Identification of the human mature B cell miRNome

Abstract

The full set of microRNAs (miRNAs) in the human genome is not known. Because presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, many tissue-specific miRNAs remain unrevealed. To understand the role of miRNAs in B cell function and lymphomagenesis, we generated short-RNA libraries from normal human B cells at different stages of development (naive, germinal center, memory) and from a Burkitt lymphoma cell line. A combination of cloning and computational analysis identified 178 miRNAs (miRNome) expressed in normal and/or transformed B cell libraries. Most notably, the B cell miRNome included 75 miRNAs which to our knowledge have not been previously reported and of which 66 have been validated by RNA blot and/or RT-PCR analyses. Numerous miRNAs were expressed in a stage- or transformation-specific fashion in B cells, suggesting specific functional or pathologic roles. These results provide a resource for studying the role of miRNAs in B cell development, immune function, and lymphomagenesis.

Figure 1

Predicted precursor and mature miRNAs. The number of predicted precursor miRNAs (pre-miR) and mature miRNAs (mature-miR) are plotted independently for each library and overall. Throughout the figures, the sequences matching miRNAs deposited in the miRBase database (v.11.0) are defined as “known” and conversely the sequences which to our knowledge have not been previously reported are named “new”.

Figure 2

Abundance and evolutionary conservation of the B-cell miRNome. (A) Frequencies of previously reported (known) and to our knowledge newly identified (new) miRNAs as occurring in naïve, centroblasts, memory and Ramos cells. Single occurrences miRNAs are not included. (B) Number of miRNAs cloned multiple times and identified in naïve, centroblasts and memory B cells. A larger overlap is observed for known compared to new miRNA (48% versus 38%). (C) Conservation analysis for orthologous miRNAs was performed in 5 mammal species for all miRNA reported in the miRBase database (miRBase-all) and for known and new mature miRNA expressed in the B-cell libraries. The percentages of miRNAs having either perfect conservation for the entire mature miRNA (top panel) or for its seeds (bottom panel) are displayed.

Figure 3

Detection of previously unreported miRNAs by RT-PCR and RNA blot. (A) Representative results of RT-PCR detection of miRNA in Ramos cell line and tonsil cells. miR-30c was used as loading control. (B) Detection of mature-miRNA species by RNA blot in Ramos cell line, centroblasts (CB) and naïve B cells isolated from human tonsils. RNU44 was used as loading control. (C) RNA blot images displaying both the mature (20–25nt) and the precursor (60–80nt) miRNA species. miRNA expression can be regulated at transcriptional level (top panel) or at the processing level (bottom panel) when intermediate forms (pre-miRNA) are generated but are not fully processed to mature miRNA. The naming of miRNAs is provisional.

Figure 4

miRNA expression profiling distinguishes developmental stages of normal as well as malignant B cells. (A) Unsupervised clustering performed using miRNA frequencies values (≥0.08) calculated as the fraction of the total pool of cloned miRNAs represented by a given miRNA in a library. (B) Unsupervised clustering of microarray-based miRNA expression profiles distinguishes centroblasts, naïve and memory B cells purified from tonsil tissue of six patients/each.