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, 8 (7), 11809-11826

Identification of microRNAs Implicated in the Late Differentiation Stages of Normal B Cells Suggests a Central Role for miRNA Targets ZEB1 and TP53

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Identification of microRNAs Implicated in the Late Differentiation Stages of Normal B Cells Suggests a Central Role for miRNA Targets ZEB1 and TP53

Giorgio Malpeli et al. Oncotarget.

Abstract

In the late B cell differentiation stages, miRNAs expression modifications promoting or inhibiting key pathways are only partially defined. We isolated 29 CD19+ human B cell samples at different stages of differentiation: B cells from peripheral blood; naïve, germinal center (GC) and subepithelial (SE) B cells from tonsils. SE cells were further split in activated and resting B cell. The miRNA expression profile of these B cells was assessed by microarray analysis and selected miRNAs were validated by quantitative RT-PCR and in situ hybridization on normal tonsils. The comparison of all samples showed changes in 107 miRNAs in total. Among 48 miRNAs differentially expressed in naïve, GC and SE cells, we identified 8 miRNAs: mir-323, mir-138, mir-9*, mir-211, mir-149, mir-373, mir-135a and mir-184, strictly specific to follicular cells that had never been implicated before in late stages of B cell development. Moreover, we unveiled 34 miRNAs able to discriminate between CD5- activated B cells and resting B cells. The miRNAs profile of CD5- resting B cells showed a higher similarity to naïve CD5+ than CD5- activated B cells. Finally, network analysis on shortest paths connecting gene targets suggested ZEB1 and TP53 as key miRNA targets during the follicular differentiation pathway. These data confirm and extend our knowledge on the miRNAs-related regulatory pathways involved in the late B cell maturation.

Keywords: B cell development; follicle; germinal centre; microRNAs; network analysis.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Expression profile of miRNAs in cell subsets representing different stages of B cell maturation
CD5+: naïve B cells from tonsils; CD23CD39: germinal centre (GC) B cells from tonsils; CD5−: subepithelial (SE) mature B cells from tonsils, subdivided in CD5 resting and CD5− activated B cells. (A) Array tree of 25 samples representing different stages of maturation of the B cells based on the expression levels of miRNAs. (B) The heat map describes the expression levels of 48 differentially expressed miRNAs in 25 samples owning to four B cell subsets isolated from tonsils (FDR 1%). Clusters 1, 2 and 3 are three miRNAs groups isolated by clustering procedures. Red, higher expression (log2, +4); green, lower expression (log2, −4).
Figure 2
Figure 2. Expression levels of top 30 differentially expressed miRNAs in cell subsets representing different stages of late B cell differentiation
Graphs report expression levels of 30 miRNAs with lower Q value among the 48 differentially expressed miRNAs in 29 samples divided in four B cell subsets. Color graphs indicate the miRNAs included in Cluster 1 (yellow), Cluster 2 (orange) and Cluster 3 (green). Data were obtained from microrray analysis after normalization and log2 transformation of the probe signal. 0: naïve B cells; 1: germinal centre B cells; 2: subepithelial activated B cells; 3: subepithelial resting B cells.
Figure 3
Figure 3. Differential expression of miRNAs in subepithelial CD5− activated and resting B cell subsets
The heat map reports the expression levels of differentially expressed miRNAs between two subepithelial (SE) CD5− B cell populations (FDR 10%): activated IgV mutated SE B cells and resting IgV unmutated SE B cells. Red, higher expression (log2, +4); green, lower expression (log2, −4).
Figure 4
Figure 4. MiR-9*, miR-29b and miR-150 distribution in normal tonsillar tissue
The expression of the three miRNAs was detectable in pharyngeal tonsils as a grainy blue cytoplasmic staining. MiR-9* was significantly overexpressed in germinal centers (GC), mantle zone (MZ), and subepithelial marginal zone (MaZ) in comparison to squamous epithelium (Sq). At higher magnification, it is evident a stronger expression in GC cells in comparison to MZ. MiR-29b was significantly overexpressed in both MZ and MaZ in comparison to GC, whereas miR-150 was significantly downregulated in GC in comparison to MZ and MaZ. Bar scale = 200 μm.
Figure 5
Figure 5
(A) Distribution 28 genes targeted by differentially expressed miRNAs in the three clusters reported in Figure 1. Venn diagram reports the distribution and intersection of target genes of differentially expressed miRNAs which belong to the gene ontology category “lymphocyte differentiation”. Two boxes indicate the miRNAs known to target ZEB1 and TP53. (B) Differential expression of miRNAs known to target ZEB1 and TP53 between naïve and GC B cells.
Figure 6
Figure 6. Network of proteins obtained by shortest path analysis among target genes of miRNA differentially expressed (Figure 1), their first interactor proteins and miRNAs targeting hub proteins and first interactors
Dark grey square: hub proteins, that is proteins nodes of the network with higher degree; light grey circle: protein first interactors of hub proteins; white hexagon: miRNA targeting hub proteins. In tables, list of hub proteins (bold letters), first interactors of hub proteins and miRNAs targeting hub proteins and their first interactors present in the network. (A) Network of target proteins of miRNA listed in Cluster 1 of Figure 1, their first interactor proteins and miRNAs targeting hub proteins and first interactors. (B) Same representation as in 6A by using the miRNAs listed in Cluster 2 of Figure 1. (C) Same representation as in 6A by using the miRNAs listed in Cluster 3 of Figure 1.

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