Identification of miR-28 targets in lymphoma B cells. (A) miR-28 microarray expression data (GSE29493) in cohorts of patients with CLL (18 samples), DLBCL (29 samples), BL (12 samples), and follicular lymphoma (FL, 23 samples). Controls were human GC B cells (CD10+ CD19+) extracted from tonsils of healthy donors. Adjusted P values were calculated with the Benjamini and Hochberg method (****FDR <10−4). (B) Ramos BL cells were transduced with scramble or miR-28 vectors, induced with Dox, and flow cytometry isolated RFP+ cells were analyzed by RNA-Seq and iTRAQ for differential transcriptome and proteome characterization, respectively. Plots are volcano representations of transcriptomic and proteomic changes in Ramos cells upon miR-28 reexpression. Dots represent mean fold change (miR-28 Ramos cells/control Ramos cells) at the level of transcripts (RNA-Seq; 6 replicates) or proteins (iTRAQ; 4 replicates) (x-axes) against the statistical significance of the change (y-axes), both in 2-base logarithmic scale. Changes were considered significant at cutoffs of P < .05 (RNA-Seq) or FDR <10% (P < .0038) (iTRAQ) (dots below red lines). (C) Gene set enrichment analysis of predicted miR-28 targets vs transcriptome analysis of miR-28–expressing BL cells (NES = −1.42; P = .004; q = 0.001). (D) qRT-PCR validation of miR-28–mediated transcript downregulation of 6 representative genes found to be significantly downregulated in the RNA-Seq analysis (*P < .05, unpaired Student t test). (E) miR-28–altered transcripts (top) and proteins (bottom) were annotated with functional gene ontology (GO) categories. The x-axes plot the number of miR-28–altered transcripts or proteins within each GO category; y-axes plot the proportion of miR-28–altered transcripts or proteins within each GO category. Circled area represents the relative contribution of each GO category to the total number of miR-28–altered transcripts or proteins. (F) The graph shows the cumulative distributions of the standardized variable at the protein level (Zqa) plotted separately for each miR-28–altered category in the iTRAQ proteomic analysis. (G) Ingenuity pathway analysis of the proteins differentially expressed upon miR-28 expression in BL cells (BCR signaling pathway enrichment, P = 10−46). Upregulated proteins are depicted in green, downregulated in red, and nodes in blue.
