Ccar1 as A Partner of Β-Catenin Correlates with Ground-State Pluripotency in Mescs

Background: Dual inhibition of extracellular signal-regulated kinase (MEK) and transforming growth factor beta (TGFβ), known as R2i, helps to maintain the mouse embryonic stem cells (mESC) under-ground state condition. To understand the molecular mechanisms of R2i, in our previous study we in-vestigated the proteome profile of mES cells that was cultured under R2i condition. According to the proteomics data, the pro-tein expression level of the cell cycle and apoptosis regulator 1 (Ccar1) as a partner of β-catenin, was significantly upregulated in R2i culture.Materials and Methods: In this experimental study, we used siRNA against Ccar1 and then we analyzed β-catenin localiza-tion and its target genes expression by immunofluorescence as-say and qRT-PCR, respectively.Results: The Immunofluorescence data showed that the reduc-ing Ccar1 level by siRNA influenced the β-catenin translocation. The qRT-PCR analysis revealed significant downregulation of the pluripotency genes as well as some cell cycle markers such as Ccar1, c-myc and Tbx3 in the siRNA treated cells. In addi-tion, the expression level of the Wnt target genes such as Cdx1, Wnt3a, Tbx1, Fgf4, Apc and Cdh1 was significantly upregu-lated by the knockdown of Ccar1.Conclusion: In conclusion, upregulating of Ccar1 under R2i culture condition, can prevent β-catenin nuclear translocation and maintains the pluripotency and self-renewal of mESCs.

Keywords:

MESCs , Pluripotency , β-catenin , Ccar1 , Wnt Signaling