MicroRNA-302/367 cluster governs hESC self-renewal by dually regulating cell cycle and apoptosis pathways.
Bottom Line: We demonstrate that in addition to its role in cell cycle regulation, miR-302/367 cluster conquers apoptosis by downregulating BNIP3L/Nix (a BH3-only proapoptotic factor) and upregulating BCL-xL expression.Furthermore, we show that butyrate, a natural compound, upregulates miR-302/367 cluster expression and alleviates hESCs from apoptosis induced by knockdown of miR-302/367 cluster.In summary, our findings provide new insights in molecular mechanisms of how miR-302/367 cluster regulates hESCs.
Affiliation: Department of Medicine and Cancer Center, Division of Hematology/Oncology, University of Illinois at Chicago, Chicago, IL 60612, USA; Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA; Berkeley Stem Cell Center, University of California at Berkeley, Berkeley, CA 94720, USA.Show MeSH
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Mentions: So far, our data demonstrated that miR-302/367 cluster is required for hESC growth (Figure 1) and can dually regulate cell cycle and apoptosis in hESCs (Figures 3 and 4). To dissect molecular pathways by which the endogenous miR-302/367 cluster regulates hESC self-renewal, we overexpressed antiapoptotic gene BCL-xL (with a mCherry marker) in the TALE1-KRAB-expressing hESCs and examined the effects of BCL-xL on apoptosis in these cells. Significantly, our data showed that overexpression of BCL-xL not only rescued hESC from apoptosis caused by knockdown of the endogenous miR-302/367 cluster but also partially blocked the spontaneous apoptosis in WT hESCs (Figure 5A). Next, we assessed effects of BCL-xL on proliferation of hESCs expressing TALE1-KRAB using the competitive growth assay as described in Figure 1B. Our data showed that hESCs expressing TALE1-KRAB have growth disadvantage compared with WT hESCs, which is similar with the result shown in Figures 1C and 1D, but forced expression of BCL-xL enables TALE1-KRAB-expressing hESCs to regain their normal growth capacity. Because it was reported that BCL-xL is also involved in cell cycle regulation (Cheng et al., 2003; Janumyan et al., 2003), we analyzed cell cycle profile of the three groups of hESCs that express control-KRAB or TALE1-KRAB or both TALE1-KRAB and BCL-xL. As shown in Figure S4, BCL-xL only inhibits apoptosis but does not affect cell cycle progression in TALE1-KRAB hESCs. Collectively, our data undoubtedly demonstrated that the endogenous miR-302/367 cluster regulates hESC self-renewal predominantly through the inhibition of apoptosis in hESCs (Figure 5B).
Affiliation: Department of Medicine and Cancer Center, Division of Hematology/Oncology, University of Illinois at Chicago, Chicago, IL 60612, USA; Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA; Berkeley Stem Cell Center, University of California at Berkeley, Berkeley, CA 94720, USA.