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Loss of non-coding RNA expression from the DLK1-DIO3 imprinted locus correlates with reduced neural differentiation
Chu-Fan Mo1, Fang-Chun Wu2, Kang-Yu Tai3,4,5
1Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan. d98642005@ntu.edu.tw.
Introduction:
Pluripotent stem cells are increasingly used to build therapeutic models, including the transplantation of neural progenitors derived from human embryonic stem cells (hESCs). Recently, long non-coding RNAs (lncRNAs), including delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) imprinted locus-derived maternally expressed gene 3 (MEG3), were found to be expressed during neural development. The deregulation of these lncRNAs is associated with various neurological diseases. The imprinted locus DLK1-DIO3 encodes abundant non-coding RNAs (ncRNAs) that are regulated by differential methylation of the locus. We aim to study the correlation between the DLK1-DIO3-derived ncRNAs and the capacity of hESCs to differentiate into neural lineages.
Methods:
We classified hESC sublines into MEG3-ON and MEG3-OFF based on the expression levels of MEG3 and its downstream microRNAs as detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A cDNA microarray was used to analyze the gene expression profiles of hESCs. To investigate the capacity of neural differentiation in MEG3-ON and MEG3-OFF hESCs, we performed neural lineage differentiation followed by neural lineage marker expression and neurite formation analyses via qRT-PCR and immunocytochemistry, respectively. MEG3-knockdown via small interfering RNA (siRNA) and small hairpin RNA (shRNA) was used to investigate the potential causative effect of MEG3 in regulating neural lineage-related gene expression.
Results:
DLK1-DIO3-derived ncRNAs were repressed in MEG3-OFF hESCs compared with those in the MEG3-ON hESCs. The transcriptome profile indicated that many genes related to nervous system development and neural-type tumors were differentially expressed in MEG3-OFF hESCs. Three independent MEG3-knockdown assays using different siRNA and shRNA constructs consistently resulted in downregulation of some neural lineage genes. Lower expression levels of stage-specific neural lineage markers and reduced neurite formation were observed in neural lineage-like cells derived from MEG3-OFF-associated hESCs compared with those in the MEG3-ON groups at the same time points after differentiation.
Conclusions:
Repression of ncRNAs derived from the DLK1-DIO3 imprinted locus is associated with reduced neural lineage differentiation potential in hESCs.
Insights
Repression of maternally expressed gene 3 (MEG3) and other non-coding RNAs (ncRNAs) from the DLK1-DIO3 locus in human embryonic stem cells (hESCs) is linked to diminished neural differentiation capacity. This finding is crucial for understanding neurological development and disease.
Area of Science:
- Stem cell biology
- Developmental neuroscience
- Epigenetics
Background:
- Human embryonic stem cells (hESCs) are vital for modeling neurological diseases and developing cell-based therapies.
- Long non-coding RNAs (lncRNAs), such as maternally expressed gene 3 (MEG3) from the DLK1-DIO3 imprinted locus, play roles in neural development.
- Dysregulation of these lncRNAs is implicated in neurological disorders.
Purpose of the Study:
- To investigate the relationship between DLK1-DIO3-derived non-coding RNAs (ncRNAs) and the neural differentiation potential of hESCs.
- To determine if MEG3 expression levels correlate with the ability of hESCs to differentiate into neural lineages.
Main Methods:
- Classification of hESC sublines into MEG3-ON and MEG3-OFF groups based on MEG3 expression.
- Analysis of gene expression profiles using cDNA microarray.
- Assessment of neural differentiation capacity via marker expression and neurite formation.
- MEG3 knockdown using siRNA and shRNA to assess its causative role.
Main Results:
- DLK1-DIO3-derived ncRNAs were significantly lower in MEG3-OFF hESCs.
- Transcriptome analysis revealed differential expression of nervous system development genes in MEG3-OFF hESCs.
- MEG3 knockdown consistently downregulated neural lineage genes.
- MEG3-OFF hESCs exhibited reduced neural lineage marker expression and neurite formation post-differentiation.
Conclusions:
- Repression of ncRNAs from the DLK1-DIO3 imprinted locus is associated with impaired neural differentiation potential in hESCs.
- MEG3 may be a key regulator of neural lineage commitment in hESCs.
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