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Cited by 31

The DOI matches its Crossref record; 49 of the 49 references it cites were found in a registry or an index.

Erythrocyte survival is controlled by microRNA-142

Rivkin, Natalia, Chapnik, Elik, Mildner, Alexander, Barshtein, Gregory, Porat, Ziv, Kartvelishvily, Elena, Dadosh, Tali, Birger, Yehudit, Amir, Gail, Yedgar, Saul, Izraeli, Shai, Jung, Steffen, Hornstein, Eran·2017·Haematologica
DOI 10.3324/haematol.2016.156109PMC PMC5395108
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Abstract

Hematopoietic-specific microRNA-142 is a critical regulator of various blood cell lineages, but its role in erythrocytes is unexplored. Herein, we characterize the impact of microRNA-142 on erythrocyte physiology and molecular cell biology, using a mouse loss-of-function allele. We report that microRNA-142 is required for maintaining the typical erythrocyte biconcave shape and structural resilience, for the normal metabolism of reactive oxygen species, and for overall lifespan. microRNA-142 further controls ACTIN filament homeostasis and membrane skeleton organization. The analyses presented reveal previously unappreciated functions of microRNA-142 and contribute to an emerging view of small RNAs as key players in erythropoiesis. Finally, the work herein demonstrates how a housekeeping network of cytoskeletal regulators can be reshaped by a single micro-RNA denominator in a cell type specific manner.

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Figures

Figure 1. Ineffective erythropoiesis in miR-142−/− animals, erythroid hyperplasia and anemia. Bar graphs are shown for measures of (A) red blood cell numbers (RBC), (B) hemoglobin (HGB), (C) hematocrit (HCT) and (D) red cell distribution width (RDW) values in six-month-old miR-142−/− mice and controls. (E) Reticulocytes (Retic) levels in three-month-old miR-142−/− mice and controls. (F) Upper panels, micrographs of miR-142−/− and WT peripheral blood smears, stained with Methylene blue, demonstrating residual RNA in reticulocytes (black arrows) and of May-Grunwald-Giemsa staining (lower panels), which depict abnormal morphology of miR-142−/− erythrocytes. Scale bars, 5 μm. Representative results from one of two independent experiments are shown (mean ± SEM) with at least five animals in each group. *P<0.05; **P<0.005; ***P<0.0005.
Figure 1. Ineffective erythropoiesis in miR-142−/− animals, erythroid hyperplasia and anemia. Bar graphs are shown for measures of (A) red blood cell numbers (RBC), (B) hemoglobin (HGB), (C) hematocrit (HCT) and (D) red cell distribution width (RDW) values in six-month-old miR-142−/− mice and controls. (E) Reticulocytes (Retic) levels in three-month-old miR-142−/− mice and controls. (F) Upper panels, micrographs of miR-142−/− and WT peripheral blood smears, stained with Methylene blue, demonstrating residual RNA in reticulocytes (black arrows) and of May-Grunwald-Giemsa staining (lower panels), which depict abnormal morphology of miR-142−/− erythrocytes. Scale bars, 5 μm. Representative results from one of two independent experiments are shown (mean ± SEM) with at least five animals in each group. *P<0.05; **P<0.005; ***P<0.0005.CC BY-NC · PubMed Central
Figure 2. Vulnerability of miR-142−/− erythrocytes to oxidative challenge and increased ROS levels. (A) Kaplan-Meier survival plot of WT (n=12) and miR 142−/−(n=13) mice, treated by two intraperitoneal injections of PHZ (48 mg/kg) on the first two days of the study (arrows). Bar graph of erythrocyte measures in peripheral blood of animal subjected to reduced PHZ dosing (single injection of 48 mg/kg, n=4 per group), including (B) blood cell numbers (RBC), (C) hemoglobin (HGB), (D) hematocrit (HCT), (E) red cell distribution width (RDW) values and (F) percentage of circulating reticulocytes (Retic). (G) In vitro hemolysis, measured as percentage of live RBCs in glass hemocytometer, 4 hrs after incubation with increasing phenylhydrazine (PHZ) concentrations. (H) ROS quantified by FACS of cells treated with CM-H2DCFDA and co-gated as Ter119HI and (I) bar graph depicting the percentage of erythroid cells with relatively high ROS, taken from top right plot quadrant and averaged for three independent experimental repeats from WT and miR-142−/− mice (n=3 per genotype). (J) In vivo eryhtorcyte lifespan, gated by streptavidin-Cy5 and Ter119HI antibodies in control and miR-142−/− mice, pulse-chased by Sulfo-NHS-Biotin labeling (n=4). *P<0.05; **P<0.005.
Figure 2. Vulnerability of miR-142−/− erythrocytes to oxidative challenge and increased ROS levels. (A) Kaplan-Meier survival plot of WT (n=12) and miR 142−/−(n=13) mice, treated by two intraperitoneal injections of PHZ (48 mg/kg) on the first two days of the study (arrows). Bar graph of erythrocyte measures in peripheral blood of animal subjected to reduced PHZ dosing (single injection of 48 mg/kg, n=4 per group), including (B) blood cell numbers (RBC), (C) hemoglobin (HGB), (D) hematocrit (HCT), (E) red cell distribution width (RDW) values and (F) percentage of circulating reticulocytes (Retic). (G) In vitro hemolysis, measured as percentage of live RBCs in glass hemocytometer, 4 hrs after incubation with increasing phenylhydrazine (PHZ) concentrations. (H) ROS quantified by FACS of cells treated with CM-H2DCFDA and co-gated as Ter119HI and (I) bar graph depicting the percentage of erythroid cells with relatively high ROS, taken from top right plot quadrant and averaged for three independent experimental repeats from WT and miR-142−/− mice (n=3 per genotype). (J) In vivo eryhtorcyte lifespan, gated by streptavidin-Cy5 and Ter119HI antibodies in control and miR-142−/− mice, pulse-chased by Sulfo-NHS-Biotin labeling (n=4). *P<0.05; **P<0.005.CC BY-NC · PubMed Central
Figure 3. miR-142−/− erythrocytes exhibit defective morphology. (A) Scanning electron microscopy reveals the stereotypic biconcave form of WT erythrocytes and atypical morphology of miR-142−/− erythrocytes (knizocytes, leptocytes). Micrograph scales noted in panels. (B) Quantification of cell surface area, (C) percentage of cells with abnormal morphology or (D) structural membrane changes in miR-142−/− (n=3) and WT cells (n=3). Representative results from one of two independent experiments (mean ± SEM) with >100 cells per group. *P<0.05; ***P<0.0005. AU: arbitrary unit.
Figure 3. miR-142−/− erythrocytes exhibit defective morphology. (A) Scanning electron microscopy reveals the stereotypic biconcave form of WT erythrocytes and atypical morphology of miR-142−/− erythrocytes (knizocytes, leptocytes). Micrograph scales noted in panels. (B) Quantification of cell surface area, (C) percentage of cells with abnormal morphology or (D) structural membrane changes in miR-142−/− (n=3) and WT cells (n=3). Representative results from one of two independent experiments (mean ± SEM) with >100 cells per group. *P<0.05; ***P<0.0005. AU: arbitrary unit.CC BY-NC · PubMed Central
Figure 4. Disturbed cytoskeletal architecture in the absence of miR-142. (A) Representative single-cell images of RBCs, obtained by ImageStreamX flow cytometer. Bright-field (BR, gray), Ter119HI (yellow), Alexa Fluor 647-Phalloidin (F-actin, red), CD71Neg (green) and HoechstNeg (purple). Scale bar, 7 μm. (B) Decreased ACTIN polarity, (C) ACTIN position in the cell and (D) low circularity in miR-142−/− RBCs relative to WT controls revealed by ImageStreamX analysis. (E) Super-resolution microscopy images of F-ACTIN depth in RBCs, top and side view. Scale bar, 2 μm. Three animals per group (mean ± SEM). *P<0.05; **P<0.005; ***P<0.0005. AU: arbitrary unit.
Figure 4. Disturbed cytoskeletal architecture in the absence of miR-142. (A) Representative single-cell images of RBCs, obtained by ImageStreamX flow cytometer. Bright-field (BR, gray), Ter119HI (yellow), Alexa Fluor 647-Phalloidin (F-actin, red), CD71Neg (green) and HoechstNeg (purple). Scale bar, 7 μm. (B) Decreased ACTIN polarity, (C) ACTIN position in the cell and (D) low circularity in miR-142−/− RBCs relative to WT controls revealed by ImageStreamX analysis. (E) Super-resolution microscopy images of F-ACTIN depth in RBCs, top and side view. Scale bar, 2 μm. Three animals per group (mean ± SEM). *P<0.05; **P<0.005; ***P<0.0005. AU: arbitrary unit.CC BY-NC · PubMed Central
Figure 5. Disturbed RBC deformability and osmotic fragility of miR-142−/−. (A) RBC deformability is described by the elongation ratio (ER) distribution in miR-142−/− (n=4) and WT (n=4) cells under shear stress of 3.0 Pa. miR-142−/− curve is shifted to the right, meaning that mutant cells are more deformable than control erythrocytes. (B) miR-142−/− osmotic fragility curve is shifted to the left of a typical WT curve. miR-142−/− RBCs are less susceptible to decreased osmotic pressure and undergo lysis only after greater reduction in NaCl concentrations, relative to WT cells. n=4 WT and 4 miR-142−/− experimental repeats. *P<0.05. RBC: red blood cell; NaCI: sodium chloride.
Figure 5. Disturbed RBC deformability and osmotic fragility of miR-142−/−. (A) RBC deformability is described by the elongation ratio (ER) distribution in miR-142−/− (n=4) and WT (n=4) cells under shear stress of 3.0 Pa. miR-142−/− curve is shifted to the right, meaning that mutant cells are more deformable than control erythrocytes. (B) miR-142−/− osmotic fragility curve is shifted to the left of a typical WT curve. miR-142−/− RBCs are less susceptible to decreased osmotic pressure and undergo lysis only after greater reduction in NaCl concentrations, relative to WT cells. n=4 WT and 4 miR-142−/− experimental repeats. *P<0.05. RBC: red blood cell; NaCI: sodium chloride.CC BY-NC · PubMed Central
Figure 6. miR-142 activity is required for normal RBC protein expression. (A) Western blots of RBC proteins probed with the indicated antibodies. Each panel is representative of results from >5 blots. (B) Bar graph represents the ratio of SPECTRIN/GAPDH. (C) GRLF1/GAPDH. (D) ADUCTIN/GAPDH. (E) ACTIN/GAPDH. (F) BAND3/GAPDH. (G) ANKYRIN/GAPDH. (H) COFILLIN/GAPDH. (I) WASL/GAPDH. *P<0.05.
Figure 6. miR-142 activity is required for normal RBC protein expression. (A) Western blots of RBC proteins probed with the indicated antibodies. Each panel is representative of results from >5 blots. (B) Bar graph represents the ratio of SPECTRIN/GAPDH. (C) GRLF1/GAPDH. (D) ADUCTIN/GAPDH. (E) ACTIN/GAPDH. (F) BAND3/GAPDH. (G) ANKYRIN/GAPDH. (H) COFILLIN/GAPDH. (I) WASL/GAPDH. *P<0.05.CC BY-NC · PubMed Central
Figure 7. miR-142 regulates ACTIN cytoskeletal architecture and dynamics in K-562 cells. Expression of miR-142 (A) and its targets (B) in K-562 cells transfected with a knockdown (KD) miR-ZIP-142 vector. Representative micrographs, obtained with ImageStreamX flow cytometer and stained with Alexa Fluor 647-Phalloidin (F-actin, red), and Hoechst (blue, C). Scale bar, 7 μm. Decreased F-ACTIN polarity (D), increased cell area (E) and decreased circularity (F) in K-562 cells after miR-142 knockdown, relative to WT controls. K-562 transduced with miR-ZIP-142 knockdown vector and concomitantly with a lentivirus encoding a shRNA directed against RFP (shRFP) or a set of shRNA vectors as indicated. ImageStreamX flow cytometery that was performed 72 hrs later revealed that knockdown of targets partially restored F-ACTIN polarity (G) and circularity (H). Representative results from one of two independent experiments (mean ± SEM), three experimental repeats per group. *P<0.05; **P<0.005; ***P<0.0005. AU: arbitrary unit; n.s: non significant; sh: short hairpin; RFP: red fluorescent protein.
Figure 7. miR-142 regulates ACTIN cytoskeletal architecture and dynamics in K-562 cells. Expression of miR-142 (A) and its targets (B) in K-562 cells transfected with a knockdown (KD) miR-ZIP-142 vector. Representative micrographs, obtained with ImageStreamX flow cytometer and stained with Alexa Fluor 647-Phalloidin (F-actin, red), and Hoechst (blue, C). Scale bar, 7 μm. Decreased F-ACTIN polarity (D), increased cell area (E) and decreased circularity (F) in K-562 cells after miR-142 knockdown, relative to WT controls. K-562 transduced with miR-ZIP-142 knockdown vector and concomitantly with a lentivirus encoding a shRNA directed against RFP (shRFP) or a set of shRNA vectors as indicated. ImageStreamX flow cytometery that was performed 72 hrs later revealed that knockdown of targets partially restored F-ACTIN polarity (G) and circularity (H). Representative results from one of two independent experiments (mean ± SEM), three experimental repeats per group. *P<0.05; **P<0.005; ***P<0.0005. AU: arbitrary unit; n.s: non significant; sh: short hairpin; RFP: red fluorescent protein.CC BY-NC · PubMed Central

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Research works printed but not linked: 1

  1. [22]Kim M Tan YS Cheng WC Kingsbury TJ Heimfeld S Civin CI MIR144 and MIR451 regulate human erythropoiesis via RAB14. Br J Haematol. 2015; 168(4): 583– 597. 25312678 PMC4314389

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Metadata

Authors
Rivkin, N., Chapnik, E., Mildner, A., Barshtein, G., Porat, Z., Kartvelishvily, E., Dadosh, T., Birger, Y., Amir, G., Yedgar, S., Izraeli, S., Jung, S., & Hornstein, E.
Venue
Haematologica
Publisher
Ferrata Storti Foundation (Haematologica)
Published
2016-12-01
Volume
102
Issue
4
Pages
676-685
ISSN
  • 0390-6078
  • 1592-8721
DOI
10.3324/haematol.2016.156109
PMC
PMC5395108
Cited by
31
References
49
Language
en

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