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• Research Paper Volume 12, Issue 8 pp 6865-6879

  Circular RNA circRGNEF promotes bladder cancer progression via miR-548/KIF2C axis regulation

  Relevance score: 9.97841

  Chen Yang, Qiong Li, Xinan Chen, Zheyu Zhang, Zezhong Mou, Fangdie Ye, Shengming Jin, Xiang Jun, Feng Tang, Haowen Jiang

  Keywords: circular RNA, circRGNEF, miR-548, bladder cancer, KIF2C

  Published in Aging on April 19, 2020

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  Circular RNAs (circRNAs) play an important role in bladder cancer (BC). Though circRNA involvement in BC has been reported, the underlying regulatory mechanisms are unknown. In this study, we performed EdU, CCK8, colony formation and Transwell assays to establish the role of circRGNEF in BC cell migration, proliferation, and invasion. We used bioinformatics and luciferase reporter experiments to investigate the regulatory mechanism. Nude mice xenografts and live imaging were used to explore the role of circRGNEF in tumor metastasis and growth. Expression profile analysis of human circRNAs in BC revealed that circRGNEF was upregulated significantly. High circRGNEF expression was correlated with aggressive BC phenotypes. The downregulation of circRGNEF suppressed BC cell metastasis and proliferation by targeting the miR-548/KIF2C axis in vitro and in vivo; these results were verified with luciferase reporter assays. Our results show that miR-548 downregulation or KIF2C overexpression restored BC cell proliferation, migration, and invasion following silencing of circRGNEF. KIF2C overexpression reversed miR-548-induced cell invasion and migration as well as growth inhibition in vitro. In summary, the data illustrate that circRGNEF suppresses BC progression by functioning as a miR-548 sponge to enhance KIF2C expression. Therefore, circRGNEF might be a candidate BC treatment target.

  

  High expression of hsa_circ_0072995 (circRGNEF) predicted an unfavorable prognosis of bladder cancer (BC). (A) RT-qPCR assay of circRGNEF in 90 paired BC tumor and adjacent non-tumor tissues. Data are means ± standard deviation (SD). ***P < 0.001 vs. normal controls. (B) Fluorescent in situ hybridization (FISH) indicates the subcellular localization of circRGNEF. (C) The prognostic significance of circRGNEF expression for BC patients was performed with FISH values using the median value as the cut-off. The observation time was 60 months. (D) Genomic loci of RGNEF and circRGNEF. The red signal indicates back splicing.

  
  
  

  

  circRGNEF expression was also increased in BC cell lines and knockdown of circRGNEF suppressed cell proliferation. (A) RT-qPCR detection of circRGNEF expression in BC cell lines EJ, T24, J82, UM-UC-3, TCC, and RT-4, and the normal cell line SV-HUC. Data are presented as the mean ± SD. ^***P < 0.001 vs. SV-HUC. (B) RT-qPCR detection shows the expression of circRGNEF in both T24 and UM-UC-3 cells following transfection with small interfering RNA targeting circRGNEF (si-circRGNEF) or negative control (NC). Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. (C) Cell cycle distribution by flow cytometry after propidium iodide staining. (D, E) CCK8 assay shows the proliferation of T24 and UM-UC-3 cells with or without circRGNEF silencing. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. (F) Colony formation assay was performed to determine the colony-forming ability of T24 and UM-UC-3 cells. (G, H) circNRIP1 silencing significantly inhibited DNA synthesis, as determined by the EdU assay. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC.

  
  
  

  

  circRGNEF silencing suppressed tumor growth of xenografts in nude mice. (A, B) Photographs of tumors and curve of T24 tumor volume growth (B) of the nude mice. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. (C) Tumor weights. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. (D) Ki67 staining of tumor tissues.

  
  
  

  

  Knockdown of circRGNEF decreased the invasion ability of BC in vitro and in vivo. (A–C) Cell migration and invasion were assessed in T24 and UM-UC-3 cells using Transwell assays. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. (D) Live imaging shows the effects of circRGNEF on metastasis of T24 cells 30 days after intravenous tail injection.

  
  
  

  

  miR-548 and KIF2C are downstream targets of circRGNEF. (A) Bioinformatics analysis (https://circinteractome.nia.nih.gov/bin/mirnasearch) and high-throughput sequencing indicated miR-548 is a target of circRGNEF. (B) RT-qPCR assay of miR-548 in 90 paired BC tumor and adjacent non-tumor tissues. Data are means ± SD. ***P < 0.001 vs. normal controls. (C) The mutated (Mut) version of circRGNEF is shown. (D) The relative luciferase activity was determined 48 h after transfection of HEK293T cells with miR-548 mimic/normal control (NC) or circRGNEF wild-type/Mut. Data are presented as the mean ± SD. ^***P < 0.001. (E) Bioinformatics analysis (http://circnet.mbc.nctu.edu.tw/ and http://www.targetscan.org/vert_71/) indicated KIF2C is a direct target of miR-548. (F) The mutated (Mut) version of the 3’UTR-KIF2C is shown. (G) The relative luciferase activity was determined 48 h after transfection of HEK293T cells with miR-548 mimic/normal control (NC) or 3’UTR-KIF2C wild-type/Mut. Data are presented as the mean ± SD. ^***P < 0.001.

  
  
  

  

  Downregulation of miR-548 or overexpression of KIF2C restored proliferation, migration, and invasion after circRGNEF silencing. (A–F) RT-qPCR shows the expression of circRGNEF (A, B), miR-548 (C, D), and KIF2C (E, F) in T24 and UM-UC-3 cells following transfection or treatment with NC, si-circRGNEF, miR-548 inhibitor, KIF2C overexpression vector (KIF2C) single or combined. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRGNEF. (G–I) Cell proliferation was analyzed by EdU assays. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRGNEF. (J–N) Cell migration and invasion were assessed in T24 and UM-UC-3 cells using Transwell assays. Data are presented as the mean ± SD. ^***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRGNEF.

  
  
  

  

  KIF2C overexpression reversed miR-548-induced cell migration, invasion, and growth inhibition in vitro. (A–D) T24 and UM-UC-3 cells were transfected with miR-548 mimics with or without the KIF2C overexpression vector. RT-qPCR shows the expression of miR-548 (A, B) and KIF2C (C, D) in T24 and UM-UC-3 cells. Data are denoted by the mean ± SD. ^***P < 0.001 vs. NC. ^###P < 0.001 vs. mimic. (E–H) EdU assay showing the proliferation of T24 (E, F) and UM-UC-3 (G, H) cells. Data are denoted by the mean ± SD. ^*P < 0.05, ^***P < 0.001. ^###P < 0.001 vs. mimic. (I–M) Cell migration and invasion were determined in T24 and UM-UC-3 cells by Transwell assays. Data are denoted by the mean ± SD. ^***P < 0.001 vs. NC. ^###P < 0.001 vs. mimic.