| Embryonic stem cells (ES cells) have two defining properties, self-renewal and pluripotency, which makes them very attractive in clinic. Fully understanding of the mechanism involved in the pluripotency and early differentiation is essential for achieving these goals. Nanog, a homeodomain-containing transcription factor, was found at 2003 and specially expressed in pluripotency cells. Nanog plays important roles in ES cells pluripotency, early differentiation and somatic cells dedifferentiation. But its mechanism is not well elucidated. Here, we aimed to illustrate the mechanism of Nanog in embryonic stem cells by studying the target genes, the related signaling pathway and the effect in the somatic cells dedifferentiation.The purpose of the first part of this paper is to investigate the target genes which be regulated by Nanog. Nanog, as a transcription factor, produces effects by regulating target genes expression. The information about these target genes is helpful for us to well known the mechanism of Nanog in embryonic stem cells self-renewal. Here, we aim to investigate the genes possibly participating in the course of self-renewal regulated by Nanog by combining RNA interference and microarray detection method. In order to down-regulate Nanog expression efficiently, four siRNAs were designed on the basis of the conserved Nanog sequence and their effects on the Nanog expression were tested by Western-Blot and Real-Time PCR. Among these four siRNAs, Nanog-siRNA-P1 was found to be most effective. The interference efficience was up to 90%. Once Nanog was down-regulated, ES cells underwent differentiation by showing morphological change and decreased proliferation rate. Microarray analysis was then used to identify the altered gene expression at 24 or 48 hours after Nanog was silenced. These changed genes in our experiment were considered as the result of Nanog downregulation, and this would help us to known better the mechanism of Nanog in the pluripotency of ES cells. In the second part, we investigated the effect of PI3K signaling transduction pathway on ES cells self-renewal and Nanog expression. We found that LY294002, a specific inhitor of PI3K pathway, induced mouse ES cells differentiation and decreased Nanog expression. To investigate the mechanism of PI3K signaling on ES cells self-renewal, we constructed a Nanog-GFP fusion protein expression plasmid, and then trasfected ES cells. After G418 selection, we got Nanog-overexpressing ES cells (Ex-Nanog-J1). Exogenous Nanog sustained mouse ES cells pluripotency independent of LIF, and alleviates the differentiation induced by LY294002. But it was insufficient to totally reverse the differentiation.In the third part, we tried to investigate the effect of Nanog in somatic cells dedifferentiation (reprogramming). We all know that ES cells have the ability to differentiate into all kind cells and tissues of three germinal layers. And, somatic cells can be dedifferentiated to stem cells by cell fusion and somatic cells nuclear transfer (SCNT). Nanog is expressed specifically in embryonic stem cells, and plays an important role in embryonic stem cells self-renewal. Then, we want to know the effect of Nanog in somatic cells dedifferentiation. To investigate the effect of Nanog in somatic cells dedifferentiation, we constructed Nanog-GFP fusion protein expression vector and then transfected it to 3T3 cells. After G418 selection, we got Nanog-expressing 3T3 cells. We observed that Nanog was located in 3T3 cells nuclear, which was similar with the location of Nanog in embryonic stem cells. Then we investigated if the 3T3 cells with Nanog-overexpression could dedifferentiate. There was no significant differentiation in the cells morphology. Cell cycle results showed that S phase was increased and G0/G1 phase and M phase were decreased in 3T3 cells with Nanog expression compared with wild-type 3T3 cells. Furthermore, we observed that Oct4 expressed in 3T3 cells with exogenous Nanog. But, other ES cells specific genes, such as Sox2, Utf1, and Ets2 were not detected in 3T3 cells with Nanog overexpression. All of these evidences indicated that Nanog might take part in the somatic cells dedifferentiation. But only exogenous Nanog was insufficient to promote somatic cells dedifferentiated totally.This investigation shows that Nanog plays a key role in ES cells self-renewal. Nanog, as a transcription factor, sustains ES cells undifferentiated state by activating pluripotency related genes expression and repressing differentiation related genes expression. PI3K signaling pathway plays a significant role in embryonic stem cells self-renewal. LY294002, a specific inhibitor of PI3K signaling, induced ES cells differentiation. Exogenous Nanog alleviates the differentiation induced by LY294002. But it's insufficient to totally reverse the differentiation. So, we think that the PI3K-dependent regulation of ES cells self-renewal is partially mediated by regulated Nanog expression. Nanog is helpful for somatic cells dedifferentiation but only Nanog overexpression was insufficient to induced somatic cells to pluripotency cells.
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