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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 the Endogenous miR-302/367 Cluster in Regulation of hESC Growth(A) qPCR analysis of mature miR-302/367 members in hESCs stably expressing control-KRAB and TALE1-KRAB. hESCs were infected with control-KRAB or TALE1-KRAB. Transcripts of miR-302/367 members were analyzed by qPCR using specific primers. Data are represented as mean ± SD of technical replicates (n = 3).(B) Scheme of a GFP fluorescence-based growth competition assay. GFP+ hESCs (control-KRAB or TALE1-KRAB) and GFP− hESCs (WT) were mixed at nearly 1:1 ratio and cultured together for two passages. The ratio of GFP+ and GFP− cells was determined before and after passaging.(C) Percentage of GFP+ cell populations in hESCs stably expressing control-KRAB or TALE1-KRAB. (Left) A representation of flow cytometric analysis of GFP+ cells before and after passage. (Right) Percentage of GFP+ cells in hESCs stably expressing control-KRAB or TALE1-KRAB before and after passage. Data are a representative of two independent experiments.(D) The effects of miR-302/367 cluster on the growth of hESCs. WT hESCs or stable expressing control-KRAB or TALE1-KRAB-hESCs were seeded alone in 12-well plate (7,000 cells per well). The cells were then counted at indicated time points. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01). See also Figure S1.(E) Pluripotency analysis of hESCs stably expressing control-KRAB or TALE1-KRAB by flow cytometry. Pluripotency of GFP+ cell population was assessed by analysis of SSEA4 expression with anti-SSEA4 antibody. Data are a representative of two independent experiments.
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fig1: Role of the Endogenous miR-302/367 Cluster in Regulation of hESC Growth(A) qPCR analysis of mature miR-302/367 members in hESCs stably expressing control-KRAB and TALE1-KRAB. hESCs were infected with control-KRAB or TALE1-KRAB. Transcripts of miR-302/367 members were analyzed by qPCR using specific primers. Data are represented as mean ± SD of technical replicates (n = 3).(B) Scheme of a GFP fluorescence-based growth competition assay. GFP+ hESCs (control-KRAB or TALE1-KRAB) and GFP− hESCs (WT) were mixed at nearly 1:1 ratio and cultured together for two passages. The ratio of GFP+ and GFP− cells was determined before and after passaging.(C) Percentage of GFP+ cell populations in hESCs stably expressing control-KRAB or TALE1-KRAB. (Left) A representation of flow cytometric analysis of GFP+ cells before and after passage. (Right) Percentage of GFP+ cells in hESCs stably expressing control-KRAB or TALE1-KRAB before and after passage. Data are a representative of two independent experiments.(D) The effects of miR-302/367 cluster on the growth of hESCs. WT hESCs or stable expressing control-KRAB or TALE1-KRAB-hESCs were seeded alone in 12-well plate (7,000 cells per well). The cells were then counted at indicated time points. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01). See also Figure S1.(E) Pluripotency analysis of hESCs stably expressing control-KRAB or TALE1-KRAB by flow cytometry. Pluripotency of GFP+ cell population was assessed by analysis of SSEA4 expression with anti-SSEA4 antibody. Data are a representative of two independent experiments.

Mentions: We previously constructed TALE-based transcriptional repressors that specifically bind to the promoter region of human miR-302/367 cluster and could efficiently inhibit the elevated expression of primary miR-302/367 during reprogramming (Zhang et al., 2013). To understand functional roles of the endogenous miR-302/367 cluster in hESCs, we first determined whether TALE1-KRAB, an miR-302/367 cluster-specific TALE-based transcriptional repressor constructed previously (Zhang et al., 2013), could efficiently knock down the expression of five mature miR-302/367 members. We generated lentiviral particles expressing TALE1-KRAB or control-KRAB (with a GFP marker) and transduced them into hESCs, respectively. We sorted GFP+-transduced hESCs and measured the expression of five mature miR-302/367 members by qPCR. As shown in Figure 1A, TALE1-KRAB evenly inhibited expressions of five mature miR-302/367 members by 80% when compared with the control-KRAB group.


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 the Endogenous miR-302/367 Cluster in Regulation of hESC Growth(A) qPCR analysis of mature miR-302/367 members in hESCs stably expressing control-KRAB and TALE1-KRAB. hESCs were infected with control-KRAB or TALE1-KRAB. Transcripts of miR-302/367 members were analyzed by qPCR using specific primers. Data are represented as mean ± SD of technical replicates (n = 3).(B) Scheme of a GFP fluorescence-based growth competition assay. GFP+ hESCs (control-KRAB or TALE1-KRAB) and GFP− hESCs (WT) were mixed at nearly 1:1 ratio and cultured together for two passages. The ratio of GFP+ and GFP− cells was determined before and after passaging.(C) Percentage of GFP+ cell populations in hESCs stably expressing control-KRAB or TALE1-KRAB. (Left) A representation of flow cytometric analysis of GFP+ cells before and after passage. (Right) Percentage of GFP+ cells in hESCs stably expressing control-KRAB or TALE1-KRAB before and after passage. Data are a representative of two independent experiments.(D) The effects of miR-302/367 cluster on the growth of hESCs. WT hESCs or stable expressing control-KRAB or TALE1-KRAB-hESCs were seeded alone in 12-well plate (7,000 cells per well). The cells were then counted at indicated time points. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01). See also Figure S1.(E) Pluripotency analysis of hESCs stably expressing control-KRAB or TALE1-KRAB by flow cytometry. Pluripotency of GFP+ cell population was assessed by analysis of SSEA4 expression with anti-SSEA4 antibody. Data are a representative of two independent experiments.
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fig1: Role of the Endogenous miR-302/367 Cluster in Regulation of hESC Growth(A) qPCR analysis of mature miR-302/367 members in hESCs stably expressing control-KRAB and TALE1-KRAB. hESCs were infected with control-KRAB or TALE1-KRAB. Transcripts of miR-302/367 members were analyzed by qPCR using specific primers. Data are represented as mean ± SD of technical replicates (n = 3).(B) Scheme of a GFP fluorescence-based growth competition assay. GFP+ hESCs (control-KRAB or TALE1-KRAB) and GFP− hESCs (WT) were mixed at nearly 1:1 ratio and cultured together for two passages. The ratio of GFP+ and GFP− cells was determined before and after passaging.(C) Percentage of GFP+ cell populations in hESCs stably expressing control-KRAB or TALE1-KRAB. (Left) A representation of flow cytometric analysis of GFP+ cells before and after passage. (Right) Percentage of GFP+ cells in hESCs stably expressing control-KRAB or TALE1-KRAB before and after passage. Data are a representative of two independent experiments.(D) The effects of miR-302/367 cluster on the growth of hESCs. WT hESCs or stable expressing control-KRAB or TALE1-KRAB-hESCs were seeded alone in 12-well plate (7,000 cells per well). The cells were then counted at indicated time points. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01). See also Figure S1.(E) Pluripotency analysis of hESCs stably expressing control-KRAB or TALE1-KRAB by flow cytometry. Pluripotency of GFP+ cell population was assessed by analysis of SSEA4 expression with anti-SSEA4 antibody. Data are a representative of two independent experiments.
Mentions: We previously constructed TALE-based transcriptional repressors that specifically bind to the promoter region of human miR-302/367 cluster and could efficiently inhibit the elevated expression of primary miR-302/367 during reprogramming (Zhang et al., 2013). To understand functional roles of the endogenous miR-302/367 cluster in hESCs, we first determined whether TALE1-KRAB, an miR-302/367 cluster-specific TALE-based transcriptional repressor constructed previously (Zhang et al., 2013), could efficiently knock down the expression of five mature miR-302/367 members. We generated lentiviral particles expressing TALE1-KRAB or control-KRAB (with a GFP marker) and transduced them into hESCs, respectively. We sorted GFP+-transduced hESCs and measured the expression of five mature miR-302/367 members by qPCR. As shown in Figure 1A, TALE1-KRAB evenly inhibited expressions of five mature miR-302/367 members by 80% when compared with the control-KRAB group.

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