The miRNA signature comprising relative and absolute expression levels of CD133+ cells was determined by microarrays, qRT-PCR, and Solexa sequencing. On this basis, we selected differentially expressed miRNAs in CD133+ cells in comparison with CD34+CD133− cells.
CD133 appear to be ancestral to CD34, as CD34+
cells can be generated in vitro from CD133+
]. To elucidate the molecular architecture of CD133+
cells and the role of miRNAs in the early steps of hematopoiesis, we compared CD133+
cells with CD34+
cells. It is noteworthy that most of the CD133+
cells (>99%) express CD34 and half of the CD34+
cells express CD133 [13
Currently, the available miRNA profiles for human HSCs and HPCs are restricted to CD34+
cells from bone marrow, peripheral blood, mobilized peripheral blood and cord blood [9
cells from cord blood [5
], and CD133+
cells from mobilized peripheral blood [39
]. To our knowledge, here, we compared for the first time CD133+
bone marrow cells from the same donor on miRNA and mRNA level. Most of the miRNAs (79%) in CD133+
cells were present with less than 200 copies per cell and the 25 highest expressed miRNAs accounted for 72.8% of the miRNA pool. A finding that correlates well with previously published sequencing data [40
]. The majority of miRNAs was expressed at low levels, whereas only a few miRNAs were expressed at high level with around 2,000 copies per cell (), for example, miR-26a amounted to 7.2% of the overall miRNA pool. Although miR-26a was expressed at high levels, it is possible that the majority of low-expressed miRNAs are of biological significance when acting in combination with other low-expressed miRNAs [41
A combined miRNA and mRNA analysis was performed to develop a model for the biological role of the 18 differentially expressed miRNAs in CD133+ stem cells (). The mRNA data used for filtering of the predicted targets was generated from the same cells as the miRNA profiles.
Notably, mRNAs expressed in CD133+
cells and predicted to be targeted by miRNAs higher expressed in CD133+
cells as compared with CD34+
cells (group 1; ) were enriched for the GO category “hematopoietic or lymphoid organ development.” Interestingly, the corresponding miRNAs have already been described to play a role in hematopoiesis. It was shown that miR-10a [42
], miR-146a [43
], and miR-125b [44
] inhibit differentiation of specific hematopoietic lineages ().
Figure 7 Model for the biological role of significantly differentially expressed miRNAs in CD133+ cells. (A): The targets of the miRNAs are highly relevant in the context of differentiation (green), apoptosis (blue), and cytoskeletal remodeling (red). Predicted (more ...)
Furthermore, p53 and the proapoptotic B-cell CLL/lymphoma 2 antagonist killer 1 (BAK1) are targeted by miR-125b, suppressing apoptosis in human cancer cells [45
]. BAK1 is also directly downregulated by miR-125a [47
]. In addition, suppression of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2) and ERBB3 by enforced expression of miR-125a or miR-125b resulted in impaired anchorage-dependent growth in breast cancer cells [48
]. As activation of ERBB2 can induce loss of cell polarity, suppression of ERBB2 by miR-125 in CD133+
cells might lead to a more polarized morphology.
Taken together, miR-146, miR-10a, and miR-125b might prevent differentiation, whereas miR-125b seems to play an antiapoptotic role in CD133+ cells as well as taking part in remodeling of the cytoskeleton ().
The second group of higher expressed miRNAs in CD133+
cells compared with CD34+
cells were also highly expressed in CD34−
cells (group 2; ) and were among others enriched for annotations related to developmental processes, negative regulation of cell migration and collagens. Roles for the respective miRNAs in developmental processes were already shown for miR-23a, miR-23b, miR-29a, and miR-24 [49
]. Furthermore, an influence of miR-29b on the regulation of the extracellular matrix and of miR-24a on the proapoptotic factor caspase 9 was demonstrated [52
The predicted targets of the lower expressed miRNAs (group 3; ) were highly enriched for the UPS and the regulation of cytoskeleton organization. Interestingly, a function of miR-142-5p in ubiquitin-mediated proteolysis and of miR-142-3p in regulation of cytoskeleton was also predicted by Tsang et al. [54
] using the computational method mirBridge.
After analyzing the coexpression of bioinformatically predicted miRNA-mRNA pairs (approach I; ), we focused on inversely correlated miRNA-mRNAs pairs (approach II; ) and analyzed some of them via luciferase assays. One of those pairs was miR-29a and TPM1, an important component of the cytoskeleton playing a fundamental role in many aspects of eukaryotic cell behavior such as cell morphology, divisions, and motility. Our results revealed that TPM1 can be regulated by miR-29a and that this interaction could lead to a lower expression of TPM1 in CD133+
cells. As tropomyosin inhibits lamellipodium formation [55
], a lower expression of TPM1 in CD133+
cells might lead to enhanced podia formation as compared with CD34+
cells. Interestingly, it was described that more primitive HPCs possess more lamellipodia with strong expression of CD133 [56
Another inversely correlated miRNA-mRNA interaction was miR-29a and FZD5, one of the seven-pass transmembrane Frizzled receptors, which bind Wnt proteins. Wnt signaling controls diverse processes such as cell proliferation and stem cell maintenance. A role for Wnt signaling in self-renewal of HSCs was demonstrated by Reya et al. [57
]. Regulation of cell polarity and migration by ß-catenin-independent Wnt pathways was also described [58
Another very interesting interaction that was validated via luciferase assay was the binding of miR-142-3p, being lower expressed in CD133+ cells than in CD34+CD133− cells, to the CD133 mRNA. Our results also provided first evidence that miR-142-3p has a negative influence on the proliferation of CD133+ cells. However, further analysis is necessary to show if the surface expression of CD133 is controlled by miRNAs.
According to the biological effects of the miRNAs summarized above (), the miRNAs higher expressed in CD133+ cells prevent differentiation, are antiapoptotic and regulate the cytoskeleton leading to enhanced podia formation and cell polarity (). The miRNAs higher expressed in CD34+CD133− cells repress the ubiquitin-proteasome system and negatively influence the proliferation of CD133+ cells. Taken together, it is proposed that the miRNAs significantly differentially expressed in CD133+ cells as compared with CD34+CD133− function by fine-tuning the expression of their targets to a precise level at which the gene will execute its specific function to prevent processes such as differentiation and apoptosis. In addition, they play a role in remodeling of the cytoskeleton. Detailed functional analysis of miRNA effects in CD133+ cells will gain further insights into the role of miRNAs in stem cells.