCa)is one of the most common urinary malignancies with high morbidity and mortality.Studies have shown that circular RNA(circ RNA)plays an important role in tumor progression,but the mechanism of its regulation in bladder cancer has not been fully clarified.Through high-throughput sequencing analysis of circ RNA expression profiles in 12 pairs of bladder cancer tissues and adjacent tissues,we found that circEPB41L2 was significantly down-expressed in bladder cancer tissues.Based on the previous results,we took circEPB41L2 as the research object to further explore the effects of circEPB41L2 on bladder cancer survival,migration,invasion in vitro,and proliferation in vivo,and reveal the molecular mechanism of circEPB41L2 in regulating the occurrence and development of bladder cancer.Methods: 1)The expression level of circEPB4L2 was verified by q RT-PCR in 50 pairs of bladder cancer tissues and adjacent normal tissues.Meanwhile,it was also detected in bladder cancer cell lines,and immortalized bladder epithelial cells SV-HUC-1.2)Lentivirus vector overexpressing circEPB41L2 was constructed and transfected into bladder cancer cell lines 5637 and T24 to obtain stable strain.The correct loop formation of circEPB41L2 lentivirus was verified by PCR,RNase R degradation assay and Sanger sequencing.The sublocation of circEPB41L2 in bladder cancer cell lines was studied by nucleus-cytoplasmic separation assay and fluorescence in situ hybridization.The effects of circEPB41L2 on the ability of viability,migration and invasion of bladder cancer cells were investigated by survival assay,migration assay and invasion assay.Si RNA was used to silence the expression of circEPB41L2 in SV-HUC-1 cells,and the effects of circEPB41L2 on the ability of viability and migration of SV-HUC-1 cells were investigated by survival assay and migration assay.3)5637 cells overexpressed with circEPB41L2 were inoculated into the right axilla subcutaneously of nude mice to construct subcutaneous tumorigenesis model.After tumor formation,tumor volume and weight were measured to investigate the effect of circEPB41L2 on bladder cancer proliferation in vivo.4)Bioinformatics,Western blot experiments and LC-MS/MS were performed to analyze and verify the ability of circEPB41L2 to encode protein,and the protein was named circEPB41L2-240 aa protein.The sublocation of circEPB41L2-240 aa in bladder cancer cells was investigated by immunofluorescence.A lentiviral vector overexpressing circEPB41L2-240 aa was constructed and transfected into bladder cancer cell lines,and the effects of circEPB41L2-240 aa on the capacity of viability,migration and invasion of bladder cancer cells were investigated by survival assay,migration assay and invasion assay.5)To further investigate whether circEPB41L2 plays a role by translating circEPB41L2-240 aa protein,we transfected the circEPB41L2-mutation lentivirus vector with the start codon mutation into bladder cancer cells.The biological function of circEPB41L2 with knockdown protein translation function in bladder cancer was investigated by survival assay,migration assay and invasion assay.Results: 1)The expression of circEPB41L2 in bladder cancer tissues was lower than that in adjacent normal tissues(P<0.01).Compared with SV-HUC-1 cells,the expression in bladder cancer cell lines was lower(P<0.05).2)PCR assay,RNase R degradation assay and Sanger sequencing confirmed that circEPB41L2 could be overexpressed and cycled correctly in bladder cancer cells.Nucleus-cytoplasmic separation assay and fluorescence in situ hybridization confirmed that circEPB41L2 was mainly located in cytoplasm.Compared to the NC group,the group transfected with circEPB41L2 overexpression lentivirus vector significantly inhibited the viability(P<0.05),migration(P<0.01)and invasion(P<0.01)of bladder cancer cells.Compared to the oligo group,the group silencing the expression of circEPB41L2 significantly promoted the ability of viability(P<0.05)and migration(P<0.01)of SV-HUC-1.3)The results of tumorigenic assay in nude mice revealed that the tumor volume(P<0.05)and tumor wight(P<0.01)of circEPB41L2 overexpression group were lower than those of the control group.4)Bioinformatics analysis showed that circEPB41L2 could encode a protein containing 240 amino acids(about 27KDa).Western blot confirmed that there were obvious bands at25-30 k Da in circEPB41L2 overexpressed cells.No bands were found in the NC group and the circEPB41L2-mutation transfection group.A specific protein sequence "VTLLDGTEYSCDLEHK" encoded by circEPB41L2 was detected in the 25～30KDa LC-MS/MS band.Immunofluorescence assay showed that circEPB41L2-240 aa was mainly distributed in cytoplasm.Survival assay,migration assay and invasion assay showed that circEPB41L2-240 aa could significantly inhibit the capacity of viability(P<0.01),migration(P<0.01)and invasion(P<0.01)of bladder cancer cells.5)Although circEPB41L2-240 aa was still able to inhibit the survival,migration and invasion of bladder cancer cells due to the loss of the ability to encode circEPB41L2-240 aa protein,its tumor suppressor effect was significantly inhibited.Conclusion: 1)circEPB41L2 was low expressed in bladder cancer tissues and bladder cancer cell lines,and could significantly inhibit the viability,migration,invasion and subcutaneous tumorigenic capacity of bladder cancer cells.2)The protein encoded by circEPB41L2,namely circEPB41L2-240 aa,significantly inhibited the viability,migration and invasion of bladder cancer cells.However,after circEPB41L2 lost its ability to encode protein,its tumor suppressor effect was weakened,suggesting that circEPB41L2-240 aa may be partly involved in the regulation of bladder cancer by circEPB41L2.