Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis

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 ofCDR1aswas 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 ofCDR1asand 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 whichCDR1asexpression was manipulated. AfterAGO2orDicerwas knocked-down to inhibit miRNA biogenesis, effects ofCDR1ason p53 expression, stability and activity were determined by immunoblot, RT-qPCR and luciferase reporter assays. Meanwhile, impacts ofCDR1ason DNA damage were evaluated by flow cytometric assays and immunohistochemistry. Tumorigenicity assays were performed to determine the effects ofCDR1ason 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). Results CDR1asis found to bind to p53 protein.CDR1asexpression decreases with increasing glioma grade and it is a reliable independent predictor of overall survival in glioma, particularly in GBM. Through a mechanism independent of acting as a miRNA sponge,CDR1asstabilizes p53 protein by preventing it from ubiquitination.CDR1asdirectly interacts with the p53 DBD domain that is essential for MDM2 binding, thus disrupting the p53/MDM2 complex formation. Induced upon DNA damage,CDR1asmay preserve p53 function and protect cells from DNA damage. Significantly,CDR1asinhibits tumor growth in vitro and in vivo, but has little impact in cells where p53 is absent or mutated. Conclusions Rather than acting as a miRNA sponge,CDR1asfunctions as a tumor suppressor through binding directly to p53 at its DBD region to restrict MDM2 interaction. Thus,CDR1asbinding disrupts the p53/MDM2 complex to prevent p53 from ubiquitination and degradation.CDR1asmay also sense DNA damage signals and form a protective complex with p53 to preserve p53 function. Therefore,CDR1asdepletion may play a potent role in promoting tumorigenesis through down-regulating p53 expression in glioma. Our results broaden further our understanding of the roles and mechanism of action of circular RNAs in general andCDR1asin particular, and can potentially open up novel therapeutic avenues for effective glioma treatment.