Patterns of microRNA expression characterize stages of human B-cell differentiation - PubMed (original) (raw)

. 2009 May 7;113(19):4586-94.

doi: 10.1182/blood-2008-09-178186. Epub 2009 Feb 6.

Dereje D Jima, Cassandra Jacobs, Randy Fischer, Eva Gottwein, Grace Huang, Patricia L Lugar, Anand S Lagoo, David A Rizzieri, Daphne R Friedman, J Brice Weinberg, Peter E Lipsky, Sandeep S Dave

Affiliations

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

Jenny Zhang et al. Blood. 2009.

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.

PubMed Disclaimer

Figures

Figure 1

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

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

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

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.

References

    1. Nutt SL, Heavey B, Rolink AG, Busslinger M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature. 1999;401:556–562. - PubMed
    1. Chang CC, Ye BH, Chaganti RS, Dalla-Favera R. BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor. Proc Natl Acad Sci U S A. 1996;93:6947–6952. - PMC - PubMed
    1. Turner CA, Jr, Mack DH, Davis MM. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell. 1994;77:297–306. - PubMed
    1. Shaffer AL, Shapiro-Shelef M, Iwakoshi NN, et al. XBP1, Downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity. 2004;21:81–93. - PubMed
    1. Schebesta M, Heavey B, Busslinger M. Transcriptional control of B-cell development. Curr Opin Immunol. 2002;14:216–223. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources