miRNA list let-7g miR-23a miR-23b miR-24 miR-26a miR-27b miR-29a miR-29b miR-29c miR-101 miR-150 miR-155 miR-181a miR-181b miR-223 Activation IgM/CD40L TLRs CLL -/ Table 1. Signature miRNA expression comparison between activated B cells and CLL cells relative to control B cells. Up and down arrows indicate upregulation or downregulation of specific miRNAs, respectively, when compared to untransformed, control B cells. A parallel line indicates no change when compared to untransformed, control B cells. miR-223, miR-29a, miR-29b and miR-29c are upregulated in response to certain B cell activators and downregulated in response to other B cell activators. miR-26a is unaffected by IgM stimulation, but upregulated in response to other B cell activators. doi:10.1371/journal.pone.0016956.t001 observations. We found that miRNAs associated with poor clinical outcomes are a part of a larger pattern of miRNA changes consistent with B cell activation. In B cells, miR-29c and miR-223 are both reduced after BCR or co-stimulatory activation. In CLL cells, decreased miR-29c and miR-223 are associated with ZAP70+ and IgVH unmutated status (Tables S4A and S4B) and shorter times to first therapy (Figure 4). We do not have access to sufficient number of fresh samples from additional patients with full clinical annotation to serve as a MicroRNA in Chronic Lymphocytic Leukemia validation set for these observations. However, these observations are consistent with previous observations that have associated reduced levels of these two miRNAs with ZAP70+ and IgVH unmutated status [11,12,32,37]. Moreover, previous studies have associated reduced miR-29c with p53 status and 17p deletion, the worst prognostic group in CLL [30,31]. We did not identify an association between reduced miR-29c and any cytogenetic abnormalities (data not shown). miR-29c and miR-223 co-vary with other clinical parameters and thus may not have independent prognostic value, but these miRNA changes may have biological function in CLL. For example, reduced levels of miR-29c have been shown to de-repress expression of MCL-1 and TCL-1, known oncogenes implicated in the CLL anti-apoptotic defect [30,31,33]. BCR activation led to downregulation of miR-29c and miR-223 (Figure S7A) and ZAP70 is associated with increased BCR signaling in CLL [38]. It is likely that miR-29c and miR-223 are negatively affected by enhanced BCR signaling mediated by ZAP70 [38]. Although the kinase domain of ZAP70 is not required for BCR pathway stimulation in CLL, the scaffolding function of ZAP70 acts as an adaptor that clusters and thereby increases signaling through BCRs in CLL [38,39]. Thus, downregulation of miR-29c and miR-223 may potentiate BCR signaling and accelerate CLL oncogenesis. Other miRNA changes consistent with B cell activation may also have an important role in CLL oncogenesis. miR-155 demonstrates an average of 50-fold increase in expression in CLL cells tested compared with control B cells (Figure 2D), is increased in multiple cancers [27,28,40], and promotes B cell lymphomagenesis [41]. Conversely, miR-181a and miR-181b demonstrate approximately a 100-fold decrease in expression in virtually all CLL cells tested as compared with control B cells (Figure 2E). PI-3K is a predicted target of miR-181a and miR- 181b [42] and the PI3K pathway has been implicated in the apoptotic defect in CLL cells [43]. These data suggest that miR- 155 has an oncogenic function whereas miR-181a and miR-181b have a tumor suppressive function in CLL. Analysis of changes in miRNA expression and their target genes during B cell activation and CLL oncogenesis will provide insights into the physiological roles of these miRNAs. Understanding the roles of miRNAs in the dysregulated gene networks that underlie the pathology of CLL could enable the application of miRNA-based therapeutics for this common leukemia. Figure 4. The expression of miR-29c and miR-233 is associated with time to first therapy in CLL. A shorter time from diagnosis to initiation of first therapy is significantly associated with reduced expression of miR-29c and miR-223. A recursive partitioning approach identifies the optimal split for each miRNA: miR-29c expression at 11.82 or lower is associated with shorter time to first therapy, q = 0.07. miR-223 expression at a level of 11.14 or lower is associated with shorter time to first therapy, q = 0.07. doi:10.1371/journal.pone.0016956.g004 PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e16956