The Mechanisms MicroRNA In Embryonic Stem Cell Differentiation

Embryonic stem cells (ES cells) are of great value due to the characters of self-renewal and pluripotency. The status of ES cells is controlled by lif/STAT3, BMP4and WNT signaling pathways.Oct4, Sox2and Nanog are core transcript factors highly expressed in ES cells. MicroRNAs are short (about22nucleotides), non-coding RNAs that negatively regulate gene expression in the post-transcriptional level mainly through binding to the3’UTR of targets. Emerging evidences have shown that microRNAs play key roles in regulating various normal physiological processes. MicroRNAs are important in sustaining ES cell states, some evidences show that microRNAs regulate ES cells self-renewal and pluripotency. ESCs lacking of key enzymes in the miRNA biogenesis pathway, such as Dicer or Dgcr8, show deficiency in self-renewal and differentiation. A number of miRNAs have been reported to participate in regulating ESC self-renewal and differentiation, and promote iPSCs generation. ESCC miRNAs were identified by screening a comprehensive miRNA mimic library in Dgcr8deficient ESCs which overcomes issues of redundancy and saturation that are inherent to the miRNA system. However, miRNAs which can suppress self-renewal and may contribute to lineage commitment of ESCs have not been fully addressed. So, we aim to uncover novel miRNAs which can silence ESC self-renewal and to better understand the intricate regulatory mechanisms of ESC differentiation.Firstly, we evaluated the microRNA expression profile of ES cells and differentiated cells, and microRNAs that targeted important transcription factors for maintaining self-renewal, such as Oct4, Sox2, Nanog, Klf4, c-myc, Rexl, Sall4, Stella and Lin28, and identified40microRNAs that might promote ES cells differentiation. Secondly, we preformed function verification in dgcr8-/-ES cells that had no endogenous microRNAs. Systematically analyzing the data from colony formation assay, AP staining, cell cycle, gene expression pattern and Oct4staining, we ranked the miRNAs mainly based on their ability to silence ESC self-renewal. The top-ranking15miRNAs, such as let-7c, miR-129, miR-9, miR-124, miR-24, were considered to be important differentiation-promoting miRNAs which can silence ESC self-renewal evidently. Among them,7microRNAs, such as let-7c, miR-300, miR-24, miR-27a, miR-124, miR-27b, miR-129, can promote differentiation in wild type ES cells. We decided to further investigate the roles and molecular mechanisms of miR-27a and miR-24, which are the products from the same miR-23a～27a～24-2cluster, and possess similar properties to inhibit self-renewal of ESCs. MiR-27a and miR-24are are restrained by c-Myc in ESC and the down-regulation of c-Myc during differentiation increases miR-27a and miR-24rapidly. They exert their roles of silencing self-renewal through directly target important pluripotency transcription factors (Oct4, Foxo1) and signal transducers (gp130, Smads). Furthermore, these pluripotency-associated factors can directly or indirectly repress c-Myc, thus forming a mutual negative feedback loop to further reinforce the differentiated state. In addition, depleting miR-27a and/or miR-24in mouse embryonic fibroblasts (MEFs) promotes their reprogramming to induced pluripotent stem cells (iPSCs).In this study, we have conducted a comprehensive screening of differentiation-promoting microRNA, and get15microRNAs who promoted differentiation signicicantly. We further investigated the roles and molecular mechanisms of miR-27a and miR-24, and clarified the mechanism of these microRNAs in regulating differentiation. It should have great reference value in ES cells differentiation research.