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that catalyzes the cleavage of pre-miR-451 hairpins.12 Inhibition of miR-144/451 blocks erythropoiesis in tissue culture models.8,10,13-15 Fewer studies have been performed with in vivo models.13-15 Moreover, the phenotypes observed after manipulating miR-144/451 expression vary according to the model used and the mode of gene manip- ulation. For example, miR-144/451 inhibition appears to exert a greater effect on erythroblasts in culture than on those in vivo, suggesting that the phenotype depends on the cell environment (X Fang et al., unpublished data, 2017). Along with others, we have also demonstrated that miR- 144/451 gene knockout (KO) mice exhibit mild baseline anemia that worsens upon oxidative stress.13,15 Similarly, loss of miR-451 in zebrafish renders erythroid precursors sensitive to oxidant stress.15 The anti-oxidant role of miR- 144/451 during erythropoiesis is at least partially depend- ent on suppression of the mRNA target Ywhaz, which encodes the cytoplasmic adaptor protein 14-3-3ζ.13,15 miR- 144/451 depletion increases 14-3-3ζ protein, which sequesters the transcription factor FoxO3 in the cyto- plasm, thereby reducing expression of several target genes that encode anti-oxidant proteins. This mechanism explains the hypersensitivity of miR-144/451−/− RBCs to oxidant stress but is unlikely to account for all the activi- ties of these miRs. For example, miR-144/451−/− mice exhibit ineffective erythropoiesis at baseline and delayed recovery after anemia caused by oxidant stress13-15 via
unknown mechanisms.
We discovered that erythroblasts isolated from
miR-144/451−/− FL during embryonic gestation or bone marrow and spleen during acute anemia exhibit increased apoptosis compared to wild-type (WT) counterparts. This effect is mediated by derepression of the direct miR-451 target mRNA Cab39 followed by activation of the down- stream LKB1/AMPK/mTOR pathway. Thus, miR-144/451 enhances physiological responses to acute anemia by pro- moting the survival of RBC precursors.
Methods
Animals
miR-144/451 KO mice were described previously.15 p53ER knock-in (KI) mice were kindly provided by Gerard Evan (University of Cambridge, UK).16
Cell culture and treatment
G1E and G1E-ER4 erythroid cells were grown in culture as pre- viously described.17 The isolation of erythroid progenitors from embryonic day 14.5 (E14.5) FLs, the growth of erythroid progeni- tors in maturation medium, and the retroviral infection of ery- throid cells in expansion medium have all been described previ- ously.18 Details of drug treatments of cells are described in the Online Supplementary Appendix.
Protein and miRNA expression
Western blot and real-time PCR analyses for gene expression were described in the Online Supplementary Appendix.
Fluorescence-activated cell sorting (FACS)
The expression of RBC surface markers and cell death were analyzed with an LSRII or LSRFortessa Cell Analyzer System (BD Biosciences). The Annexin V Early Apoptosis Detection Kit (cat. n. 553786) was obtained from BD Biosciences. The nucleation of erythroid cells was quantitated by staining with Hoechst 33342
(Sigma); cell viability was quantitated by staining cells with death markers 7AAD, propidium iodide (PI) or Live/Dead® Near-IR Fixable Dead Cell Stain (Invitrogen). Erythroid subpopulations were sorted on the basis of CD71/Ter119 expression (BD Biosciences).
Dual-luciferase reporter assay
Construction of the plasmids for luciferase assays is described in the Online Supplementary Appendix. The dual-luciferase reporter assay was performed as previously described.15
Retroviral shRNA delivery
The construction of retroviral plasmids and transduction of cells are described in the Online Supplementary Appendix.
Hematologic analysis
For hematocrit (HCT) and reticulocyte counts, blood from adult mice was sampled retro-orbitally, anticoagulated with EDTA, and analyzed on a Hemavet HV950FS analyzer (Drew Scientific, Dallas, TX, USA). Heparinized glass microhematocrit tubes (Globe Scientific, Paramus, NJ, USA) were used for manual spun hematocrits. Reticulocytes were counted using Retic-COUNT reagent (BD Biosciences) or Ter119/CD71 staining and analyzed on a FACSCalibur Flow Cytometer (BD Biosciences).
For phenylhydrazine (PHZ) treatment, mice were injected intraperitoneally (63 mg/kg). HCT and reticulocyte counts were analyzed for ten consecutive days thereafter.
haematologica | 2018; 103(3)
miR-451 inhibits Cab39 for stress erythropoiesis
To eradicate erythroid progenitors in adult mice, 5-FU (Sigma- Aldrich) at a single dose of 150 mg/kg was injected intraperitoneal- ly, and the HCT and reticulocyte counts were analyzed for 25 con- secutive days thereafter. Animal survival was monitored every day.
To generate acute anemia by bleeding, 400 μL of blood was drawn retro-orbitally every day. The HCT and reticulocyte counts were analyzed three days later.
Microarray analysis
CD71+/Ter119+/FSChigh nucleated bone marrow cells from miR-144/451−/− mice and WT controls were purified by flow cytometry, and samples were processed for microarray analysis using the GeneChip Mouse Genome 430 2.0 Array (Affymetrix), as described previously.15 The database for G1E-ER4 erythroid maturation has been described in an earlier paper.19
Statistical analysis
Statistical analyses were performed using Microsoft Office Excel 2011 (Microsoft Corporation, Redmond, WA, USA). Graphs were created using Adobe Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA). Data from triplicate experiments or 3 differ- ent samples are presented as the mean ± standard deviation. The differences were assessed by two-tailed Student t-tests. P<0.05 was considered statistically significant. All experiments were repeated at least 3 times.
Results
Increased apoptosis of miR-144/451−/− erythroblasts during erythropoietic stress
To study the effects of miR-144/451 on erythropoiesis, we cultured equal numbers of FL erythroid precursors from embryonic day (E) 14.5 KO or WT embryos in media that facilitated their expansion or terminal maturation18 (Figure 1A). After 48 hours (h) in expansion medium, miR-144/451−/− erythroblasts exhibited reduced cell num-
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