miR-144-Nrf2 regulatory axis plays a partial role in the alleviation of anemia in th3+/− mice. (A) Microarray transcriptome analysis of BM erythroblasts (Ter119+/CD71+/FSCHi) from th3+/− (βhet) and mKO/th3+/− (mKO/βhet) mice. Samples were analyzed for messenger RNA (mRNA) expression using Affymetrix Gene Chips. (B) Gene set enrichment analysis showing that Nrf2-related genes are upregulated in mKO/th3+/− BM erythroblasts compared with that in th3+/− BM erythroblasts. (C) Targetscan nucleotide sequence alignments showing the complementarity of 3' untranslated region (3′UTR) of Nrf2 mRNA and miR-144. There are 2 miR-144–binding sites in both mouse and human Nrf2 mRNAs. (D) Nrf2 mRNA levels in BM erythroblasts from mKO/th3+/− mice quantitated via real-time polymerase chain reaction. Experiments were repeated at least 3 times. (E) Western blot showing Nrf2 protein levels in BM erythroblasts from different groups of mice. Experiments were repeated at least 3 times. (F) Firefly luciferase reporter assay showing the direct binding of miR-144 to Nrf2 mRNA. 3′UTR of Nrf2 (WT) or its mutant version (mut, an 8-base pair replacement within the region complementary to miR-144 seed sequence) were cloned to luciferase reporter construct. Luciferase activities were determined 24 hours after transfection in 293T cells. (G-H) Flow cytometry analysis of the percentages of reticulocytes in peripheral blood from different group of mice. R1 (Ter119+CD71Hi), early-stage reticulocytes; R2 (Ter119+CD71Med), late-stage reticulocytes. (I) Automated hematology analysis revealing more RBCs in miR-144 KO/th3+/− blood and lower RDW in comparison to th3+/− mice. (J-K) Flow cytometry analysis of the percentages of peripheral blood reticulocytes by Retic-count (thiazole orange) reagent. (L-M) Flow cytometry analysis showing the frequencies of peripheral blood reticulocytes from WT, miR-144 KO, th3+/−, and miR-144 KO/th3+/− mice. The percentage of early-stage reticulocytes (R1) declines and mature RBCs (R3) increases in miR-144 KO/th3+/− blood compared with that in th3+/− mice. (I-M) All experiments were repeated at least 3 times. ∗P < .05; ∗∗P < .01. RDW, red cell distribution width; WT, wild-type; PB, peripheral blood; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

miR-144-Nrf2 regulatory axis plays a partial role in the alleviation of anemia in th3+/− mice. (A) Microarray transcriptome analysis of BM erythroblasts (Ter119+/CD71+/FSCHi) from th3+/− (βhet) and mKO/th3+/− (mKO/βhet) mice. Samples were analyzed for messenger RNA (mRNA) expression using Affymetrix Gene Chips. (B) Gene set enrichment analysis showing that Nrf2-related genes are upregulated in mKO/th3+/− BM erythroblasts compared with that in th3+/− BM erythroblasts. (C) Targetscan nucleotide sequence alignments showing the complementarity of 3' untranslated region (3′UTR) of Nrf2 mRNA and miR-144. There are 2 miR-144–binding sites in both mouse and human Nrf2 mRNAs. (D) Nrf2 mRNA levels in BM erythroblasts from mKO/th3+/− mice quantitated via real-time polymerase chain reaction. Experiments were repeated at least 3 times. (E) Western blot showing Nrf2 protein levels in BM erythroblasts from different groups of mice. Experiments were repeated at least 3 times. (F) Firefly luciferase reporter assay showing the direct binding of miR-144 to Nrf2 mRNA. 3′UTR of Nrf2 (WT) or its mutant version (mut, an 8-base pair replacement within the region complementary to miR-144 seed sequence) were cloned to luciferase reporter construct. Luciferase activities were determined 24 hours after transfection in 293T cells. (G-H) Flow cytometry analysis of the percentages of reticulocytes in peripheral blood from different group of mice. R1 (Ter119+CD71Hi), early-stage reticulocytes; R2 (Ter119+CD71Med), late-stage reticulocytes. (I) Automated hematology analysis revealing more RBCs in miR-144 KO/th3+/− blood and lower RDW in comparison to th3+/− mice. (J-K) Flow cytometry analysis of the percentages of peripheral blood reticulocytes by Retic-count (thiazole orange) reagent. (L-M) Flow cytometry analysis showing the frequencies of peripheral blood reticulocytes from WT, miR-144 KO, th3+/−, and miR-144 KO/th3+/− mice. The percentage of early-stage reticulocytes (R1) declines and mature RBCs (R3) increases in miR-144 KO/th3+/− blood compared with that in th3+/− mice. (I-M) All experiments were repeated at least 3 times. ∗P < .05; ∗∗P < .01. RDW, red cell distribution width; WT, wild-type; PB, peripheral blood; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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