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: Our data established that knockdown of the endogenous miR-302/367 cluster impairs hESC self-renewal by triggering apoptosis. We thus predicted that increasing miR-302/367 cluster expression would restore normal growth of hESCs by alleviating them from apoptosis. Recently, we found that butyrate, a natural compound and histone deacetylase inhibitor, can enhance the expression of primary miR-302/367 during reprogramming process (Zhang and Wu, 2013). Thus, we hypothesized that butyrate might rescue, partially if not completely, hESC apoptosis induced by knockdown of endogenous miR-302/367 cluster. To test this hypothesis, we first tested the optimal concentrations of butyrate for modulating expression of miR-302/367 cluster because butyrate concentrations commonly used in cell culture (>1.0 mM) were toxic to hESCs. We found that lower concentrations of butyrate (0.25 to 0.5 mM) enhanced the expression of five mature miR-302/367 members (Figure S5). Interestingly, a low concentration of butyrate (0.5 mM) was previously shown to promote hESCs self-renewal in the absence of basic fibroblast growth factor (bFGF) (Ware et al., 2009). As expected, forced expression of TALE1-KRAB decreased the expression of miR-302/367 cluster by 2.5-fold, and butyrate induced expression of pri-miR-302/367 cluster in the control group of hESCs expressing control-KRAB by 2-fold (Figure 6A). Interestingly, butyrate treatment elevated expression of pri-miR-302/367 in hESCs expressing TALE1-KRAB to a level similar with that in the control group before butyrate treatment. Next, we measured by qPCR expression of BNIP3L/Nix, a target gene of miR-302/367 cluster in these cell groups. In agreement with our result (Figure 4), upregulation of miR-302/367 cluster by butyrate treatment downregulated BNIP3L/Nix expression to a level, which was comparable with the control groups (Figure 6B). Together, our data indicated that butyrate is able to suppress BNIP3L/Nix expression by upregulating the expression of endogenous miR-302/367 cluster.
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.