Nitroxoline suppresses metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway

Abstract

Bladder cancer is one of the most common and deadly cancer worldwide. Current chemotherapy has shown limited efficacy in improving outcomes for patients. Nitroxoline, an old and widely used oral antibiotic, which was known to treat for urinary tract infection for decades. Recent studies suggested that nitroxoline suppressed the tumor progression and metastasis, especially in bladder cancer. However, the underlying mechanism for anti-tumor activity of nitroxoline remains unclear. Methods: CircRNA microarray was used to explore the nitroxoline-mediated circRNA expression profile of bladder cancer lines. Transwell and wound-healing assay were applied to evaluate the capacity of metastasis. ChIP assay was chosen to prove the binding of promotor and transcription factor. RNA-pulldown assay was performed to explore the sponge of circRNA and microRNA. Results: We first identified the circNDRG1 (has_circ_0085656) as a novel candidate circRNA. Transwell and wound-healing assay demonstrated that circNDRG1 inhibited the metastasis of bladder cancer. ChIP assay showed that circNDRG1 was regulated by the transcription factor EGR1 by binding the promotor of host gene NDRG1. RNA-pulldown assay proved that circNDRG1 sponged miR-520h leading to the overexpression of smad7, which was a negative regulatory protein of EMT. Conclusions: Our research revealed that nitroxoline may suppress metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway.

Keywords: Bladder cancer; metastasis circNDRG1; microRNA; nitroxoline.

Figure 1

Nitroxoline suppressed the metastasis of bladder cancer in vitro and in vivo. (A and B) Transwell assay and wound-healing assay were performed to evaluate migration of bladder cancer cells. (C) Luciferase-labelled UM-UC-3 cells were injected into nude mice by tail veins for experimental metastasis. (D) Bioluminescent signals were measured by the IVIS imaging system. *P<0.05, **P<0.01 and ***P<0.001.

Figure 2

CircNDRG1 was up-regulated after nitroxoline treatment. (A) Venn diagram of the intersection among up-regulated circRNAs in 4 bladder cancer cells. (B) Heatmap of circRNA-seq analysis showed top 31 differentially high expressed circRNAs of 4 bladder cancer cells. (C) Schematic illustration demonstrates circNDRG1 formation via exons 4-7 circularization of NDRG1. (D) RT-qPCR was utilized to analyze the circNDRG1 expression. (E) RT-qPCR was used to determine the abundances of circNDRG1 and linear mRNA NDRG1 in T24 and UM-UC-3 cells treated with RNase R. (F) Transcription half-life of circNDRG1 and linear mRNA NDRG1 was demonstrated at the indicated times after adding actinomycin D. (G) Southern-blotting was used to analyze the amplification of divergent (◀▶) and convergent (▶◀) primers in cDNA and gDNA. (H and I) Nuclear-cytoplasm separation RT-qPCR assay showed circNDRG1 was predominantly localized in cytoplasm. *P<0.05, **P<0.01 and ***P<0.001.

Figure 3

CircNDRG1 inhibited the metastasis of bladder cancer in vitro and in vivo. (A) The silence efficiency of circNDRG1 and linear mRNA NDRG1 by circNDRG1-si. (B and C) Transwell and wound-healing assay indicated the silence of circNDRG1 promoted the migration of bladder cancer cells. Cells were pretreated with nitroxoline (10 µM) for 24 hours. (D) The overexpression efficiency of circNDRG1 was determined by RT-qPCR. (E and F) Transwell and wound-healing assay showed the overexpression of circNDRG1 inhibited the migration of bladder cancer. Cells were pretreated with nitroxoline (10 µM) for 24 hours. (G and H) UM-UC-3 cells stably transfected with circNDRG1-sh and vector were injected into nude mice by tail veins and bioluminescent signals were measured by the IVIS imaging system. *P<0.05, **P<0.01 and ***P<0.001.

Figure 4

Predicting EGR1 as upstream transcription factor of circNDRG1. (A and B) Venn diagram representations of mRNA-seq in 4 bladder cancer cells and the intersection of predicted transcription factor from the JASPAR database. (C) The sequences binding with EGR1. (D and E) RT-qPCR and western-blotting assay to measure EGR1 mRNA and protein level after nitroxoline treatment. (F and G) EGR1 mRNA and protein measured by RT-qPCR and western-blotting assay after knocking down EGR1 by siRNA. (H and I) RT-qPCR showed the down-regulation of EGR1 contributed to the reduce of circNDRG1 and mRNA NDRG1. (J and K) EGR1-OE increased the mRNA and protein of EGR1 by RT-qPCR and western-blotting. (L and M) RT-qPCR showed overexpression of EGR1 activated circNDRG1 and mRNA NDRG1 expression. (N) ChIP assay of the binding of EGR1 to the promoter of host gene NDRG1. (O and P) Transwell and wound-healing assay showed that EGR1 inhibited the capacity of migration in bladder cancer. *P<0.05, **P<0.01 and ***P<0.001.

Figure 5

CircNDRG1 acted as a sponge for miR-520h in bladder cancer. (A) Venn diagram of the intersection among predicted microRNA by Starbase, CircInteractome and CircBank. (B) RT-qPCR showed nitroxoline suppressed the expression of miR-520h. (C) RIP experiments were carried out using an AGO2 antibody. (D and E) Biotin-labelled circNDRG1 probe was used to bind circNDRG1 through streptavidin agarose and miR-520h were sponged by circNDRG1. (F) Dual-luciferase reporter assay validated the binding between circNDRG1 and miR-520h. (G and H) Transwell and wound-healing assay showed miR-520h minic promoted the migration of bladder cancer cells, while miR-520h inhibitor inhibited the migration. *P<0.05, **P<0.01 and ***P<0.001.

Figure 6

Smad7 was the target of miR-520h and regulated the EMT. (A) Venn diagram of the intersection among target genes by miRmap, Starbase, TargetScan and miRDB. (B) RT-qPCR assay demonstrated that nitroxoline led to increased smad7 expression. (C) Western-blotting assay demonstrated that nitroxoline up-regulated the expression of smad7, contributing to the repression of smad2/3 and EMT. (D) Dual luciferase assay showed the direct binding of miR-520h and 3'UTR of smad7 mRNA. (E) The exogenous dysregulation of miR-520h influence the expression of smad7, smad2/3 and EMT markers. (F and G) RT-qPCR and western blotting assay indicated the inhibition of smad7 after the transfection of smad7-si. (H and I) The capacity of migration was rescued by the decrease of smad7, which was inhibited by miR-520h-inhibitor. *P<0.05, **P<0.01 and ***P<0.001.

Figure 7

CircNDRG1 regulated smad7 expression and inhibited metastasis by targeting miR-520h. (A and B) Transwell and wound-healing assay demonstrated nitroxoline could inhibit the migration of bladder cancer cells, circNDRG1-si further promoted the migration, which was significantly rescued by preventing the expression of miR-520h. (C) Western-blotting assay showed that alteration of the expression levels of smad7, smad2/3 and the EMT-associated markers E-cadherin and N-cadherin, caused by nitroxoline plus circNDRG1-si treatment, were rescued by transfection with the miR-520h inhibitor. *P<0.05, **P<0.01 and ***P<0.001.

Figure 8

A schematic diagram depicting the mechanism of nitroxoline-mediated EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway in bladder cancer.