CircCPSF6 Promotes The Proliferation And Migration Of Bladder Cancer Cells Via MiR-3909/YKT6 Axis

Objective:(1)to verify the loop formation of circCPSF6.(2)to verify the expression of circCPSF6 in bladder cancer tissues and cells.(3)to explore the localization of circCPSF6 in bladder cancer cells(4)to predict the downstream target gene of circCPSF6(5)to explore the effect of circCPSF6 on the phenotype of bladder cancer cells.(6)to explore the downstream miRNA molecules regulated by circCPSF6.(7)to elucidate the specific mechanism of circCPSF6 on the progression of pancreatic cancer.(8)to explore the effect of circCPSF6 on the growth of bladder cancer cells in vivo.Methods:(1)circCPSF6 cycling was verified by Sanger sequencing,RNase R digestion and agarose gel electrophoresis.(2)the expression of circCPSF6 was evaluated by q PCR.(3)FISH and nuclear and cytoplasmic separation experiments were used to understand the localization of circCPSF6 in bladder cancer cells.(4)predict the potential target genes downstream of circCPSF6 through online database(5)verify the binding of circCPSF6 with miR-3909 and miR-3909 with YKT6 by RNAPULLDOWN test and double luciferase report experiment.(6)the expression of circCPSF6 and miR-3909 was changed by transfection of small interference and overexpression plasmids,and the effects of circCPSF6 and miR-3909 on the phenotype of bladder cancer cells were investigated by cck-8,Edu,transwell and scratching.(7)Western blotting and immunohistochemistry were used to detect the changes of protein level.Results:(1)circCPSF6 was formed by reverse cutting of exon 2-10 of linear CPSF6.(2)q RT-PCR assay showed that circCPSF6 was relatively highly expressed in bladder cancer tissues and bladder cancer cells.(3)the results of FISH and nuclear-cytoplasmic separation experiments showed that circCPSF6 mainly existed in the cytoplasm.(4)knockdown of circ RNA inhibits the proliferation and migration of bladder cancer cells,and overexpression of circ RNA promotes the proliferation and migration of bladder cancer.It is suggested that circCPSF6 is an oncogenic gene.(5)the results of RNAPULLDOWN assay and double luciferase report assay showed that circCPSF6 was targeted to miR-3909 and miR-3909 to YKT6.(6)overexpression of miR-3909 inhibits the proliferation and migration of bladder cancer cells,while knocking down miR-3909 promotes the proliferation and migration of bladder cancer cells.(7)overexpression of YKT6 can promote the proliferation and migration of bladder cancer cells,and inhibit the proliferation and migration of bladder cancer cells after knocking down YKT6.(8)the recovery experiment showed that knocking down circCPSF6 could reverse the enhancement of proliferation and migration of bladder cancer cells caused by the decrease of miR-3909 expression.(9)tumorigenesis in nude mice showed that knockdown /overexpression of circCPSF6 could affect the proliferation of bladder cancer cells in vivo.Conclusion:(1)as an oncogene,circCPSF6 promotes the proliferation and migration of bladder cancer.(2)miR-3909 acts as a tumor suppressor gene to inhibit the proliferation and migration of bladder cancer.(3)circCPSF6 can target miR-3909/YKT6 and regulate the proliferation and migration of bladder cancer.(4)circCPSF6 may be a potential biomarker and target for the diagnosis of bladder cancer.