Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation

Abstract

Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1-S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells. Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.

Figure 1

Screening for miRNAs that rescue the proliferation defects of Dgcr8 Δ/Δ ES cells. (a) Screening strategy. Proliferation of ES cells transfected with individual miRNA mimics was first evaluated by the MTT assay. The positive hits were then assessed for their ability to rescue the G1 accumulation defects of Dgcr8 Δ/Δ ES cells. (b) Z-scores for individual miRNA mimics. Shown are average Z-scores from triplicates. Error bar indicates the range of triplicates. (c) Top 14 positive hits with Z-score > 3 (P value < 0.001). The growth rate was normalized to mock transfected DGCR8 Δ/Δ ES cells. (d) 11 positive hits share similar seed sequence. Seed sequences are highlighted in gray box.

Figure 2

Rescue of proliferation and G1 accumulation defects by representative miRNAs. (a) Resynthesized screen positive miRNAs promoted Dgcr8 null ES cell proliferation. Growth rates were normalized to mock transfected Dgcr8 Δ/Δ ES cells. miR-1 served as a control. Error bars represent s.d. n=6. (b) ES cell specific miRNAs rescued G1 accumulation of Dgcr8 Δ/Δ ES cells. Shown is flow cytometry analysis of propidium iodide stained cells. Results are shown as means±s.d except for miR-1, for which is shown as mean±range. n=2 for miR-1. n=4 or 5 for rest of samples. *, P value < 0.01; **, P value < 0.001. The P value was calculated based on the 2-tailed t-test.

Figure 3

Expression of Cdkn1a mRNA and protein upon introduction of ESCC miRNAs. (a) Quantatitive PCR. mRNA expression was normalized to mock transfected Dgcr8 Δ/Δ ES cells. Error bars indicate s.d except for miR-1, for which the error bar indicates range. n=2 for miR-1. n=3 for wild type and n=4 for ESCC miRNAs. (b) Representative Western blot of Cdkn1a protein. (c) Densitometry for Western analysis of Cdkn1a protein. Data was normalized to mock transfected Dgcr8 Δ/Δ ES cells. Numbers of independent transfection experiments performed are indicated on the top of each bar. Error bars indicate the range.

Figure 4

Luciferase reporter assay indicates that ESCC miRNAs directly interact with 3' UTR of Cdkn1a. (a) Firefly luciferase reporter constructs. The 3' UTR of Cdkn1a (∼1.3 kb) was cloned downstream of firefly in the pGL3-control vector. Two predicted targeting sites and the corresponding mutations are listed. All experiments were normalized to cotransfected renilla luciferase. (b) Relative luciferase activity between wild-type and Dgcr8 knockout cells following transfection of wild-type and mutant reporters. Error bars indicate s.d. n=8. (c) Cotransfection of wild-type reporter with ESCC miRNAs. Data was normalized to miRNA mock transfected Dgcr8 Δ/Δ ES cells. Error bars indicate s.d. n=4 for wild type ES cells; n=8 for Dgcr8 Δ/Δ ES cells. (d) Cotransfection of ESCC miRNAs with mutant reporter constructs. Each reporter was normalized to miRNA mock transfected Dgcr8 Δ/Δ ES cells. Error bars indicate s.d. n=4. (e) Cell cycle profile of wild type ES cells with ectopic expression of Cdkn1a. n=6 for wild type and Dgcr8 Δ/Δ ES cells. n=12 (from 3 independent transfections) for wild type ES cells with GFP or Cdkn1a overexpression.

Figure 5

Additional inhibitors of the Cyclin E/Cdk2 are regulated by miRNAs in ES cells. (a) Affymetrix Mouse Gene 1.0 ST arrays were probed with RNA from wild-type and Dgcr8 Δ/Δ ES cells. Shown are the RMA normalized array signals for six inhibitors of the Cyclin E/Cdk2 induced transition from G1 to S. n=3. (b) qRT-PCR confirmation of array data. Shown is the relative expression of each gene to the average of wild-type samples. Each represents the average of a single biological replicate done in triplicate. (c) Diagrammatic representation of Targetscan predicted 3'UTR sites for the ESCC miRNAs and relative luciferase activity between wild-type and Dgcr8 knockout cells following transfection of respective reporters. Error bars indicate s.d. n=8. (d) Diagram of Cyclin E/CdK2 pathway with inhibitors regulated by ESCC miRNAs boxed.