This study demonstrates the feasibility to perform in vivo
miRNA screens in live mice with a barcoded library, and the power to uncover regulators of complex physiological processes. The finding through the screen that miR-150 impaired hematopoietic injury response contrasts the miRNA expression profiling results comparing bone marrow samples with or without 5-FU treatment, which found 92 out of 168 (55%) miRNAs with significant expression changes (Fig. S1A
). Interestingly, miR-150 was among the 45% of miRNAs that were not significantly changed after 5-FU-treatment (Table S1
). These results reiterate usual problems associated with gene profiling studies, that the resulting miRNA changes are often too numerous to study. On the other hand, functionally important genes may be missed with profiling. Even after we identified miR-150, measuring miR-150 expression in Kit+
or other potential bone marrow progenitor populations did not yield intuitive expression patterns associated with its functional importance: miR-150 expression was higher and c-myb lower in post-5-FU samples than those without treatment (Fig S6C
). Similarly, the complex nature of our measured hematopoietic injury response, which reflects the clinically relevant rebound of circulating mature cells within the peripheral blood after acute injury, can be challenging to model in vitro
. We performed two experiments to measure the effect of forced miR-150 expression in cultured Ficoll-purified bone marrow cells, but did not observe any differential competitive expansion/survival disadvantage in the presence of varying amounts of 5-FU (data not shown). We only detected changes in apoptosis when starting with purified HSPCs, suggesting that it would have been difficult to discover the role of miR-150 in hematopoietic injury response in a culture dish without the initial in vivo
The bead-based detection system for genetic barcodes is specific and reproducible for functional screens. Compared to next generation sequencing, it is inexpensive, flexible to design and more readily accessible. The inexpensiveness makes it more attractive to run small sample batches, such as those during assay set up, or screens with a medium-sized library. It is likely less sensitive compared to next generation sequencing and less powerful for large genetic libraries, but our detection rate in mouse (92% of the library at 3 weeks) suggests that sufficient sensitivity of detection can be achieved for libraries of ~100 to 200 genes in complexity.
The data presented in this study highlight the role of miR-150 in controlling hematopoietic recovery after injury. Even though the functional screen was designed to uncover regulatory miRNAs that control short-term peripheral blood recovery after acute injury, which is highly relevant to clinical management of patients receiving chemotherapy, the effects with miR-150 overexpression and knockout studies could be seen within the short-term window of 1–3 weeks and also beyond. The miR-150 overexpression model showed weak but significant lymphoid recovery changes, which was not seen in the miR-150 knockout model. This difference may be due to forcing miR-150 expression in cell types that normally do not express miR-150. Our finding, that miR-150 suppresses multiple types of hematopoietic progenitor activities, including both immature and more mature myeloid progenitors upon 5-FU treatment, provides one explanation for the observed short-term and longer-term effects of miR-150. It is important to note that this progenitor cell effect of miR-150 does not contradict previous knowledge of miR-150 in specific lineage differentiation (Lu et al., 2008
; Barroga et al., 2008
; Xiao et al., 2007
; Bezman et al., 2011
; Zheng et al., 2012
). For example, even though the forced expression of miR-150 elevates platelet production in comparison to myeloid cells (Fig. S4C
) under homeostatic conditions as expected, upon hematopoietic recovery from acute injury, miR-150 affected more upstream progenitors which largely masked this platelet production bias.
Mechanistically, we show that hematopoietic-intrinsic heterozygous loss of c-myb produced similarly delayed recovery after 5-FU. It is known that c-myb regulates proliferation and apoptosis related genes, such as c-myc, cyclins, KIT, Bcl-2 and Bcl-XL
(Ramsay et al., 2003
; Ramsay and Gonda, 2008
), some of which may mediate c-myb s function in hematopoietic recovery. The delayed recovery in the c-myb model was both shorter and weaker than the gain-of-function of miR-150, and did not affect the lymphoid lineages. We speculate that additional targets may also be involved, or the difference in phenotype may be due to quantitative differences in c-myb suppression in the two models.
It is intriguing that loss of miR-150 had mild effects on steady state hematopoiesis (other than B cell subsets, NK and iNKT cells), yet knockout cells displayed the beneficial effect of accelerated rate of hematopoietic recovery in the peripheral blood. This is somewhat puzzling given that miR-150 is evolutionarily conserved from human to frog, although it is possible that the roles of miR-150 in specialized immune cell production, or anti-cancer effect in lymphoma (Chang et al., 2008
) contribute to the pressure for genetic conservation. Nevertheless, our data suggest that an interesting avenue to explore in the future will be to temporarily suppress miR-150 activity for the clinically beneficial outcomes of faster platelet and myeloid cell recovery. Since mice null of miR-150 show largely normal behaviors, it suggests that inhibition of miR-150 may have limited side-effects. The recent progress on in vivo
delivery of miRNA inhibitors (Stenvang et al., 2012
) will, in the future, help to test this concept in a more translational setting.