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miR-127 controls HSC maintenance
similar to that of cells recently infected, and inferior to that of miR-16 (Online Supplementary Figure S5C), thus excluding effects due to supra-physiological expression.
The observed lymphopenia was not accompanied by myeloproliferation or splenomegaly, as expected if 127OE were to lead to HSC expansion. Indeed, 127OE did not affect maintenance of stem and progenitor cells (Figure 6C). This is similar to what has been described for miR-99, which did not cause HSC defects when over-expressed, despite its function in regulating HSC self-renewal being revealed after downregulation.20
To test whether miR-127OE HSC show an advantage when subjected to challenging conditions, transduced Lin- cells were transplanted in competition with un-transduced cells at different ratios, and the presence of OFP+ donor cells was monitored over time on myeloid cells in the PB, as described for the sponge experiments, as well as in BM and spleen at necropsy (Online Supplementary Figure S5D). 127OE OFP+ cells did not expand in primary recipients (Figure 6D and E). Donor-derived OFP+ stem cells were functionally competent, since they engrafted, expanded and gave rise to multi-lineage progeny in secondary recip- ients (Figure 6F and G), independently from the overex- pression of miR-127. However, lower lymphoid reconsti- tution was detected in secondary recipients (Figure 6G).
Taken together, these results suggest that miR-127-3p maintains the HSC pool by limiting premature differenti- ation, and must be down-regulated upon HSC maturation to maintain correct homeostasis of the hematopoietic sys- tem.
Discussion
In this report we demonstrate that miR-127-3p is an important novel regulator of the balance between self- renewal and differentiation in HSC.
The miR-127 gene is encoded in a large evolutionary conserved miRNA gene cluster located within an imprint- ed domain (14q32 in human, 12qF in mouse),28 the Dlk1- Gtl2 locus. On the maternally imprinted allele, this locus encodes multiple long ncRNA, snoRNA and a mega-clus- ter of miRNA organized in two groups, among which the miR-127/miR-136 cluster composed of 7 miRNA genes. RNA-seq data showed that the entire locus, which is reg- ulated by a common cis-element, is specifically tran- scribed in CD49blo long-term HSC compared to all other BM progenitor and mature cells.28 However, according to our profiling, of the miR-127/miR-136 cluster, only miR- 127 and miR-434 are expressed as mature forms in adult HSC. Similarly, of all the other miRNA included in the Dlk1-Gtl2 locus beside the miR-127/miR-136 cluster, only miR-411 is expressed in HSC. Of note, three out of the four top miRNA in our HSC-to-MPP list are part of this locus (Online Supplementary Table S2), in line with previous data showing that ncRNA within the Dlk1-Gtl2 locus can be considered HSC markers.28 The miR-127 gene is embedded in a CpG island; therefore, it is conceivable that the rapid downregulation in the earliest step of hematopoietic differentiation is due to epigenetic silenc- ing. Indeed, the expression of miR-127 was experimental- ly induced in cancer cell lines by chromatin-modifying drugs, which did not affect expression of the other mem- bers of the miRNA gene cluster.29
The entire locus has been inactivated in the maternally-
derived allele.30,31 Due to embryonic lethality, the conse- quences on hematopoiesis were studied using fetal liver stem and progenitor cells. The phenotype observed pre- sented features similar to what we observed in mice trans- planted with 127DR Lin– cells, such as reduction of HSC despite normal progenitor and lineage cell counts, and defective long-term reconstitution capacity.28 Altered PI3K- mTOR pathway affecting mitochondrial metabolism explained this phenotype,28 although other molecular mechanisms cannot be excluded. Our data on ROS pro- duction and apoptosis do not support the hypothesis that miR-127-3p is involved in regulating oxidative stress in HSC, and indeed miR-127-3p was not one of the miRNA for which a target within the PI3K-mTOR pathway was confirmed.28 A mouse model with specific deletion of miR-127 was generated;32 however, the effects on hematopoiesis have not been investigated.
MiR-127 is aberrantly expressed in the context of sever- al solid tumors, potentially acting as either an oncogene (oncomiR)33-35 or as a tumor suppressor.36-41 Within hematopoietic malignancies, miR-127 was found up-regu- lated, together with other miRNA included within the 14q32 domain, in the acute promyelocytic leukemia (APML) due to PML-RARa translocation.42 It would be worth investigating whether miR-127 aberrant expression contributes to the differentiation block in APML myeloid progenitors. Although miR-127-3p overexpression did not lead to myelo- or lymphoproliferation, we cannot exclude that, in the presence of other genetic abnormalities, miR- 127-3p may act as an oncomiR.
A number of miR-127-3p targets have been found in cancer cells,21,33,35-39,41 in other cell lines,43,44 or during devel- opment,32 but they are either not expressed in hematopoi- etic progenitors, or their expression does not change in the HSC-to-MPP transition. Our search for predicted miR- 127-3p targets within DE mRNA in the HSC-to-MPP tran- sition and in Pbx1-deficient HSC revealed two potential targets, Gp1bb and Nek2 (Online Supplementary Table S3). We confirmed by qRT-PCR their upregulation in the phys- iological HSC-to-MPP transition (data not shown); however, the increase in Gp1bb expression was extremely mild, whereas Nek2 seed-sequence is located within an alterna- tively spliced, non-coding portion of the transcript. It is, therefore, unlikely that Nek2 protein expression is directly regulated by miR-127-3p. Since miR-127-3p binding to its target mRNA might result in translation inhibition rather than mRNA degradation, a global proteomic approach would be required to gain insights on miR-127-3p func- tional targets mediating the observed phenotype, although protein analysis on rare primary HSC is techni- cally challenging, especially in a model in which stem cells are reduced.
The physiological downregulation of miR-127-3p observed in the transition from HSC to MPP coincides with the loss of self-renewal ability that is known to occur during this differentiation step. Moreover, miR-127-3p is absent in HSC obtained from Pbx1-cKO mice, which dis- play a profound self-renewal defect.4 This stimulates future studies to investigate if Pbx1, together with its homeobox partners, directly regulates miR-127 expres- sion, or if miR-127 OE can rescue part of the severe Pbx1- cKO phenotype. Our data strongly suggest that HSC must maintain proper levels of miR-127-3p to preserve their long-term self-renewal capacity. We also found that miR- 29a, miR-99 and miR-126a are expressed in HSC but also
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