MicroRNAs (or miRNAs) are small noncoding RNAs (21 to 25 nucleotides) that are processed from longer hairpin RNA precursors and are believed to be involved in a wide range of developmental and cellular processes, by either repressing translation or triggering mRNA degradation (RNA interference). By using a computer-assisted approach, we have identified 46 potential miRNA genes located in the human imprinted 14q32 domain, 40 of which are organized as a large cluster. Although some of these clustered miRNA genes appear to be encoded by a single-copy DNA sequence, most of them are arranged in tandem arrays of closely related sequences. In the mouse, this miRNA gene cluster is conserved at the homologous distal 12 region. In vivo all the miRNAs that we have detected are expressed in the developing embryo (both in the head and in the trunk) and in the placenta, whereas in the adult their expression is mainly restricted to the brain. We also show that the miRNA genes are only expressed from the maternally inherited chromosome and that their imprinted expression is regulated by an intergenic germline-derived differentially methylated region (IG-DMR) located ∼200 kb upstream from the miRNA cluster. The functions of these miRNAs, which seem only conserved in mammals, are discussed both in terms of epigenetic control and gene regulation during development.

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.