The reintroduction of wild-type Dgcr8 into Dgcr8 knockout ES cells rescued the proliferation and cell cycle defects, proving these defects are not due to the secondary and irreversible cellular events. In addition, the reversibility suggested that it may be possible to rescue these defects by reintroducing individual miRNAs. To this end, a screening strategy was established where chemically synthesized miRNA duplexes, called miRNA mimics, were individually transfected into the Dgcr8 knockout cells (19
). The transfected cells were then evaluated for changes in their rate of cell proliferation. Using a colorimetric based assay in 96 well plates, it was possible to expand this screen to hundreds of miRNA mimics. This unbiased screening approach identified multiple miRNAs that partially rescued the proliferation defect. Most of these miRNAs shared a common seed sequence “AAGUGCU”. These miRNAs include members of the miR-290 cluster (miR-291-3p, miR-294, and mir-295) and the miR-302 cluster, and those with the slightly different seed sequence “AAAGUGC” including miR-20, miR-93 and miR-106 belonging to the miR-17/20/106 family. Similar seed sequences suggest similar sets of genes are regulated by these miRNAs and, therefore, there is a high degree of redundancy among these miRNAs. All these miRNAs are expressed in wild type ES cells. The mir-290 cluster alone makes up greater than 70% of the total quantity of miRNAs in ES cells (20
). Members of this cluster are co-transcribed as a single transcript (21
), suggesting synergistic regulation by these miRNAs. Furthermore, expression of this cluster is rapidly downregulated upon differentiation, coincident with the elongation of the cell cycle. Members from the miR-17/20/106 family are analogously highly expressed in many cancers promoting their growth (22
Further characterization showed that the miR-291-3p, miR-294 and miR-295 fully rescued the G1 accumulation phenotype suggesting they were acting to promote the G1/S transition (). To confirm this hypothesis it was essential to identify the targets of these miRNAs. The previous work on the ES cell cycle had provided important hints to what these targets may be (14
). Specifically, the miRNAs are presumably acting through the Cdk2–Cyclin E pathway as this is the key G1/S promoting pathway in ES cells. Moreover, a previous report showed p21 which inhibits the Cdk2-Cyclin E activity is post-transcriptionally regulated during ES cell differentiation with p21 protein, but not mRNA, levels dramatically increasing as the G1 phase extended (24
). Indeed, p21 levels were elevated in the Dgcr8 knockout cells, while introduction of the rescuing miRNAs reduced p21 to the wild-type level. p21 was then confirmed as a direct target of the miRNAs by luciferase assays. However, miRNAs are unlikely to function through a single target as they are known to influence the levels of numerous proteins(25
). Indeed, the overexpression of p21 in wild-type ES cells only partially phenocopied the G1 accumulation seen in the Dgcr8 knockout cells. Further analysis using mRNA profiling data, target prediction, and luciferase assays identified additional inhibitors of the Cdk2–Cyclin E pathway including p130 (Rbl2) and Lats2 as direct targets of these miRNAs (). These results show that the members of the miR-290 cluster act to suppress several well known inhibitors of the G1/S transition thereby modulating the cell cycle structure of ES cells. Interestingly, mir-106b has recently been reported to promote cell cycle progression in a breast cancer cell line (27
) by mechanisms very similar to the mir-290 cluster in ES cells reflecting parallels in the molecular control of the cell cycle of embryonic and cancer cells.
The miR-290 family suppresses multiple inhibitors of the G1/S transition to enable a short G1 phase in ES cells. During differentiation, the expression level of the miR-290 family is downregulated and the G1 phase of the cell cycle is elongated.
The screening approach in the miRNA deficient background provides a powerful strategy to identify functions of individual miRNAs. Widespread redundancy of miRNAs often impedes assigning biological functions to specific miRNAs by reverse genetic approaches. Starting with cells deficient for all miRNAs and reintroducing individual miRNAs overcomes this problem. For example, besides the miR-290 cluster, miRNAs from the miR-302 cluster and the miR-17/20/106 family are also expressed in ES cells (20
). All together more than ten miRNAs containing the seed sequence “AAGUGCU” or “AAAGUGC” make up a majority of the total miRNA population in ES cells. This degree of redundancy makes it very difficult to use a reverse knockdown approach. Indeed, knockdown of a number of these miRNAs individually or even in combination did not produce a significant phenotype (19