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Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos
Published on: October 9, 2020
MicroRNA-125b/Lin28 pathway contributes to the mesendodermal fate decision of embryonic stem cells
1Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Abstract:
MicroRNAs (miRNAs) are important regulators of cell fate decisions, while the miRNAs and their targets in the regulation of stem cell differentiation are largely unidentified. Here we report novel functions of miR-125b/Lin28 axis in the regulation of mouse embryonic stem cell (mESC) lineage specification and cardiomyocyte differentiation. With a MicroRNA Array screen, we identified a number of miRNAs significantly changed during ESC differentiation, among which miR-125b showed a marked reduction during early differentiation. The abundantly expressed miR-125b in undifferentiated mESCs was dramatically downregulated to a level hardly detected during differentiation day 3 to 5, with a concomitant upregulation of Lin28. Ectopically expressing miR-125b did not alter characteristics of undifferentiated mESCs, whereas it impaired the endoderm and mesoderm development, but not the ectoderm, and inhibited cardiomyocyte formation. We further demonstrate that miR-125b targeted the 3'-untranslated region of Lin28 and reduced the abundance of Lin28 at both mRNA and protein levels. Moreover, phenotypes of miR-125b overexpressing cells were mimicked by downregulation of Lin28 and rescued by reintroduction of Lin28. In addition, the impaired cardiogenesis in miR-125b-introduced cells was greatly recovered when mimicking endoderm environment by cultivation with the condition medium from a visceral endoderm-like cell line, END-2. These results reveal that the miR-125b/Lin28 axis is an important regulator of early lineage specification and cardiomyocyte differentiation of ESCs.
Insights
The miR-125b/Lin28 axis regulates mouse embryonic stem cell (mESC) differentiation. Upregulating miR-125b inhibits endoderm, mesoderm, and cardiomyocyte development by downregulating Lin28.
Area of Science:
- Stem cell biology
- Molecular and developmental biology
Background:
- MicroRNAs (miRNAs) are key regulators of cell fate, but their roles in stem cell differentiation are not fully understood.
- Identifying specific miRNAs and their targets is crucial for understanding stem cell differentiation processes.
Purpose of the Study:
- To investigate the function of the miR-125b/Lin28 axis in mouse embryonic stem cell (mESC) lineage specification and cardiomyocyte differentiation.
- To elucidate the regulatory mechanism of miR-125b and Lin28 in early embryonic stem cell development.
Main Methods:
- MicroRNA Array screening to identify differentially expressed miRNAs during ESC differentiation.
- Overexpression and downregulation experiments of miR-125b and Lin28 in mESCs.
- Analysis of lineage-specific differentiation and cardiomyocyte formation.
- mRNA and protein level analysis of Lin28.
Main Results:
- miR-125b expression significantly decreased during early mESC differentiation, inversely correlating with Lin28 upregulation.
- Ectopic miR-125b expression inhibited endoderm and mesoderm development, and cardiomyocyte differentiation, but not ectoderm.
- miR-125b directly targets Lin28, reducing its mRNA and protein levels. Lin28 downregulation mimicked miR-125b overexpression phenotypes, which were rescued by Lin28 reintroduction.
- Impaired cardiogenesis due to miR-125b was partially rescued by mimicking an endoderm environment.
Conclusions:
- The miR-125b/Lin28 axis plays a critical role in regulating early lineage specification and cardiomyocyte differentiation in ESCs.
- This axis provides a novel regulatory mechanism controlling stem cell fate decisions during early development.
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