p-Value 4.31E-20 1.64E-21 7.17E-08 8.52E-09 3.05E-06 1.20E-05 1.18E-21 5.34E-18 2.24E-21 3.21E-09 4.05E-06 1.63E-16 3.86E-4 1.99E-08 1.26E-07 miR-150 miR-029a miR-142-3p miR-146b miR-155 miR-532 miR-019a miR-140 miR-021 miR-374 miR-181d miR-345 let-7g miR-015a miR-019b miR-142-5p miR-106b miR-016 miR-026b miR-195 Table 3. Comparison of miRNA expression between plasma microvesicles and PBMC. Expression in Plasma Microvesicles Fold Change Expression in PBMC Fold Change miR-486 miR-328 miR-183 miR-032 miR-574 miR-027b miR-222 miR-197 miR-151 miR-199a* miR-133b miR-320 miR-096 miR-103 miR-017-5p 6.742 6.602 5.979 4.530 4.390 4.208 3.995 3.783 3.482 3.454 3.348 2.637 2.270 2.190 2.037 59.93 40.31 23.49 22.85 9.252 4.730 4.682 4.219 4.211 3.769 3.713 3.670 3.247 3.226 3.213 2.671 2.491 2.392 2.307 2.243 miRNAs in Blood Microvesicles p-Value 7.00E-21 9.69E-28 1.90E-22 1.81E-22 6.61E-23 6.14E-10 2.85E-12 2.87E-17 4.23E-08 8.00E-15 5.30E-07 1.25E-08 4.99E-09 3.24E-07 7.11E-12 9.83E-08 1.28E-07 1.35E-10 5.44E-06 5.50E-5 In order to test the differences of miRNA expression between plasma microvesicles and PBMC, data were analyzed using linear mixed models. The p-values were generated from the model based on the estimated difference and sample variation. A bonferroni adjustment for multiple comparisons (72 comparisons) to control type I error to reduce the number of false positives was performed, therefore ‘‘p-value’’ was considered as significant if p-value,0.05/72 = 0.0006. Fold-change was calculated based on the estimated mean difference (2^(2DCT)). Shown are miRNAs that were significantly expressed at greater than 2-fold. doi:10.1371/journal.pone.0003694.t003 Many miRNAs expressed in the plasma microvesicles also regulate the progression of the cell cycle proteins [55,56]. miR-222 targets p27Kip1 [55] while miR-24 suppresses p16 (INK4a) [56]. Increased expression of miR-16 results in the accumulation of cells in G0/G1 phase of the cell cycle [47]. To date, there are no known functions for miR-484 which is the second highest expressed miRNA in the plasma microvesicles fraction based on normalized expression (Table 1). Using IPA analysis, miR-484 appears to regulate hematopoiesis, as well as cellular differentiation, proliferation and growth similar to many of 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. doi:10.1371/journal.pone.0003694.g005 the highly expressed miRNAs in the plasma microvesicles. Another of the highly expressed miRNAs, miR-146a is predicted to modulate NK cell signaling and IL-4 signaling pathways. Similarly, miR-146a is also predicted to regulate immune functions [57,58]. Based on IPA analysis examining gene ontology of targets, the top associated networks predicted to be influenced by miR-146a expression are cellular proliferation, immune and lymphatic system development and function. In addition, this miRNA is predicted to regulate innate immune responses. From the analysis, we found that LPS/IL-1 and toll-like receptor signaling are among the top five canonical pathways predicted to be regulated by miR-146a. Comparison of differentially expressed miRNAs between the plasma microvesicles and the PBMC indicated that miR-486 was the highest differentially expressed in the plasma microvesicles (Table 3). Little is known about this miRNA, except that it is expressed in glioblastoma cells and may function in brain cell differentiation [59]. Analyzing the predicted targets from Sanger miRBase with the IPA software, the top five canonical pathways predicted to be regulated by miR-486 are those in phenylalanine and cyanoamino acid metabolism, insulin receptor signaling, antigen presentation, and pentose phosphate pathways. Another miRNA differentially expressed in the plasma microvesicles is miR-328. Recently, Wang and colleagues reported that miR-328 is important in regulating tumor development and invasion by targeting CD44 expression in epithelial carcinoma cell lines [60]. Overexpression of miR-328 inhibits cell migration and adhesion and preventing them to expand into the tumor. The majority of the plasma microvesicles from normal individuals are derived from blood cells. We did detect a small PLoS ONE | www.plosone.org 8 November 2008 | Volume 3 | Issue 11 | e3694