MicroRNA-200a Regulates Grb2and Suppresses Differentiation Of Mouse Embryonic Stem Cells Into Endoderm And Mesoderm

Embryonic stem cell (ESC) is derived from the inner cell mass (ICM) of blastocysts,and can maintain unlimited symmetrical self-renewal and the capacity for differentiationinto multilineage. They enables development into all types of cells in the body. Theresearches of ESC show a wide range of application prospects in research of development,tissue regeneration, organ reconstruction, disease research, cell-based therapy and drugdiscovery.In early embryogenesis, ESC generates the three germ layers including endoderm,mesoderm and ectoderm, as well as germ cells. These mass of cells will construct thedifferent cell fates towards some kinds of tissue and organ.Endoderm forms the gastrointestinal tract, respiratory tract, intestine epithelial andendocrine glands. Mesoderm forms the blood, lymph, bone, muscle and mesenchyme.Ectoderm forms the epidermis and the nervous system. In this process, the mechanism inlayer formation is not clear. This work tends to reveal a regulation of it.miRNA plays an essential role in fate decisions in embryonic development, but itparticipates layer decision needs to be explained completely. miRNA is a class ofnon-coding RNA which is length in20-25nucleotide. It has the capable of specificallytarget at3’untranslated regions of mRNA, leading to degradation of the target mRNA orrepression of the target mRNA translation. We found that miR-200a expression wassignificantly decreased in the process of differentiation of embryonic stem cells.Exogenous expression of miR-200a results in suppression of endoderm and mesodermdifferentiation in mouse embryonic stem cells. This suggested that miR-200a was involvedin three layers differentiation.With predicting in bioinformatics websites, we found that thegrowth factor receptor-bound protein2(Grb2) will be miR-200a target for its3’untranslated regions exist binding sites. Dual luciferase reporter and proteinimmunoblotting proved that miR-200a significantly down-regulated Grb2expression inESC. On the other hand, knockdown of Grb2showed the same phenotype inoverexpression of miR-200a, supressing endoderm and mesoderm. This indicated that thebiological effects in miR-200a and Grb2are relevant.Erk signaling pathway plays an important role in the specific differentiation ofembryonic stem cells and germ layers. Low levels of activity in Erk miantains self-renewal and impedes differentiation in ESC, and reinforcement in Erk activity prompted theprimitive endoderm differentiation of ESC. It is clear that Erk signaling pathway mediatedby the adapter molecule Grb2to transmiss signaling. While knockdown of Grb2exerted inESC, the Erk activity came down. Exogenous overexpression of miR-200a had the sameeffect. But exogenous Grb2-expression plus expressing miR-200a restored the effects ofmiR-200a in Erk activity. Totally, miR-200a targets Grb2mRNAand inhibit translation,and then adjusts the activation of the Erk. miR-200a is involved in endoderm andmesoderm development in mouse ESC fate decisions.In summary, the study found that miR-200a regulated Erk signaling pathway by Grb2,thus regulated endoderm and mesoderm development. It revealed miR-200a as regulater inthe differentiation of ESC, and further clarified the molecular mechanisms in Erk signalingpathway in the early developmental events.