Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis
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RESEARCH
Open Access
Circular RNA CDR1as disrupts the p53/ MDM2 complex to inhibit Gliomagenesis Jiacheng Lou1†, Yuchao Hao1†, Kefeng Lin1,2, Yizhu Lyu1,3, Meiwei Chen1,3, Han Wang1, Deyu Zou1, Xuewen Jiang1, Renchun Wang4, Di Jin1, Eric W.-F. Lam5, Shujuan Shao1,6, Quentin Liu1, Jinsong Yan3*, Xiang Wang1, Puxiang Chen2*, Bo Zhang1,7* and Bilian Jin1*
Abstract Background: Inactivation of the tumor suppressor p53 is critical for pathogenesis of glioma, in particular glioblastoma multiforme (GBM). MDM2, the main negative regulator of p53, binds to and forms a stable complex with p53 to regulate its activity. Hitherto, it is unclear whether the stability of the p53/MDM2 complex is affected by lncRNAs, in particular circular RNAs that are usually abundant and conserved, and frequently implicated in different oncogenic processes. Methods: RIP-seq and RIP-qPCR assays were performed to determine the most enriched lncRNAs (including circular RNAs) bound by p53, followed by bioinformatic assays to estimate the relevance of their expression with p53 signaling and gliomagenesis. Subsequently, the clinical significance of CDR1as was evaluated in the largest cohort of Chinese glioma patients from CGGA (n = 325), and its expression in human glioma tissues was further evaluated by RNA FISH and RT-qPCR, respectively. Assays combining RNA FISH with protein immunofluorescence were performed to determine co-localization of CDR1as and p53, followed by CHIRP assays to confirm RNA-protein interaction. Immunoblot assays were carried out to evaluate protein expression, p53/MDM2 interaction and p53 ubiquitination in cells in which CDR1as expression was manipulated. After AGO2 or Dicer was knocked-down to inhibit miRNA biogenesis, effects of CDR1as on p53 expression, stability and activity were determined by immunoblot, RT-qPCR and luciferase reporter assays. Meanwhile, impacts of CDR1as on DNA damage were evaluated by flow cytometric assays and immunohistochemistry. Tumorigenicity assays were performed to determine the effects of CDR1as on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on tumor volume/weight and survival of nude mice xenografted with GBM cells (in vivo). (Continued on next page)
* Correspondence: [email protected]; [email protected]; [email protected]; [email protected] † Jiacheng Lou and Yuchao Hao contributed equally to this work. 3 Department of Hematology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116044, Liaoning, People’s Republic of China 2 Department of Obstetrics and Gynecology, The Second Xiangya Hospital, Central South University, Changsha 410011, Hunan, People’s Republic of China 1 Department of Neurosurgery, The Second Affiliated Hospital; Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, Dalian 116044, Liaoning, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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