Detection of microRNA expression in human peripheral blood microvesicles

Abstract

Background: MicroRNAs (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 findings: Microvesicles 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.

Conclusions: This 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.

Figure 1. Analysis of the origin of peripheral blood microvesicles.

Peripheral blood microvesicles from healthy donors (n = 10) were analyzed by flow cytometry. (A) The size of the microvesicles was determined by FSC vs. SSC. The gate was set according to 2 micron beads (inset). (B) To determine cell origin, microvesicles were stained for CD3, CD202b (Tie-2), CD66b, CD79a, or CD41a to determine those that originated from T-cells, endothelial cells, neutrophils, B-cells, or platelets. Mononuclear phagocyte-derived microvesicles were positive for CD14, CD206, CCR3, CCR2, or CCR5. Shown is the average % maximum of total gated events±S.E.M.

Figure 2. Distribution of miRNA expression.

The box plots were generated to demonstrate the distribution of miRNA expression from each PBMC and plasma microvesicle sample. The bottom and top of the boxes are the 25^th and 75^th percentile (the lower and upper quartiles, respectively), and the band near the middle of the boxes are the 50^th percentile (the median). The ends of the whiskers represent the minimum and maximum of the data. (A) Raw C_T score of the data from individual donor were plotted. (B). 18s standardized data (ΔC_T ) were plotted: ΔC_T = raw C_T of PBMC or plasma microvesicles – C_T of 18S. (C). Median normalized data of each donor was used.

Figure 3. miRNA expression from peripheral blood microvesicles and PBMC.

Hierarchical cluster analysis for microvesicles and PBMC is shown based on filtering criteria. The (*) denotes the three PBMC samples which clustered with the plasma microvesicle samples.

Figure 4. Expression profile of common miRNAs.

Heat-maps demonstrating the expression profile for microvesicles (A) and PBMC (B) were generated for miRNAs that were commonly expressed. (C) The number of shared and specific miRNAs for each sample group is shown.

Figure 5. Venn diagram comparing miRNA expression in platelets and plasma microvesicles.

The number of shared and specific miRNAs for each sample group is shown.