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MicroRNA-302/367 cluster governs hESC self-renewal by dually regulating cell cycle and apoptosis pathways.

Zhang Z, Hong Y, Xiang D, Zhu P, Wu E, Li W, Mosenson J, Wu WS - Stem Cell Reports (2015)

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.

View Article: PubMed Central - PubMed

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.

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Role of miR-302/367 Cluster in Regulation of Cell Cycle and Apoptosis of hESCs(A) Cell cycle analysis of hESCs-expressing control-KRAB or TALE1-KRAB by flow cytometry. Cells were dissected and stained by Vybrant Dyecycle according to manufacturer’s instructions. A representative graph of analyzing cell cycle processed with the FlowJo program was shown in the left, and analysis of cell cycle phase distribution was shown in the right. Data are represented as mean ± SD of three independent experiments (∗p < 0.05, ∗∗p < 0.01).(B) Analysis of proliferating hESCs by EdU staining. hESCs expressing control-KRAB or TALE1-KRAB were cultured in 24-well plate overnight and then followed by the addition of EdU solution for 1 hr. Cells were dissected for EdU detection using the Click-iT detection kit. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(C) Flow cytometric analysis of apoptotic hESCs. Control-KRAB- or TALE1-KRAB-expressing hESCs were stained with Annexin V-APC and then analyzed by flow cytometry (left). The percentage of Annexin V+ cells was determined (right). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
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fig2: Role of miR-302/367 Cluster in Regulation of Cell Cycle and Apoptosis of hESCs(A) Cell cycle analysis of hESCs-expressing control-KRAB or TALE1-KRAB by flow cytometry. Cells were dissected and stained by Vybrant Dyecycle according to manufacturer’s instructions. A representative graph of analyzing cell cycle processed with the FlowJo program was shown in the left, and analysis of cell cycle phase distribution was shown in the right. Data are represented as mean ± SD of three independent experiments (∗p < 0.05, ∗∗p < 0.01).(B) Analysis of proliferating hESCs by EdU staining. hESCs expressing control-KRAB or TALE1-KRAB were cultured in 24-well plate overnight and then followed by the addition of EdU solution for 1 hr. Cells were dissected for EdU detection using the Click-iT detection kit. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(C) Flow cytometric analysis of apoptotic hESCs. Control-KRAB- or TALE1-KRAB-expressing hESCs were stained with Annexin V-APC and then analyzed by flow cytometry (left). The percentage of Annexin V+ cells was determined (right). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).

Mentions: Our data showed that knockdown of the endogenous miR-302/367 cluster impairs hESC self-renewal capacity (Figure 1); we thus hypothesized that miR-302/367 cluster regulates cell cycle and/or apoptosis in hESCs. To test our hypothesis, we first assessed the effects of the endogenous miR-302/367 cluster on the hESC cell cycle. We dissociated stable hESC lines, incubated them with Vybrant DyeCycle Violet, and followed by flow cytometric analysis. Our data show that indeed hESCs expressing TALE1-KRAB accumulated in cell cycle G0/G1 phase, with a concomitant decrease in the fraction of cells in S or G2/M phrase (Figure 2A). Next, we measured proliferation of hESCs in the two groups by performing EdU incorporation assay. As shown in Figure 2B, EdU incorporation rate was significantly reduced in hESCs expressing TALE1-KRAB when compared with control-KRAB group, indicating that knockdown of the endogenous miR-306/367 cluster decreases hESC proliferation. Furthermore, we decided to determine whether knockdown of the endogenous miR-302/367 cluster causes apoptosis in hESCs during cell culture. To address this question, we dissociated hESCs expressing control-KRAB or TALE1-KRAB, stained them with Annexin V-APC, and performed analysis by flow cytometry. As shown in Figure 2C, hESCs expressing TALE1-KRAB exhibited a significantly higher apoptotic rate when compared with hESCs expressing control-KRAB. These data indicate that the endogenous miR-302/367 cluster is required to prevent hESCs from apoptosis during cell culture.


MicroRNA-302/367 cluster governs hESC self-renewal by dually regulating cell cycle and apoptosis pathways.

Zhang Z, Hong Y, Xiang D, Zhu P, Wu E, Li W, Mosenson J, Wu WS - Stem Cell Reports (2015)

Role of miR-302/367 Cluster in Regulation of Cell Cycle and Apoptosis of hESCs(A) Cell cycle analysis of hESCs-expressing control-KRAB or TALE1-KRAB by flow cytometry. Cells were dissected and stained by Vybrant Dyecycle according to manufacturer’s instructions. A representative graph of analyzing cell cycle processed with the FlowJo program was shown in the left, and analysis of cell cycle phase distribution was shown in the right. Data are represented as mean ± SD of three independent experiments (∗p < 0.05, ∗∗p < 0.01).(B) Analysis of proliferating hESCs by EdU staining. hESCs expressing control-KRAB or TALE1-KRAB were cultured in 24-well plate overnight and then followed by the addition of EdU solution for 1 hr. Cells were dissected for EdU detection using the Click-iT detection kit. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(C) Flow cytometric analysis of apoptotic hESCs. Control-KRAB- or TALE1-KRAB-expressing hESCs were stained with Annexin V-APC and then analyzed by flow cytometry (left). The percentage of Annexin V+ cells was determined (right). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
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fig2: Role of miR-302/367 Cluster in Regulation of Cell Cycle and Apoptosis of hESCs(A) Cell cycle analysis of hESCs-expressing control-KRAB or TALE1-KRAB by flow cytometry. Cells were dissected and stained by Vybrant Dyecycle according to manufacturer’s instructions. A representative graph of analyzing cell cycle processed with the FlowJo program was shown in the left, and analysis of cell cycle phase distribution was shown in the right. Data are represented as mean ± SD of three independent experiments (∗p < 0.05, ∗∗p < 0.01).(B) Analysis of proliferating hESCs by EdU staining. hESCs expressing control-KRAB or TALE1-KRAB were cultured in 24-well plate overnight and then followed by the addition of EdU solution for 1 hr. Cells were dissected for EdU detection using the Click-iT detection kit. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(C) Flow cytometric analysis of apoptotic hESCs. Control-KRAB- or TALE1-KRAB-expressing hESCs were stained with Annexin V-APC and then analyzed by flow cytometry (left). The percentage of Annexin V+ cells was determined (right). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
Mentions: Our data showed that knockdown of the endogenous miR-302/367 cluster impairs hESC self-renewal capacity (Figure 1); we thus hypothesized that miR-302/367 cluster regulates cell cycle and/or apoptosis in hESCs. To test our hypothesis, we first assessed the effects of the endogenous miR-302/367 cluster on the hESC cell cycle. We dissociated stable hESC lines, incubated them with Vybrant DyeCycle Violet, and followed by flow cytometric analysis. Our data show that indeed hESCs expressing TALE1-KRAB accumulated in cell cycle G0/G1 phase, with a concomitant decrease in the fraction of cells in S or G2/M phrase (Figure 2A). Next, we measured proliferation of hESCs in the two groups by performing EdU incorporation assay. As shown in Figure 2B, EdU incorporation rate was significantly reduced in hESCs expressing TALE1-KRAB when compared with control-KRAB group, indicating that knockdown of the endogenous miR-306/367 cluster decreases hESC proliferation. Furthermore, we decided to determine whether knockdown of the endogenous miR-302/367 cluster causes apoptosis in hESCs during cell culture. To address this question, we dissociated hESCs expressing control-KRAB or TALE1-KRAB, stained them with Annexin V-APC, and performed analysis by flow cytometry. As shown in Figure 2C, hESCs expressing TALE1-KRAB exhibited a significantly higher apoptotic rate when compared with hESCs expressing control-KRAB. These data indicate that the endogenous miR-302/367 cluster is required to prevent hESCs from apoptosis during cell culture.

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.

View Article: PubMed Central - PubMed

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.

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Related in: MedlinePlus