miRNAs are short non coding RNAs
miRNAs are short non-coding RNAs that can modulate gene expression by binding to the complementary sequences on target sites in the 3′ untranslated regions (UTRs) of messenger RNA (Bartel, 2004). There is strong evidence that miRNAs play an important role in the maintenance of ES cells (Pauli et al., 2011). In ES cells, a majority of the miRNA species are produced by 4 miRNA loci (the miR-21, the miR-17-92 cluster, the miR-15b-16 cluster and the miR-290-295 cluster) that regulate ionophores progression and oncogenesis (Calabrese et al., 2007). During differentiation, a number of miRNAs exhibit distinct expression patterns and have been shown to fine tune or restrict cellular identities by targeting important transcription factors or key pathways (Stefani and Slack, 2008). For example, miRNA-145 has been shown to repress Oct4, Sox2, and Klf4 during differentiation of human ES cells into mesoderm and ectoderm lineages (Xu et al., 2009). Furthermore, miR-134, miR-296, and miR-470 target the transcription factors Nanog, Oct4, and Sox2 to promote retinoic-acid-induced differentiation of mouse ES cells (Tay et al., 2008). The contribution of additional miRNAs involved in the direct repression of ES cell transcription factors during neuronal differentiation remains unknown. We searched for miRNAs that show increased expression levels during neuronal differentiation and are predicted to target ES cell genes. The expression of miR-137 has been found to increase during differentiation of neural stem cells into neurons (Sun et al., 2011b; Szulwach et al., 2010). Moreover, miR-137 is one of the two miRNAs in ES cells that are co-occupied by the key transcription factors: Oct4, Sox2, and Nanog (Boyer et al., 2005). These findings suggest that miR-137 may be an important component of the transcriptional regulatory circuitry, and that temporal expression of miR-137 may influence proliferation and differentiation of ES cells.
Here, we show that miR-137 is mainly expressed in ES cells at mitotic stages of the cell cycle and is significantly upregulated when ES cells are differentiated into neuronal lineage. We demonstrated that miR-137 directly targets the ES cell transcription factors, Klf4 and Tbx3, as well as a known miR-137 target, lysine specific demethylase 1 (LSD1) (Balaguer et al., 2010; Sun et al., 2011b). The repressive effect of miR-137 is mediated by its binding to the target sites present in the 3′ untranslated regions (3′UTRs) of the mRNAs. Furthermore, we found that miR-137 disrupts ES cell self-renewal and accelerates differentiation of ES cells in vitro.
Materials and methods
Discussion In the present study, we have shown that miR-137, a neuronally-enriched miRNA, is expressed in ES cells at the mitotic phase of the cell cycle and its expression is highly upregulated during differentiation into the neuronal linage. We uncovered a direct link between miR-137 and ES cell transcription factors, Klf4 and Tbx3. It appears that miR-137 has a dual function in ES cells depending on their expression status. While its expression in the mitotic phase indicates that miR-137 is involved in cell division, increased levels of the miRNA during differentiation appear to be involved in the maturation of ES cell-derived neurons. The data could indicate that the dynamic responses of miR-137 seem to be involved in mediating the balance between self-renewal and differentiation by regulating the expression levels of its direct targets, including Kfl4, Tbx3, and LSD1; and indirectly Nanog, Sox2, and possibly others. Our findings are consistent with the reports that miRNAs promote mouse ES cell differentiation through posttranscriptional attenuation of key ES cell transcription factors (Tay et al., 2008; Xu et al., 2009). miR-137 is a known regulator of neuronal development and its expression has been found to increase during neuronal differentiation (Ripke et al., 2011; Silber et al., 2008; Smrt et al., 2010; Sun et al., 2011a). This study demonstrates that miR-137 has a role in ES cell proliferation and differentiation. We have shown that miR-137 is expressed in ES cells in a cell cycle-dependent manner and is highly upregulated during differentiation of ES cells into neuronal linage. These results suggest that miR-137 may be involved in cell cycle progression or differentiation depending on the cellular context it is expressed in. This finding is consistent with previous reports, which show that miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells (Silber et al., 2008; Sun et al., 2011b). miR-137 has been shown to mediate differentiation by targeting many genes including CDK6, a regulator of the cell cycle and differentiation (Silber et al., 2008); Ezh2, a histone methyltransferase (Szulwach et al., 2010); Jarid1b, a histone H3 lysine 4 demethylase (Tarantino et al., 2010); Mib1, a ubiquitin ligase (Smrt et al., 2010); and TLX, an orphan nuclear receptor (Sun et al., 2011b). In this study, we showed that miR-137 is also able to target the 3′UTRs of Klf4 and Tbx3 and regulate self-renewal and differentiation of ES cells.