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Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells
Published on: December 27, 2017
circFUT8 promotes proplatelet formation by interacting with IGF2BP2 and stabilizing TNS1 mRNA in megakaryocytes
Huang Wu1, Yao Lu1, Denglian Sun1
1Department of Transfusion Medicine, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University, Chongqing, China.
Abstract:
During thrombopoiesis, megakaryocytes (MKs) transform their cytoplasm into proplatelets through complex cytoskeletal rearrangements. The shear force of blood flow releases newly formed platelets from the proplatelets into the bloodstream. Defects at any phase of this process can impair platelet production. Although various noncoding RNAs have been identified as regulators of platelet production, the regulatory mechanisms of thrombopoiesis remain to be further investigated. Despite the high abundance of circular RNAs (circRNAs) in platelets, their role in platelet production is unclear. In this study, using RNA sequencing and bioinformatics analysis, we identified a circular RNA from the FUT8 gene (circFUT8) as a novel circRNA that increases as hematopoietic stem cells from human umbilical cord blood differentiate into mature MKs, showing high expression in these mature cells. Knockdown of circFUT8 led to diminished proplatelet formation (PPF) and abnormal demarcation membrane system formation in human cultured MKs. In addition, inhibition of circFut8 in vivo decreased murine platelet counts. circFut8 deficiency reduced the number of MKs in contact with sinusoids. Mechanistically, we revealed that circFUT8 interacts with insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 2 to stabilize tensin-1 (TNS1) mRNA in an m6A-dependent manner. In human cultured MKs, TNS1 knockdown resulted in defective filamentous actin polymerization and assembly, impaired spreading on extracellular matrix proteins, and decreased PPF. Taken together, our research reveals the crucial functions of circRNAs in platelet production and has significant implications for the development of therapeutic strategies for thrombocytopenia and bleeding disorders.
Insights
Circular RNAs regulate platelet production. This study identifies circFUT8 as crucial for megakaryocyte differentiation and proplatelet formation, offering therapeutic potential for bleeding disorders.
Area of Science:
- Hematology
- Molecular Biology
- RNA Biology
Background:
- Thrombopoiesis involves megakaryocyte (MK) differentiation and proplatelet formation, essential for platelet production.
- Non-coding RNAs, including circular RNAs (circRNAs), are implicated in platelet production, but their specific roles are largely unknown.
- circRNAs are abundant in platelets, yet their function in thrombopoiesis requires elucidation.
Purpose of the Study:
- To investigate the role of circRNAs in megakaryocyte differentiation and platelet production.
- To identify novel circRNAs involved in thrombopoiesis.
- To elucidate the molecular mechanisms by which circRNAs regulate megakaryocyte function.
Main Methods:
- RNA sequencing and bioinformatics analysis to identify circRNAs in differentiating hematopoietic stem cells and MKs.
- In vitro knockdown of circFUT8 in human cultured MKs to assess its effect on proplatelet formation (PPF) and demarcation membrane system (DMS) development.
- In vivo inhibition of circFut8 in mice to evaluate its impact on platelet counts and MK localization.
- Mechanistic studies involving RNA immunoprecipitation and mRNA stability assays to determine circFUT8 interactions and targets.
Main Results:
- circFUT8 was identified as a novel circRNA upregulated during MK differentiation.
- Knockdown of circFUT8 impaired PPF and DMS formation in human MKs and reduced platelet counts in mice.
- circFUT8 deficiency led to fewer MKs in contact with sinusoids.
- circFUT8 was found to interact with IGF2BP2, stabilizing TNS1 mRNA in an m6A-dependent manner.
- TNS1 knockdown resulted in defective F-actin polymerization, impaired cell spreading, and reduced PPF.
Conclusions:
- circFUT8 plays a critical role in megakaryocyte differentiation and proplatelet formation.
- The circFUT8/IGF2BP2/TNS1 axis is a key regulatory pathway in thrombopoiesis.
- circRNAs represent important regulators of platelet production with therapeutic implications for thrombocytopenia and bleeding disorders.
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