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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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miR-302/367 Cluster Dually Regulates Cell Cycle and Apoptosis of hESC in a Dose-Dependent Manner(A) Diagram of the gating strategy for hESC populations with different expression of TALE1-KRAB. hESCs were gated based GFP expression level (negative, low, medium, and high) and the following corresponding four populations were gated: P-I, GFPnegative; P-II, GFPlow; P-III, GFPmedium; P-IV, GFPhigh. Low, Med, and High were hESCs with low GFP fluorescence, medium GFP fluorescence, and high GFP fluorescence, respectively.(B) qPCR analysis of pri-miR-302/367 in four populations of hESCs expressing different level of TALE1-KRAB indicated by GFP fluorescence. Data are represented as mean ± SD of technical replicates (n = 3).(C) A representative flow cytometry analysis of cell cycle and apoptosis in the four populations of cells with different degree of GFP fluorescence (P-I, P-II, P-III, P-IV).(D) Cell cycle phase distribution of hESCs expressing different level of TALE1-KRAB. The four cell populations (P-I, P-II, P-III, P-IV) were gated as shown in (A) and analyzed as shown in (C), and their cell cycle distribution was calculated and shown as a percentage. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(E) Percentage of apoptotic cells in hESCs with a decreased expression level of miR-302/367 cluster. The apoptotic population of cells was represented by the sub-G0/G1 fraction of events in the four populations (P-I, P-II, P-III, P-IV). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
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fig3: miR-302/367 Cluster Dually Regulates Cell Cycle and Apoptosis of hESC in a Dose-Dependent Manner(A) Diagram of the gating strategy for hESC populations with different expression of TALE1-KRAB. hESCs were gated based GFP expression level (negative, low, medium, and high) and the following corresponding four populations were gated: P-I, GFPnegative; P-II, GFPlow; P-III, GFPmedium; P-IV, GFPhigh. Low, Med, and High were hESCs with low GFP fluorescence, medium GFP fluorescence, and high GFP fluorescence, respectively.(B) qPCR analysis of pri-miR-302/367 in four populations of hESCs expressing different level of TALE1-KRAB indicated by GFP fluorescence. Data are represented as mean ± SD of technical replicates (n = 3).(C) A representative flow cytometry analysis of cell cycle and apoptosis in the four populations of cells with different degree of GFP fluorescence (P-I, P-II, P-III, P-IV).(D) Cell cycle phase distribution of hESCs expressing different level of TALE1-KRAB. The four cell populations (P-I, P-II, P-III, P-IV) were gated as shown in (A) and analyzed as shown in (C), and their cell cycle distribution was calculated and shown as a percentage. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(E) Percentage of apoptotic cells in hESCs with a decreased expression level of miR-302/367 cluster. The apoptotic population of cells was represented by the sub-G0/G1 fraction of events in the four populations (P-I, P-II, P-III, P-IV). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).

Mentions: Collectively, our data show that the endogenous miR-302/367 cluster is essential for both hESC self-renewal and apoptosis (Figures 1 and 2). Now, a key question arises: how can this cluster regulate both self-renewal and apoptosis pathways? Based on miRNAs-specific modulation mechanism, one miRNA can regulate multiple target genes, and one gene can be targeted by multiple miRNAs. A previous study also showed that gene dose of each miRNA affects target selection and regulation (Shu et al., 2012). Therefore, we hypothesized that miR-302/367 cluster can dually regulate cell cycle and apoptosis pathways in hESCs in gene dose-dependent manner. To prove this hypothesis, we expanded hESCs expressing TALE1-KRAB and sorted four populations of hESCs based on GFP fluorescence: population I (P-I, GFPnegative), population II (P-II, GFPlow), population III (P-III, GFPmedium), and population IV (P-IV, GFPhigh). The three populations (P-II, P-III, P-IV) of hESCs express three different levels of GFP fluorescence: low, medium, and high. We extracted total RNAs from these four hESC populations and analyzed the transcripts of primary miR-302/367 by qPCR. Our data showed that the expression levels of pri-miR-302/367 in cell populations expressing low, medium, and high level of GFP was reduced to 70%, 40%, and 20% of the level in the control group (P-I), respectively (Figure 3B). These data indicated that the GFP fluorescence level is reversely correlated with the expression of the endogenous miR-302/367 cluster (Figures 3A and 3B). Next we stained these cells with both Vybrant DyeCycle Violet and Annexin V-APC and then analyzed cell cycle and apoptosis in each of the sorted populations by flow cytometry (Figures 3C–3E). Compared with the group with 70% of expression level (P-II), the group with 40% of expression level of miR-302/367 (P-III) accumulated in G0/G1-phase and had a decreased frequency of S-phase cells (Figure 3D). Interestingly, when the expression level of the miR-302/367 cluster was reduced to 20% (P-IV), we found a sharp decrease of hESCs in cell cycle G0/G1 and S phases and a dramatic increase of hESCs in G2/M phase (Figure 3D).


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)

miR-302/367 Cluster Dually Regulates Cell Cycle and Apoptosis of hESC in a Dose-Dependent Manner(A) Diagram of the gating strategy for hESC populations with different expression of TALE1-KRAB. hESCs were gated based GFP expression level (negative, low, medium, and high) and the following corresponding four populations were gated: P-I, GFPnegative; P-II, GFPlow; P-III, GFPmedium; P-IV, GFPhigh. Low, Med, and High were hESCs with low GFP fluorescence, medium GFP fluorescence, and high GFP fluorescence, respectively.(B) qPCR analysis of pri-miR-302/367 in four populations of hESCs expressing different level of TALE1-KRAB indicated by GFP fluorescence. Data are represented as mean ± SD of technical replicates (n = 3).(C) A representative flow cytometry analysis of cell cycle and apoptosis in the four populations of cells with different degree of GFP fluorescence (P-I, P-II, P-III, P-IV).(D) Cell cycle phase distribution of hESCs expressing different level of TALE1-KRAB. The four cell populations (P-I, P-II, P-III, P-IV) were gated as shown in (A) and analyzed as shown in (C), and their cell cycle distribution was calculated and shown as a percentage. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(E) Percentage of apoptotic cells in hESCs with a decreased expression level of miR-302/367 cluster. The apoptotic population of cells was represented by the sub-G0/G1 fraction of events in the four populations (P-I, P-II, P-III, P-IV). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
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fig3: miR-302/367 Cluster Dually Regulates Cell Cycle and Apoptosis of hESC in a Dose-Dependent Manner(A) Diagram of the gating strategy for hESC populations with different expression of TALE1-KRAB. hESCs were gated based GFP expression level (negative, low, medium, and high) and the following corresponding four populations were gated: P-I, GFPnegative; P-II, GFPlow; P-III, GFPmedium; P-IV, GFPhigh. Low, Med, and High were hESCs with low GFP fluorescence, medium GFP fluorescence, and high GFP fluorescence, respectively.(B) qPCR analysis of pri-miR-302/367 in four populations of hESCs expressing different level of TALE1-KRAB indicated by GFP fluorescence. Data are represented as mean ± SD of technical replicates (n = 3).(C) A representative flow cytometry analysis of cell cycle and apoptosis in the four populations of cells with different degree of GFP fluorescence (P-I, P-II, P-III, P-IV).(D) Cell cycle phase distribution of hESCs expressing different level of TALE1-KRAB. The four cell populations (P-I, P-II, P-III, P-IV) were gated as shown in (A) and analyzed as shown in (C), and their cell cycle distribution was calculated and shown as a percentage. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).(E) Percentage of apoptotic cells in hESCs with a decreased expression level of miR-302/367 cluster. The apoptotic population of cells was represented by the sub-G0/G1 fraction of events in the four populations (P-I, P-II, P-III, P-IV). Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
Mentions: Collectively, our data show that the endogenous miR-302/367 cluster is essential for both hESC self-renewal and apoptosis (Figures 1 and 2). Now, a key question arises: how can this cluster regulate both self-renewal and apoptosis pathways? Based on miRNAs-specific modulation mechanism, one miRNA can regulate multiple target genes, and one gene can be targeted by multiple miRNAs. A previous study also showed that gene dose of each miRNA affects target selection and regulation (Shu et al., 2012). Therefore, we hypothesized that miR-302/367 cluster can dually regulate cell cycle and apoptosis pathways in hESCs in gene dose-dependent manner. To prove this hypothesis, we expanded hESCs expressing TALE1-KRAB and sorted four populations of hESCs based on GFP fluorescence: population I (P-I, GFPnegative), population II (P-II, GFPlow), population III (P-III, GFPmedium), and population IV (P-IV, GFPhigh). The three populations (P-II, P-III, P-IV) of hESCs express three different levels of GFP fluorescence: low, medium, and high. We extracted total RNAs from these four hESC populations and analyzed the transcripts of primary miR-302/367 by qPCR. Our data showed that the expression levels of pri-miR-302/367 in cell populations expressing low, medium, and high level of GFP was reduced to 70%, 40%, and 20% of the level in the control group (P-I), respectively (Figure 3B). These data indicated that the GFP fluorescence level is reversely correlated with the expression of the endogenous miR-302/367 cluster (Figures 3A and 3B). Next we stained these cells with both Vybrant DyeCycle Violet and Annexin V-APC and then analyzed cell cycle and apoptosis in each of the sorted populations by flow cytometry (Figures 3C–3E). Compared with the group with 70% of expression level (P-II), the group with 40% of expression level of miR-302/367 (P-III) accumulated in G0/G1-phase and had a decreased frequency of S-phase cells (Figure 3D). Interestingly, when the expression level of the miR-302/367 cluster was reduced to 20% (P-IV), we found a sharp decrease of hESCs in cell cycle G0/G1 and S phases and a dramatic increase of hESCs in G2/M phase (Figure 3D).

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