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MicroRNA-22 promotes megakaryocyte differentiation through repression of its target, GFI1.

ABSTRACT: Precise control of microRNA expression contributes to development and the establishment of tissue identity, including in proper hematopoietic commitment and differentiation, whereas aberrant expression of various microRNAs has been implicated in malignant transformation. A small number of microRNAs are upregulated in megakaryocytes, among them is microRNA-22 (miR-22). Dysregulation of miR-22 leads to various hematologic malignancies and disorders, but its role in hematopoiesis is not yet well established. Here we show that upregulation of miR-22 is a critical step in megakaryocyte differentiation. Megakaryocytic differentiation in cell lines is promoted upon overexpression of miR-22, whereas differentiation is disrupted in CRISPR/Cas9-generated miR-22 knockout cell lines, confirming that miR-22 is an essential mediator of this process. RNA-sequencing reveals that miR-22 loss results in downregulation of megakaryocyte-associated genes. Mechanistically, we identify the repressive transcription factor, GFI1, as the direct target of miR-22, and upregulation of GFI1 in the absence of miR-22 inhibits megakaryocyte differentiation. Knocking down aberrant GFI1 expression restores megakaryocytic differentiation in miR-22 knockout cells. Furthermore, we have characterized hematopoiesis in miR-22 knockout animals and confirmed that megakaryocyte differentiation is similarly impaired in vivo and upon ex vivo megakaryocyte differentiation. Consistently, repression of Gfi1 is incomplete in the megakaryocyte lineage in miR-22 knockout mice and Gfi1 is aberrantly expressed upon forced megakaryocyte differentiation in explanted bone marrow from miR-22 knockout animals. This study identifies a positive role for miR-22 in hematopoiesis, specifically in promoting megakaryocyte differentiation through repression of GFI1, a target antagonistic to this process.

SUBMITTER: Weiss CN

PROVIDER: S-EPMC6325298 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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microRNA-22 promotes megakaryocyte differentiation through repression of its target, <i>GFI1</i>.

Weiss Cary N CN Ito Keisuke K

Blood advances 20190101 1

Precise control of microRNA expression contributes to development and the establishment of tissue identity, including in proper hematopoietic commitment and differentiation, whereas aberrant expression of various microRNAs has been implicated in malignant transformation. A small number of microRNAs are upregulated in megakaryocytes, among them is microRNA-22 (miR-22). Dysregulation of miR-22 leads to various hematologic malignancies and disorders, but its role in hematopoiesis is not yet well es ...[more]

PMID: 30617215

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Project description:Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3'UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/?-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.

Dataset Information

MicroRNA-22 promotes megakaryocyte differentiation through repression of its target, GFI1.

Publications

microRNA-22 promotes megakaryocyte differentiation through repression of its target, <i>GFI1</i>.

Similar Datasets

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