Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis
Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis
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环状RNA CDR1as破坏p53/MDM2复合物以抑制胶质瘤发生
DOI：
10.1186/s12943-020-01253-y
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发表时间：
2020-09-07
期刊：
影响因子：
37.3
通讯作者：
Jin, Bilian
中科院分区：
文献类型：
--
作者：
Lou, Jiacheng;Hao, Yuchao;Jin, Bilian
背景肿瘤抑制基因p53的失活在胶质瘤，特别是多形性胶质母细胞瘤（GBM）的发病机制中至关重要。MDM 2是p53的主要负调节因子，与p53结合并形成稳定的复合物以调节其活性。然而，目前还不清楚p53/MDM 2复合物的稳定性是否受到lncRNA的影响，特别是通常丰富和保守的环状RNA，并且经常涉及不同的致癌过程。方法采用RIP-seq和RIP-qPCR方法检测p53结合的lncRNA（包括环状RNA），并采用生物信息学方法检测其表达与p53信号转导和胶质瘤发生的相关性。随后，在CGGA最大的中国胶质瘤患者队列（n = 325）中评估CDR 1as的临床意义，并分别通过RNA FISH和RT-qPCR进一步评估其在人脑胶质瘤组织中的表达。进行RNA FISH与蛋白质免疫荧光相结合的测定以确定CDR 1as和p53的共定位，然后进行CHIRP测定以确认RNA-蛋白质相互作用。用免疫印迹法检测CDR 1as表达调控后的细胞中蛋白表达、p53/MDM 2相互作用和p53泛素化。通过免疫印迹、RT-qPCR和荧光素酶报告基因分析，检测AGO 2或Dicer对p53表达、稳定性和活性的影响。同时采用流式细胞术和免疫组化方法检测CDR 1对DNA损伤的影响。进行致瘤性测定以确定CDR 1对集落形成、细胞增殖、细胞周期和凋亡（体外）以及对移植有GBM细胞的裸鼠的肿瘤体积/重量和存活（体内）的影响。结果CDR 1asis与p53蛋白结合，随着胶质瘤分级的升高，CDR 1asis表达逐渐降低，CDR 1asis是预测胶质瘤尤其是GBM患者总生存率的独立指标。CDR 1as通过一种不依赖于miRNA海绵的机制，通过阻止p53蛋白的泛素化来稳定p53蛋白，CDR 1as直接与MDM 2结合所必需的p53 DBD结构域相互作用，从而破坏p53/MDM 2复合物的形成。在DNA损伤后，CDR 1as可以保护p53的功能并保护细胞免受DNA损伤。值得注意的是，CDR 1as在体外和体内抑制肿瘤生长，但在p53缺失或突变的细胞中几乎没有影响。结论CDR 1as不是miRNA海绵，而是通过直接与p53的DBD区结合来限制MDM 2的相互作用，从而发挥肿瘤抑制作用。因此，CDR 1as结合破坏了p53/MDM 2复合物，阻止了p53的泛素化和降解，CDR 1as还可以感知DNA损伤信号，并与p53形成保护性复合物，保护p53的功能。因此，CDR 1as缺失可能通过下调p53表达而促进胶质瘤的发生。我们的研究结果进一步拓宽了我们对环状RNA和CDR 1as的作用和作用机制的理解，并可能为有效治疗胶质瘤开辟新的治疗途径。
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.