Hsa＿circ＿0001361 Promotes Bladder Cancer Invasion And Metastasis Through MiR-491-5p/MMP9 Axis

Objective: Our previous studies found that hundreds of circular RNAs(circ RNAs)were aberrantly expressed in bladder cancer(BC)by high-throughput sequencing.In this study,we focused on the upregulated circ RNAs and identified a novel circ RNA,hsa＿circ＿0001361(circ0001361),and further explore its applications value as a therapeutic target and roles in BC occurrence and progression.Methods: 1.The expression of circ0001361 in 69 cases of clinical BC tissues with different stages and their corresponding adjacent normal bladder mucosa tissues were evaluated by q RT-PCR.2.RNA FISH was conducted to obtain the cell distribution of circ0001361.3.The circ0001361 knockdown and overexpression plasmid were designed and stably transfected into T24 T and UMUC3 cells.4.Transcriptome analysis was performed to identify differentially expressed genes between circ0001361 overexpression and corresponding control cells.5.Multiple databases(Target Scan and RNAhybrid)were used to predict the target binding sites between circ0001361 and miRNAs.RNA pull-down and FISH colocalization assays were performed to fish out the bona fide miRNAs that interacted with circ0001361 in BC cells.6.Dual-luciferase reporter assays were performed to determine the target gene of circ001361 and miRNA.Results: 1.Circ0001361 was expressed at high level in BC tissues and cell lines,and it was positively corelated with pathologic grade and muscle invasion.Moreover,Kaplan–Meier survival analysis implied that BC patients with high circ0001361 expression level had a poor overall survival.2.RNA FISH indicated that circ0001361 mainly localized in the cytoplasm.3.Stable overexpression or knockdown of circ0001361 strikingly facilitated and reduced the migration and invasion of BC cells in vitro and stable knockdown of circ0001361 in BC cells led to a significant decrease lung metastatic count.4.MMP9 expression was upregulated after circ0001361 was overexpressed in BC cells.5.Dual-luciferase reporter assay indicated that circ0001361 promoted MMP9 expression by enhancing MMP9 3’-UTR activity.6.Circ0001361 directly interacts with miR-491-5p in BC cells.7.miR-491-5p can directly bind to MMP9 3 ’UTR region with an inhibitory effect on its activity.8.The inhibitory effect of cell migration and invasion induced by miR-491-5p could be partly reversed by circ0001361 overexpression.Conclusion: Circ0001361 was significantly up-regulated in BC tissues and cell lines,which was mainly distributed in the cytoplasm.The expression of circ0001361 was closely related to clinical pathological staging,grading,vascular invasion and poor prognosis of patients.Circ0001361 acted as an oncogenic circ RNA through miR-491-5p/MMP9 axis,and provided a new target for the diagnosis and treatment of BC.