Division of Oncology, Department of Surgery and Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, London, UK.
Division of Oncology, Department of Surgery and Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, London, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S15. doi: 10.1016/S0140-6736(15)60330-0.
DNA damage transactivates tumour protein p53 (TP53)-regulated surveillance, crucial in suppressing tumorigenesis. TP53 mediates this process directly by transcriptionally modulating gene and microRNA (miRNA) expression and indirectly by regulating miRNA biogenesis. However, the role of TP53 in regulating miRNA-AGO2 loading and global changes in AGO2 binding to its gene targets in response to DNA damage are unknown. These processes might be novel mechanisms by which TP53 regulates miRNAs in response to DNA damage.
To show the network of miRNA-mRNA interactions that occur in response to DNA damage, we stimulated TP53 wild-type and null cell-lines with doxorubicin and performed RNA sequencing from total RNA (RNA-Seq) and AGO2-immunoprecipitated RNA (AGO2-RIP-Seq). We used a combined AGO2 RIP-seq and AGO2 PAR-CLIP-seq (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) approach to determine the exact sites of interaction between the AGO2-bound miRNAs and their mRNA targets.
TP53 directly associated with AGO2, and induced and reduced loading of a subset of miRNAs, including the lethal 7 (let-7) miRNA family members, onto AGO2 in response to DNA damage. Although mutated TP53 maintained its capacity to interact with AGO2, it mediated unloading instead of loading of let-7 family miRNAs, thereby reducing their activity. We determined the miRNA-mRNA interaction networks involved in the response to DNA damage both in the presence and absence of TP53. Furthermore, we showed that miRNAs whose cellular abundance or differential loading onto AGO2 was regulated by TP53 were involved in an intricate network of regulatory feedback and feedforward circuits that fine-tuned gene expression levels in response to DNA damage to permit the repair of DNA damage or initiation of programmed cell death.
Control of AGO2 loading by TP53 is a new mechanism of miRNA regulation in carcinogenesis.
UK Medical Research Council, Action Against Cancer.
DNA 损伤可激活肿瘤蛋白 p53（TP53）调节的监测，这对于抑制肿瘤发生至关重要。TP53 通过转录调节基因和 microRNA（miRNA）表达直接介导这一过程，通过调节 miRNA 生物发生间接介导。然而，TP53 在调节 DNA 损伤后 miRNA-AGO2 加载和 AGO2 与基因靶标结合的全局变化中的作用尚不清楚。这些过程可能是 TP53 调节 DNA 损伤反应中 miRNA 的新机制。
为了展示 DNA 损伤后发生的 miRNA-mRNA 相互作用网络，我们用阿霉素刺激 TP53 野生型和缺失细胞系，并从总 RNA（RNA-Seq）和 AGO2 免疫沉淀 RNA（AGO2-RIP-Seq）中进行 RNA 测序。我们使用 AGO2 RIP-seq 和 AGO2 PAR-CLIP-seq（光激活核苷酸增强交联和免疫沉淀）相结合的方法来确定 AGO2 结合的 miRNA 与其 mRNA 靶标之间的确切相互作用位点。
TP53 直接与 AGO2 结合，并在 DNA 损伤后诱导和减少包括致死 7（let-7）miRNA 家族成员在内的一组 miRNA 加载到 AGO2 上。尽管突变型 TP53 保持与 AGO2 相互作用的能力，但它介导了 let-7 家族 miRNA 的卸载而不是加载，从而降低了它们的活性。我们确定了在存在和不存在 TP53 的情况下参与 DNA 损伤反应的 miRNA-mRNA 相互作用网络。此外，我们表明，细胞丰度或 AGO2 差异加载受 TP53 调节的 miRNA 参与了精细的调节反馈和前馈回路网络，以微调基因表达水平，以允许 DNA 损伤的修复或程序性细胞死亡的启动。
TP53 对 AGO2 加载的控制是致癌作用中 miRNA 调节的新机制。
英国医学研究理事会，抗癌行动。
