MicroRNA-200a regulates Grb2 and suppresses differentiation of mouse embryonic stem cells into endoderm and mesoderm

Abstract

The mechanisms by which microRNAs (miRNAs) affect cell fate decisions remain poorly understood. Herein, we report that miR-200a can suppress the differentiation of mouse embryonic stem (ES) cells into endoderm and mesoderm. Interestingly, miR-200a directly targets growth factor receptor-bound protein 2 (Grb2), which is a key adaptor in the Erk signaling pathway. Furthermore, high levels of miR-200a dramatically decrease Grb2 levels and suppress the appearance of mesoderm and endoderm lineages in embryoid body formation, as well as suppressing the activation of Erk. Finally, Grb2 supplementation significantly rescues the miR-200a-induced layer-formation bias and the Erk suppression. Collectively, our results demonstrate that miR-200a plays critical roles in ES cell lineage commitment by directly regulating Grb2 expression and Erk signaling.

Figure 1. Effects of miR-200a in ES cells and ES cell differentiation.

(A) The expression of miR-200a, miR-200b and miR-429 in ES cell diferentiation. (B) Brightfield images and alkaline phosphatase staining of ES cells without LIF at 72 hours post-transfection with miRNA-200a. The scale bar represents 100 µm. (C) Relative levels of Oct4, Nanog, Sox2 and Rex1 mRNA in control or miR-200a-transfected ES cells. (D) Representative immunofluorescence images of control and miR-200a overexpression after 10 days of EB formation. Red, layer markers; blue, nuclei. The scale bar represents 100 µm. (E) Expression levels of genes associated with the differentiated state in EBs in response to miR-200a expression. All data are shown as the means ± SD. Statistical significance was assessed by the two-tailed Student’s t test. ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Figure 2. miR-200a targets Grb2 at the protein level.

(A) Outline of the interaction of miR-200a with the Grb2 3’ UTR. (B) Repression of luciferase activity validates the interaction between miR-200a and the specific predicted sites in the Grb2 3’ UTR. (C) Overexpression of miR-200a downregulates Grb2 expression, and sponge 200a rescues Grb2 expression in ES cells. (D) The expression of Grb2 in ES cell diferentiation. All data are shown as the means ± SD. Statistical significance was assessed by the two-tailed Student’s t test. **, p < 0.01.

Figure 3. Knockdown of Grb2 during EB formation.

(A) Western blotting analysis demonstrates that shGrb2 knocks down Grb2 protein levels in ES cells. (B) Relative levels of layer markers are varied in shGrb2-treated EBs. (C) The in situ expression of layer markers in day 10-differentiated EBs. Red, layer markers; blue, nuclear DNA staining. The scale bar represents 100 µm. (D) Erk activity decreases in response to Grb2 knockdown. All data are expressed as the means ± SD. Statistical significance was assessed by the two-tailed Student’s t test. ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Figure 4. Grb2 can rescue cells from the effects of miR-200a.

(A) Brightfield images and AP staining of wild-type ES cells, miR-200a-expressing ES cells and exogenous Grb2-expressing ES cells plus expressing miR-200a. The scale bar represents 100 µm. (B) Expression levels of genes associated with the differentiated state in EBs. (C) Immunofluorescence shows that Grb2 reversed the miR-200a-induced failures in endoderm and mesoderm differentiation. The scale bar represents 100 µm. (D) Erk activity can be rescued by Grb2 in miR-200a-expressing ES cells. All data are expressed as the means ± SD. Statistical significance was assessed by one-way ANOVA, followed by Tukey’s post-test. ***, p < 0.001; **, p < 0.01; *, p < 0.05.