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X. Fang et al.
(PHZ)-induced hemolytic anemia was delayed in miR- 144/451−/− mice.15
To examine further the effects of erythropoietic stress on adult miR-144/451−/− mice, we treated them with a sin- gle dose of 5-fluorouracil (5-FU), which destroys commit- ted hematopoietic progenitors, including erythroblasts. Compared to WT mice, miR-144/451−/− mice exhibited increased mortality after 5-FU treatment (55.6% vs. 86.8% survival) (Figure 2A), which was associated with a greater decline in their hematocrit (Figure 2B). During the recov- ery phase after 5-FU treatment, the mutant mice exhibited higher levels of late stage circulating erythroid precursors (reticulocytes) (Figure 2C), probably in response to the more severe anemia (Figure 2B). However, the time to maximal reticulocyte response was delayed by several days in KO mice compared to WT controls (Figure 2C). Similarly, the emergence of bone marrow erythroid pre- cursors was delayed in KO mice (Figure 2D-G). Moreover, bone marrow erythroblasts of miR-144/451−/− mice with 5- FU treatment exhibited increased apoptosis compared to WT controls (Figure 2H and I). We also observed increased apoptosis of miR-144/451−/− bone marrow and splenic ery- throblasts during recovery from PHZ-induced anemia (Figure 3A and B) or phlebotomy (Figure 3C and D). Thus, miR-144/451−/− erythroblasts exhibit increased apoptosis
under conditions of increased physiological demand for RBC production, i.e. under erythropoietic stress.
miR-451 targets Cab39 mRNA in erythroblasts
Several mRNAs previously identified as miR-451 target mRNAs, including Myc, Ywhaz/14-3-3ζ, and Cab39 (Figure 4A), encode general regulators of cell survival, pro- liferation, and maturation.13,15,21 Cab39 is an obligatory co- factor for the serine/threonine kinase LKB1, a tumor sup- pressor that regulates responses to metabolic stress, in part by activating AMP-activated protein kinase (AMPK).22 miR-451 drives human glioma cell expansion by inhibiting this pathway via direct repression of Cab39.21 Therefore, we investigated whether miR-451 repression of Cab39 reg- ulates erythroblast survival during erythropoietic stress. Compared to controls, Cab39 mRNA and protein were up-regulated in miR-144/451−/− erythroblasts in spleen, bone marrow (Figure 4B and C) and FL (Figure 4D) com- pared to WT erythroblasts in the same tissues. Retroviral vector-mediated expression of miR-451 in the erythroid cell line G1E17 reduced Cab39 protein by approximately 50% (Figure 4E). The seed sequence of miR-451 is comple- mentary to a conserved sequence within the 3′ untranslat- ed region (UTR) of human and mouse Cab39 mRNA (Figure 4A). To verify whether miR-451 inhibited Cab39
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Figure 3. Apoptosis of miR-144/451−/− bone marrow and spleen erythroblasts is increased under other stress conditions. (A and B) Results of flow cytometric analy- sis of nucleated erythroid cell apoptosis in spleen (A) and bone marrow (B) after phenylhydrazine (PHZ) administration. miR-144/451−/− mice (n=5) and wild-type (WT) littermates (n=5) were treated with PHZ (120 mg/kg) on days 0 and 1. Splenocytes and bone marrow cells were harvested four days after PHZ injection. Ter119+Hoechst+ cells are nucleated erythroid cells. Annexin V+PI–near IR− cells are early apoptotic cells. Ter119– cells are non-erythroid cells. **P<0.01 (t-test). (C and D) Flow cytometric analysis of early apoptosis rates of spleen (C) and bone marrow (D) nucleated erythroblasts after bleeding. miR-144/451−/− mice (n=5) and WT littermates (n=5) had 0.4 mL of blood drawn on two consecutive days. Splenocytes and bone marrow cells were harvested on day 3. **P<0.01 (t-test). Note: more early apoptotic erythroblasts were seen in miR-144/451−/− mice.
haematologica | 2018; 103(3)