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MicroRNA-21 maintains HSC homeostasis
quiescence is closely connected with these cells’ long-term reconstituting capability.6,29 To study this, we first conduct- ed non-competitive bone marrow transplantation (BMT) assays (Figure 4A). Mice transplanted with miR-21D/D BM cells died earlier than those transplanted with miR-21fl/fl BM cells in secondary non-competitive BMT (Figure 4B, C). To further determine the function of miR-21D/D HSC, we next performed competitive BMT assays (Figure 4D). It was observed that miR-21 deficiency led to a reduced percentage of donor-derived cells in recipients’ PB and LSK compartments after primary and secondary transplants, accompanied by myeloid-biased hematopoietic differenti- ation (Figure 4E-H; Online Supplementary Figure S4A, B).
To substantiate these findings, we performed another competitive BMT. BM cells from miR-21fl/fl;Mx1-Cre- or miR-21fl/fl;Mx1-Cre+ mice which were not administered pIpC were transplanted, and at 4 weeks after transplanta- tion, miR-21 ablation was induced by injecting pIpC into the recipients (Figure 4I). Consistently, the compromised function and biased differentiation were also observed (Figure 4J, K). Furthermore, the reciprocal BMT experi- ment indicates that BM microenvironmental changes are not enough to mediate the functional defects in miR-21D/D HSC (Online Supplementary Figure S4C-E). Collectively, these data confirm that miR-21 intrinsically modulates the function of HSC.
Specific knockout of miR-21 dramatically decreases the NF-κB pathway in hematopoietic stem cells
To gain insight into the underlying mechanisms by which miR-21 regulates HSC homeostasis and function, we conducted a microarray analysis of sorted LSK from miR-21fl/fl and miR-21D/D mice. As shown in Figure 5A, we identified 1,050 differentially expressed genes, of which 495 genes were upregulated and 555 genes were downreg- ulated in HSC when miR-21 was deleted. GO enrichment analysis showed that the upregulated genes were marked- ly enriched in nucleosome assembly, mitotic nuclear divi- sion, cell division, DNA replication-dependent nucleo- some assembly, cell cycle and chromosome segregation (Figure 5B), which was in accordance with the findings that miR-21 knockout reduced the quiescence and pro- moted the proliferation of HSC. Besides, consistent with the myeloid bias that manifested in miR-21D/D mice, we noticed a robust increase in the expression of myeloid dif- ferentiation genes, such as Mpo, Fcgr3, Csf1r, Igsf6, Ly6c1 and Ms4a3,30,31 in HSC after miR-21 knockout (Online Supplementary Figure S5).
Importantly, KEGG pathway analysis revealed that miR-21 ablation dramatically decreased the NF-κB path- way in HSC (Figure 5C), which has been reported to play a critical role in maintaining hematopoietic homeosta- sis.30,32 In fact, a marked downregulation of NF-κB down- stream genes was observed in miR-21D/D LT-HSC (Figure 5D), including p21 (Figure 2C). The attenuation of NF-κB activity was further verified by western blotting, immuno- fluorescence and flow cytometry (Figure 5E-G). Notably, the defects displayed in miR-21D/D mice, including myeloid bias, accumulation of HSC in the BM and spleen, and reduced quiescence and impaired reconstituting capacity, resemble such aspects of hematopoietic p65-null mice.30 Altogether, these results suggest that miR-21 deficiency inhibits the NF-κB pathway in HSC, which may con- tribute to the defects in HSC.
The upregulation of PDCD4 is responsible for the defects in miR-21-null hematopoietic stem cells
Considering that miRNA negatively regulate gene expression at the post-transcriptional level, we then ana- lyzed the expression of miR-21 target genes throughly. Of the miR-21 target genes that have a potential role in affect- ing NF-κB activity,25,33-35 PDCD4, but not PTEN, Spry1 or Spry2, was markedly upregulated in HSC with miR-21 deficiency (Figure 6A-C; Online Supplementary Figure S6A). The binding between miR-21 and the PDCD4 3’ untrans- lated region was then confirmed by luciferase reporter assay (Online Supplementary Figure S6B). Intriguingly, we observed that PDCD4 expression was abundant in HSC in mouse BM (Online Supplementary Figure S6C), which is consistent with the specific role of miR-21 in HSC.
We next sought to determine whether the upregulation of PDCD4 in HSC contributes to the decreased NF-κB activity, and defective phenotype and function observed in miR-21D/D mice. For this purpose, we transduced normal HSC from WT mice with a lentivirus expressing PDCD4 and found that NF-κB activity was significantly inhibited after overexpression of PDCD4 (Figure 6D). As a conse- quence, ectopic expression of PDCD4 reduced the quies- cence and long-term repopulating potential of HSC, and also biased differentiation (Figure 6E, F; Online Supplementary Figure S6D, E). To further verify this notion, miR-21D/D HSC were transduced with a lentivirus carrying shRNA against PDCD4 (Online Supplementary Figure S6F). As expected, knockdown of PDCD4 partly rescued the defects in miR-21-null HSC (Figure 6H, I; Online Supplementary Figure S6G-I). These data underscore a crit- ical role of miR-21 in supporting the NF-κB pathway by targeting PDCD4, which is essential for the maintenance of HSC homeostasis.
miR-21 protects hematopoietic stem cells from irradiation-induced damage by activating the NF-κB pathway
Evidence has indicated a vital role of NF-κB signaling in mitigating irradiation-induced hematopoietic injury,36-38 which led us to speculate that miR-21 may affect irradia- tion-induced biological processes in HSC. To confirm this, miR-21fl/fl and miR-21D/D mice were simultaneously subjected to ionizing radiation. We found that the loss of miR-21 led to distinctly decreased DNA damage repair, accompanied by increased apoptosis, in HSC exposed to irradiation (Figure 7A, B; Online Supplementary Figure S7A). Moreover, several NF-κB target genes (Ier3, Xrcc5 and Gadd45b) involved in DNA damage repair were sig- nificantly decreased in HSC with miR-21 deficiency (Figure 7C). Additionally, a more serious decrease in the number of HSC and white blood cells and an increased death rate were observed in miR-21D/D mice after irradia- tion (Figure 7D, E; Online Supplementary Figure S7B). Based on the finding that NF-κB signaling is implicated in thrombopoietin-promoted DNA damage repair in HSC, we administered thrombopoietin to the mice and observed that it did not work in miR-21D/D HSC (Online Supplementary Figure S7F, G).
Finally, we determined whether the addition of exoge- nous miR-21 can protect HSC from irradiation by activat- ing the NF-κB pathway. Treatment of mice with a miR-21 agomir, which is an engineered miRNA mimic, increased NF-κB activity in HSC by suppressing PDCD4 (Figure
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