Patterns of microRNA expression characterize stages of human B-cell differentiation

Abstract

Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

Figure 1

Mature B-cell subsets demonstrate distinct miRNA profiles. (A) Overall schema of mature B-cell differentiation. (B) Selection of the B-cell subsets with the use of flow cytometry. Cells were previously gated on CD19⁺ cells. Naive and memory B cells were distinguished from GC and plasma cells based on surface CD38 and IgD expression. (C) Distinction of naive and memory B cells based on IgD and CD27 expression with the use of flow cytometry. (D) Relative expression of miRNA in the naive to GC B-cell transition. miRNAs that were, on average, at least 2-fold differentially expressed at a false discovery rate of less than 5% are shown according to the color scale. (E) Relative expression of mRNA in the naive to GC B-cell transition. mRNAs that were, on average, at least 2-fold differentially expressed at a false discovery rate of less than 1% are shown according to the color scale. (F) Relative expression of miRNA in the GC B-cell to plasma-cell transition. miRNAs that were, on average, at least 2-fold differentially expressed at a false discovery rate of less than 5% are shown according to the color scale. (G) Relative expression of mRNA in the GC B-cell to plasma-cell transition. mRNAs that were, on average, at least 2-fold differentially expressed at a false discovery rate of less than 1% are shown according to the color scale. (H) Relative expression of miRNA in the GC B cell to memory B–cell transition. miRNAs that were, on average, at least 2-fold differentially expressed at a false discovery rate of less than 5% are shown according to the color scale. (I) Relative expression of mRNA in the GC B-cell to memory B–cell transition. mRNAs that were, on average, at least 2-fold differentially expressed at a false discovery rate of less than 1% are shown according to the color scale. (J) Expression of key miRNA processing genes DGCR8, DICER1, EIF2C2, DROSHA, and XPO5 is unchanged among the B-cell subsets (P > .1 in all cases).

Figure 2

Experimental validation of the interaction of miR-223, which is expressed highly in naive and memory B cells compared with GC B cells, and targets the transcription factor LMO2. (A) Base-pairing of the 3′UTR LMO2 gene with nucleotides 1-8 of miR-223. This 8-mer is highly conserved across several species and serves as a potential binding site for miR-223. (B) Effects of overexpression of miR-223 in GC lymphoma-derived BJAB cells in 3 separate experiments. The dark gray bars depict expression of LMO2 24 hours after transfection with a scrambled control that does not possess complementarity to the human genome. The light gray bars depict the expression of LMO2 24 hours after transfection with a precursor for miR-223. The expression of LMO2 was consistently lower in the cells treated with the miR-223 precursor (P < .05 in all cases). (C) Relative LMO2 protein expression from a representative experiment (from 3 replicates) transfecting a scrambled control versus a precursor for miR-223 in BJAB cells. (D) Average expression of LMO2 relative to actin over 3 Western blots of BJAB transfected with a scrambled control versus a precursor for miR-223. LMO2 expression is lower in cells treated with miR-223 (P < .05). (E) Luciferase-expressing vectors were coupled to the 3′UTR of the LMO2 gene. The seed sequence mutant construct had consistently diminished miR-223 repression compared with the wild-type construct in 5 separate experiments (P < .05).

Figure 3

Experimental validation of the interaction of miR-9 and miR-30, which are expressed highly in GC B cells compared with plasma cells and target the transcription factor PRDM1. (A) Base-pairing of the 3′UTR of PRDM1 gene with the 5′ seed region of miR-9 and the miR-30 family. The miR-30 regions include 3 sites complementary to nucleotides 2-8 (UTR position 408), nucleotides 1-8 (UTR position 2370), and nucleotides 2-8 (UTR position 2383) on the miRNA, respectively. The miR-9 regions include 3 sites complementary to nucleotides 1-7 (UTR position 1459), nucleotides 2-8 (UTR position 2108), and nucleotides 2-8 (UTR position 2323) on the miRNA, respectively. These sites are highly conserved across several species, with the exception of one miR-9 site (UTR position 1459) that is present only in humans. (B) Effects of overexpression of miR-9 and 2 members of the miR-30 family, miR-30b and miR-30d, in plasma cell myeloma-derived U266 cells in 3 separate experiments. Expression of PRDM1 was measured 24 hours after transfection with a scrambled control with no complementarity to the human genome, a hairpin precursor for miR-30b, a hairpin precursor for miR-30d, or a hairpin precursor for miR-9. (C) Relative PRDM1 protein expression from a representative experiment (from 3 replicates) transfecting a scrambled control versus a precursor for miR-9, miR-30b, and miR-30d in U266 cells. (D) Average expression of PRDM1 relative to actin over 3 Western blots of U266 cells transfected with a scrambled control versus a precursor for miR-9, miR-30b, and miR-30d. P < .05 for miR-30b and miR-30d; P = .08 for miR-9. (E) A luciferase-expressing vector was coupled to the 3′UTR of PRDM1. Repression of luciferase activity was observed upon overexpression of miR-30b, miR-30d, and miR-9 (P < .05 in all 3 cases) but rescued to the activity level of the empty vector control (P > .5 in all 3 cases) when the seed sequence of the miRNAs was mutated. Displayed is the average of 3 separate experiments.

Figure 4

Expression of miRNAs expressed in normal B cells is conserved in B-cell malignancies. (A) A predictor constructed of miRNAs differentially expressed in the normal naive B cells and GC B cells (miRNAs depicted in Figure 1D) was used to predict the normal counterpart B cell of both IgV mutated and unmutated chronic lymphocytic leukemia, GC B cell–derived DLBCL, and Burkitt lymphoma. The accuracy was greater than 95% in all cases. (B) Expression of miRNAs expressed in B cells that also were present and detectably measured on the microarrays (103/113) was examined in the B-cell malignancies (n = 70) and normal lymph nodes (n = 5) with use of the Student t test. miRNAs that were differentially expressed (P < .05) at greater levels in malignant cells, normal cells, as well as the miRNAs that were not differentially expressed are shown. (C) Cloning frequency of miRNAs was compared between unselected mature B cells (n = 3) compared with several B-cell malignancies (n = 42) from a previously published study (“sequencing data”) with use of the Student t test. miRNAs that were differentially expressed (P < .05) at greater levels in malignant and normal cells, as well as the miRNAs that were not differentially expressed, are shown. (D) Differentially expressed miRNAs that distinguish Burkitt lymphoma, activated B cell–like (ABC) diffuse large B-cell lymphoma (DLBCL), GC-like DLBCL (GCB DLBCL), and chronic lymphocytic leukemia. Predictor miRNAs from each pairwise comparison that distinguish each entity are shown in the boxes.