Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes
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(2) Chapter 5. Differential expression of miR-148 in hematopoietic thymic subsets and its potential function
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(22) % & MicroRNAs (miRNAs) have been implicated in several cellular processes, such as cell proliferation, differentiation and apoptosis. miR-148 was found expressed in the spleen and enriched in human cell lines of hematopoietic origin. Interestingly, we identified Dnmt3b and MLL (mixed lineage leukaemia) as possible miR-148 targets and they both appear to be involved in T cell development and survival. To study whether miR-148 mediated repression of Dnmt3b and MLL plays a role in human lymphoid development, we determined the expression of miR-148, Dnmt3b and MLL in different lymphoid subsets isolated from human postnatal thymus. Whereas miR-148 is highly expressed in CD34+CD1a- early thymic progenitor cells and remains expressed in all subsets of T cell committed lineages, its expression is significantly decreased in natural killer (NK) cells and plasmacytoid dendritic cells (pDCs). However, we found that Dnmt3b and MLL mRNA levels are reduced in these subsets as well. This suggests that relieve of miR-148 mediated repression of Dnmt3b and MLL is not a key step in pDC and NK development. Instead, miR148 might play a role in fine-tuning the expression of these targets in lymphoid development. To test whether reduction of miR-148 level is essential for pDC development we exogenously expressed miR-148 in mTPCs and performed in vitro pDC differentiation assays. Interestingly, miR-148 overexpression resulted in a dramatic increase in both percentage and number of pDCs.. Introduction *
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(275) Differential expression of miR-148. A. miR-148. CD123. miR-147. BDCA2. B relative expansion. 40.0. 30.0. 20.0. 10.0. 0.0. hTR. miR-1 147. miR-1 148. Figure 5. miR-148 overexpression results in an increased number of pDCs. A hTR, miR-147 and miR-148 transduced CD34+CD1a- cells were cocultered with OP9 cells in the presence of IL-7 and Flt3L and analysed after 11 days for the presence of pDCs (CD123highBDCA2+) B Relative expansion of pDC cells. Fold expansion in absolute cell numbers of the pD subset in the transduced population was calculated on basis of total numbers of cells harvested from the cultures after 11 days, percentages of transduced cells, and percentages of each population corrected for the number of input cells. Whether this effect is due to enhanced differentiation, proliferation or survival of pDCs remains to be determined. Furthermore, these results imply that miR148 is down-regulated in pDCs to limit the number of pDCs in the human thymus. The main function of pDCs in the immune response is their capacity to produce high levels of type I interferons, which directly inhibits viral replication and triggers T cell mediated responses (Blom et al., 2002; Kadowaki et al., 2000). However, in the thymus pDC activation has recently been shown to impair thymic T cell development (Schmidlin et al., 2006). Therefore, miR-148 downregulation in pDCs could ensure proper thymic T cell development by keeping the numbers of pDCs low. In case miR-148 expression leads to increased survival and/or proliferation of pDCs it would be interesting to test pDC leukemic cells for. expression of miR-148 (Chaperot et al., 2001). What remains to be determined is via which targets miR-148 exerts this function in pDCs and whether reduced Dnmt3b1 or MLL are involved. Furthermore, considering the similar expression profile of miR-148 and its targets in pDCs and NK cells it would be interesting to see whether miR-148 has a similar stimulatory effect on NK cell development as it has on pDCs.. Material and Methods Constructs All miRNAs were expressed from a retroviral miR-Vec vector expressing Blastacidin or YFP as described before (Voorhoeve et al., 2006). The MLL 3’UTR was cloned downstream the luciferase gene in AatII and AgeI sites generated in the pGL3 constructs. The following primers were used to clone MLL from genomic DNA:. 75.
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