Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma
Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma
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DOI：
10.1073/pnas.1019055108
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发表时间：
2011-03-22
影响因子：
11.1
通讯作者：
Tewari, Muneesh
中科院分区：
文献类型：
--
作者：
Arroyo, Jason D.;Chevillet, John R.;Tewari, Muneesh
microRNA（miRNAs）以高度稳定的细胞外形式在血流中循环，并正在开发为癌症和其他疾病的血液生物标志物。然而，其在富含RNA酶的血液环境中显着稳定性的机制尚未得到很好的理解。文献中的当前模型假定循环的miRNA通过在膜结合囊泡（例如外来体）中的包封来保护，但这尚未被系统地研究。我们使用差速离心和尺寸排阻色谱作为正交方法来表征人血浆和血清中的循环miRNA复合物。令人惊讶的是，我们发现大多数循环中的miRNA与蛋白质复合物而不是囊泡共分馏。miRNA还对血浆的蛋白酶处理敏感，表明蛋白质复合物保护循环miRNA免受血浆RNA酶的影响。进一步的表征显示，Argonaute 2（Ago 2），miRNA介导的沉默的关键效应蛋白，存在于人血浆中，并在尺寸排阻色谱中用血浆miRNA洗脱。此外，从血浆中免疫沉淀Ago 2容易回收非囊泡相关的血浆miRNA。研究的大多数miRNA与Ago 2核糖核蛋白复合物共纯化，但少数特异性miRNA主要与囊泡相关。我们的研究结果揭示了两个群体的循环miRNA，并表明循环Ago 2复合物是负责血浆miRNA稳定性的机制。我们的研究对基于捕获和分析循环miRNA的生物标志物方法的发展具有重要意义。此外，血浆中胞外Ago 2-miRNA复合物的鉴定提高了细胞释放功能性miRNA诱导的沉默复合物进入循环的可能性。
MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.