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M. Hu et al.
Figure S3B, C), which is in line with the expression pattern of miR-21. Moreover, qRT-PCR data showed that cyclin- dependent kinase inhibitors (p21 and p27) were downregu- lated, whereas cell cycle-associated genes (cyclin E1, cyclin E2, cyclin A2 and CDK6) were upregulated in LT-HSC in the absence of miR-21 (Figure 3C).
Given that proliferating active cells are highly sensitive to genotoxic stress,29 we therefore administered miR-21fl/fl and miR-21D/D mice with a single dose of 5-fluorouracil. As expected, miR-21D/D mice displayed more severe myelosup-
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pression compared with that of control mice (Figure 3D, E). Likewise, the survival rate of miR-21D/D mice was signifi- cantly reduced after weekly 5-fluorouracil injections (Figure 3F). Taken together, these data demonstrate that miR-21 is required to maintain HSC in a quiescent state.
miR-21 deficiency intrinsically compromises hematopoietic stem cell function in long-term reconstitution
It has been well accepted that the maintenance of HSC
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Figure 3. Loss of miR-21 impairs the quiescence and facilitates the proliferation of hematopoietic stem cells. (A) Cell cycle analysis of Lin-Sca1+c-Kit+ (LSK) cells and long-term hematopoietic stem cells (LT-HSC) in the bone marrow (BM) of miR-21fl/fl and miR-21∆/∆ mice (n=6 mice per group). (B) miR-21fl/fl and miR-21∆/∆ mice were intraperitoneally injected with 5-bromodeoxyuridine (BrdU: 1 mg/6 g mouse weight). Twelve hours later, BrdU incorporation into LSK and LT-HSC from these mice was analyzed by flow cytometry (n=6 mice per group). (C) Quantitative real-time polymerase chain reaction analysis of the relative expression of cyclin-depen- dent kinase inhibitors and cell cycle-associated genes in sorted miR-21fl/fl and miR-21∆/∆ LT-HSC (n=3 mice per group). (D, E) The number of BM cells (D) and LSK (E) from miR-21fl/fl and miR-21∆/∆ mice after a single injection of 5-fluorouracil (5-FU) (n=6 mice per group). (F) The survival rates of miR-21fl/fl and miR-21∆/∆ mice after sequential 5-FU injections (n=10 mice per group). All data are shown as means ± standard deviation. *P<0.05, **P<0.01.
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haematologica | 2021; 106(2)