miR-294/miR-302 promotes proliferation, suppresses G1-S restriction point, and inhibits ESC differentiation through separable mechanisms

Abstract

The miR-294 and miR-302 microRNAs promote the abbreviated G1 phase of the embryonic stem cell (ESC) cell cycle and suppress differentiation induced by let-7. Here, we evaluated the role of the retinoblastoma (Rb) family proteins in these settings. Under normal growth conditions, miR-294 promoted the rapid G1-S transition independent of the Rb family. In contrast, miR-294 suppressed the further accumulation of cells in G1 in response to nutrient deprivation and cell-cell contact in an Rb-dependent fashion. We uncovered five additional miRNAs (miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218) that silenced ESC self-renewal in the absence of other miRNAs, all of which were antagonized by miR-294 and miR-302. Four of the six differentiation-inducing miRNAs induced an Rb-dependent G1 accumulation. However, all six still silenced self-renewal in the absence of the Rb proteins. These results show that the miR-294/miR-302 family acts through Rb-dependent and -independent pathways to regulate the G1 restriction point and the silencing of self-renewal, respectively.

Figure 1

miRNAs suppress the G1 restriction point in mouse ESCs. (A) Cell cycle profile of mock or miR-294 transfected Dgcr8 knockout and triple Rb, Dgcr8 knockout ES cells. Shown is flow cytometry analysis of propidium iodide stained cells. (B) Cell cycle profile of wild type and Dgcr8 knockout ESCs before and after serum starvation. (C) Fraction of cells in the G0/G1 phase for wild type and Dgcr8 knockout ESCs at increasing densities. (D) Cell cycle profile of Bak^−/−/Bax^−/flox ESCs before and after serum starvation. Representative experiments are shown here. All results shown as mean±s.d., n=3. See also Figures S1-S4.

Figure 2

ESCC miRNAs suppress the G1 restriction point in ESCs. (A) Cell cycle profile of mock and miR-294 transfected Dgcr8 knockout ESCs before and after serum starvation. (B) Fraction of cells in the G0/G1 phase for Dgcr8 knockout ESCs transfected with control mimics or miR-294 at increasing densities. Representative experiments are shown here. All results shown as mean±s.d., n=3. See also Figure S2.

Figure 3

The ESCC miRNAs act through the Rb pathway to suppress G1 restriction point in ESCs. (A) Quantatitive PCR analysis of Rb family genes in wild type and Dgcr8 knockout ESCs before and after serum starvation. Rpl7 gene was used as loading control. mRNA expression was normalized to wild type ESCs grown at standard culture conditions. Error bars indicate s.d. n=6. (B) mRNA expression of Rb family genes in mock and miR-294 transfected Dgcr8 knockout ESCs. Left panel shows the microarray result of cells in standard culture conditions (p<0.001). Right panel shows qPCR results in serum starved cells(Cdkn1a, Rbl1, and Rbl2, p<0.02; Rb1, p=0.31). Error bars indicate s.d. n=3. (C) Cell cycle profile of Rb family knockout ESCs before and after serum starvation. (D) Fraction of cells in the G0/G1 phase for Rb family knockout ESCs and controls at increasing densities. Representative experiments are shown here. All results shown as mean±s.d., n=3. See also Figure S6.

Figure 4

Screening identifies multiple miRNAs that silence ESC self-renewal in Dgcr8 knockout ESCs. (A) A bar graph depicting fraction of cells in G0/G1 for Dgcr8 knockout ESCs mock transfected, transfected with control mimics, or with let-7c in combination with control mimics or miR-294. Results shown as mean±s.d., n=3. (B) A schematic of the screening strategy. (C) miRNA screen data plotted for individual miRNAs with the error representing the range of scores for n = 3. (D) A scatterplot depicting the results for individual miRNAs based on miRNA array data in mouse ESCs 4 days after LIF withdrawal or in 1 micromolar all-trans retinoic acid (n = 3 for each condition). Red dots show miRNAs with a screen score greater than or equal to 6. (E) A heatmap depicting miRNA expression changes in mouse NPC and MEF relative to mouse ESC, human EB differentiation, and mouse –LIF and RA differentiation. MiRNAs labeled in green were previously implicated in ESC differentiation while those in red were chosen for further investigation in this study. See also Figure S7 and Tables S1 and S2.

Figure 5

ESCC miRNAs antagonize differentiation-inducing miRNAs from silencing ESC self-renewal. (A) Representative alkaline phosphatase staining in Dgcr8 knockout (i) and wild type (ii) ESCs after transfection with let-7c, miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218 alone or in combination with miR-294, mutant-miR-294 or miR-302b. (B) qRT-PCR for Pou5f1/Oct4, Sox2, and Nanog normalized first to beta-actin then to mock transfection after miRNA introduction as in A. Results shown as mean±s.d., n=2.

Figure 6

ESCC miRNAs act independently of the Rb pathway to antagonize other miRNAs from silencing ESC self-renewal. (A) A bar graph depicting fraction of cells in the G0/G1 phase after transfection of Dgcr8 knockout ESCs with let-7c, miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218 alone or in combination with miR-294. Mean±s.d. for n = 2-5. **, p<0.001. (B) A bar graph depicting fraction of cells in the G0/G1 phase after transfection of Dgcr8 knockout and Rb family knockout and Dgcr8 knockout ESCs with let-7c, miR-26a, miR-99b, miR-193, miR-199a-5p, miR-218, and miR-294. Mean±s.d. for n = 4. *, p<0.05. (C) qRT-PCR for Pou5f1/Oct4, Sox2, and Nanog normalized first to beta-actin then to mock transfection after miRNA introduction in (i) near Rb family and Dgcr8 knockout and (ii) Rb family and Dgcr8 knockout ESCs. n ≧ 3

Figure 7

ESCC miRNAs suppress the R point and silencing of self-renewal through different mechanisms. ESCC miRNAs suppress the R point and G1 accumulation induced by differentiation-inducing miRNAs through targeting the Rb pathway. However, knocking out Rb family proteins is not sufficient to prevent silencing of self-renewal by differentiation-inducing miRNAs. Therefore, other pathways must be regulated by ESCC miRNAs to antagonize silencing of self-renewal by differentiation-inducing miRNAs. Additionally, ESCC miRNAs regulate Rb-independent pathways to promote G1/S transition and proliferation at standard culture conditions, since triple knockout of Rb family proteins neither prevent G1 accumulation nor promote proliferation rate in Dgcr8 knockout ES cells.