Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2011;6(10):e24429. doi: 10.1371/journal.pone.0024429. Epub 2011 Oct 11.
Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In this study we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53(-/-) colon cancer cell line (HCT116) or ATM(-/-) human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53(+/+) cells but not HCT116 p53(-/-) cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.
遗传毒性应激源，如辐射，会诱导细胞损伤，激活预先设定的修复途径，其中一些涉及 microRNA（miRNA），可改变基因表达。let-7 家族 miRNA 调节多种细胞过程，包括细胞分裂和 DNA 修复途径。然而，let-7 基因在应激反应中的调控作用及其机制尚未阐明。在这项研究中，我们证明了在暴露于诱导应激的试剂（包括电离辐射）后，let-7a 和 let-7b 的表达显著降低。这种表达的降低在体外依赖于 p53 和 ATM，并且在 p53(-/-)结肠癌细胞系（HCT116）或 ATM(-/-)人成纤维细胞中观察不到。染色质免疫沉淀（ChIP）分析显示，p53 在暴露于辐射后与 let-7 基因的上游区域结合。荧光素酶瞬时转染表明，这种 p53 结合位点是辐射诱导 let-7 表达降低所必需的。体内辐射敏感组织也观察到 let-7a 和 let-7b 表达的辐射诱导下降，并且与 p53 调节的促凋亡信号通路中的蛋白质表达改变相关。相比之下，在 p53 敲除小鼠中未观察到这种表达降低，表明 p53 直接抑制 let-7 表达。外源性表达 let-7a 和 let-7b 增加了 HCT116 p53(+/+)细胞中辐射诱导的细胞毒性，但在 HCT116 p53(-/-)细胞中则不然。这些结果首次证明了辐射诱导的应激反应与 miRNA 调节以及辐射诱导的细胞毒性之间的机制联系，并表明该过程可能是抗癌药物的分子靶点。
