Detection of microRNA Expression in Human Peripheral Blood Microvesicles

BackgroundMicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.Methodology/Principal FindingsMicrovesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function.ConclusionsThis study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA.

背景：微小核糖核酸（microRNAs, miRNA）是一类小型非编码RNA，可调控信使核糖核酸（mRNA）及蛋白质的翻译过程。miRNA的表达缺失或上调与包括癌症在内的多种疾病密切相关。然而，目前对于健康个体循环miRNA的鉴定特征尚未得到充分阐明。微囊泡（microvesicles，亦称外泌体或微粒）可在外周血中循环，并能激活细胞信号通路。本研究假设，在正常健康状态下，微囊泡携带有miRNA，这一过程有助于维持生物体内稳态。
方法/主要结果：研究人员从健康个体的血浆中分离得到微囊泡。分别从微囊泡及配对的单核细胞中提取RNA，并通过实时荧光定量PCR（real-time PCR）对420种已知成熟miRNA开展表达谱分析。对数据集进行层级聚类（hierarchical clustering）分析后发现，外周血单核细胞（peripheral blood mononuclear cells, PBMC）与血浆微囊泡的miRNA表达水平存在显著差异。本研究发现，微囊泡与PBMC间共有71种miRNA呈共表达状态。值得注意的是，我们分别在血浆微囊泡与单核细胞中发现了33种和4种存在显著差异表达的miRNA。研究人员对本次检测到的miRNA所调控的基因靶点及相关生物学通路开展了预测分析。预测结果显示，血液微囊泡中表达的绝大多数miRNA可调控血细胞的细胞分化及代谢通路。有趣的是，另有少量miRNA被预测可作为免疫功能的重要调节因子。
结论：本研究首次在正常受试者的循环血浆微囊泡中鉴定并明确了miRNA的表达谱。本研究产生的数据可为后续探索外周血miRNA特征在人类疾病中的预测作用提供研究基础，并有助于阐明这些miRNA所调控的生物学过程。