MicroRNA profiling of the murine hematopoietic system

Abstract

Background: MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene expression during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete.

Results: We used oligonucleotide arrays to analyze miRNA expression in the murine hematopoietic system. Complementary oligonucleotides capable of hybridizing to 181 miRNAs were immobilized on a membrane and probed with radiolabeled RNA derived from low molecular weight fractions of total RNA from several different hematopoietic and neuronal cells. This method allowed us to analyze cell type-specific patterns of miRNA expression and to identify miRNAs that might be important for cell lineage specification and/or cell effector functions.

Conclusion: This is the first report of systematic miRNA gene profiling in cells of the hematopoietic system. As expected, miRNA expression patterns were very different between hematopoietic and non-hematopoietic cells, with further subtle differences observed within the hematopoietic group. Interestingly, the most pronounced similarities were observed among fully differentiated effector cells (Th1 and Th2 lymphocytes and mast cells) and precursors at comparable stages of differentiation (double negative thymocytes and pro-B cells), suggesting that in addition to regulating the process of commitment to particular cellular lineages, miRNAs might have an important general role in the mechanism of cell differentiation and maintenance of cell identity.

Figure 1

Design and reproducibility of microarrays. (a) Examples of microarrays: three membranes were used for each biological sample; arrays for Pu.1-/- cells, BMMC and hippocampus are shown. On the left, ethidium bromide staining of total RNA run on a denaturing gel for RNA quantification and quality control. (b) Plot of the standard deviation over replicates versus the mean of each replicate. The red line is a lowest fit to the distribution and the blue dotted line is twice the value of the red one. Points below the blue curve are considered good replicates; those above it are filtered out as too noisy. For this dataset, 86% of the spots were considered as good. BMMC, bone marrow-derived mast cells.

Figure 2

Comparison of miR-150 expression by arrays and Northern blotting. (a) Array data show that miR-150 (arrows) is highly expressed in spleen B and naïve T cells, but not in pro-B cells or fully differentiated Th1 and Th2 clones. (b) Northern blot analysis for miR-150 in various lymphoid and non-lymphoid tissues and cell types. U6 RNA levels are shown as loading control. (c) Northern blots of different cell types unstimulated or stimulated for the indicated amounts of time with either PMA and ionomycin (BMMC) or anti-CD3 and anti-CD28 (Th1 and Th2 primary cells). Preliminary data obtained both in Northern blot and arrays showed no difference in miRNA expression between BMMC left untreated or treated with cyclosporin A (CsA) (not shown). (d) Ethidium bromide staining of gel of total RNA from samples used in Figure 2c, showing equivalent RNA amounts. BMMC, bone marrow-derived mast cells; MEF, mouse embryo fibroblast.

Figure 3

Microarrays and Northern blots correlate qualitatively and quantitatively. Northern blots for miRNA expression in mast cells (left panels), or T cells (right panels). BMMC were treated with cyclosporin A or PMA and ionomycin for the indicated amounts of time. Loading control is the same as Figure 2d. First row underneath the panels: ratio between the intensities of the Northern blot bands as assessed by phosphorimager and quantified by ImageQuant; all the samples are compared with Pu.1-/- cells except for miR-223, where each sample is compared with BMMC. Second row: these are also ratios between the intensities of the Northern blot bands, but the T cell samples are compared directly to each other. This allows a better direct comparison with the numbers on the third row, which are the ratios of BMMC versus Pu.1-/- cells (left panels) and D5 versus D10 versus naïve T cells as obtained from the arrays (right panels). BMMC, bone marrow-derived mast cells; CsA, cyclosporin A; n.d., not detectable.

Figure 4

Additional Northern blots showing miRNA expression in various tissues and cell types. Shown are Northern blot data for the indicated miRNAs in different unstimulated cell types. Asterisks indicate bands of the size of pre-miRNA (60-70 nucleotides), which were detected in Northern blot for only a subset of the miRNAs analyzed. There was a good correlation overall between Northern blot data and expression data from the arrays (see heat map in Figure 5 and Table 2), with some exceptions, as discussed in the text. BMMC, bone marrow-derived mast cells; DN T, double-negative thymocyte.

Figure 5

Analysis of microarray data. (a) Heat map of miRNAs expressed at least three times over the background for at least one of the samples. (b) Hierarchical clustering of hematopoietic samples (see analysis in Table 3). DN T, double-negative thymocyte; MEF, mouse embryo fibroblast.

Figure 6

MiRNA expression and lineage commitment. Expression of miRNA in the hematopoietic system changes depending on the differentiation status. Part of the hematopoietic differentiation tree is represented: myeloid and lymphoid progenitors derive from a common progenitor, which is represented by the model Pu.1-/- cell line (see text). The common lymphoid progenitor gives rise to B and T lymphocytes and the common myeloid progenitor gives rise to mast cells and other cells types. Superimposed on this diagram are examples of miRNAs that were discovered to be differentially expressed between the indicated precursor/progeny pairs using array analysis with confirmation by Northern blot. DN, double-negative.