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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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Target Genes of miR-302/367 Cluster Associated with Cell Cycle and Apoptosis in hESCs(A) qPCR analysis of potential targets of miR-302/367 cluster regulating cell cycle in hESCs. Data are represented as mean ± SD of technical replicates (n = 3). See also Figure S2 and Table S1.(B) Screening of apoptosis-related target genes of the miR-302/367 cluster. Patterns of gene expression were compared between the hESCs expressing control-KRAB or TALE1-KRAB using Apoptosis PCR Array. See also Table S2.(C) qPCR analysis of BNIP3L and BCL-xL gene expression in control-KRAB- and TALE1-KRAB-expressing hESCs. Data are represented as mean ± SD of technical replicates (n = 3).(D) Western blot analysis of BINP3L/Nix and BCL-xL in control-KRAB- and TALE1-KRAB-expressing hESCs. BINP3L/Nix and BCL-xL were detected by western blot analysis with each specific antibody. Relative expression of the BINP3L/Nix and BCL-xL was quantified by Image J (NIH) and normalized by G6PD.(E) Schematic representation of the reporter construct containing the luciferase-coding sequence fused to the BNIP3L/Nix 3′UTR. Two predicted targeting sites for miR-302/367 cluster were designated as BS1 and BS2. See also Figure S3.(F) BNIP3L/Nix 3′UTR luciferase reporter assay. 293T cells were transfected with the reporter plasmid pGL3-BNIP3L/Nix carrying mutations BS1 or BS2 or both, together with pMIGR1 (vector control) or pMIGR1_miR-302/367 and then cultured for 72 hr before luciferase activity assay. pCMV-LacZ was included in each transfection as an internal control to normalize luciferase activity. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
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fig4: Target Genes of miR-302/367 Cluster Associated with Cell Cycle and Apoptosis in hESCs(A) qPCR analysis of potential targets of miR-302/367 cluster regulating cell cycle in hESCs. Data are represented as mean ± SD of technical replicates (n = 3). See also Figure S2 and Table S1.(B) Screening of apoptosis-related target genes of the miR-302/367 cluster. Patterns of gene expression were compared between the hESCs expressing control-KRAB or TALE1-KRAB using Apoptosis PCR Array. See also Table S2.(C) qPCR analysis of BNIP3L and BCL-xL gene expression in control-KRAB- and TALE1-KRAB-expressing hESCs. Data are represented as mean ± SD of technical replicates (n = 3).(D) Western blot analysis of BINP3L/Nix and BCL-xL in control-KRAB- and TALE1-KRAB-expressing hESCs. BINP3L/Nix and BCL-xL were detected by western blot analysis with each specific antibody. Relative expression of the BINP3L/Nix and BCL-xL was quantified by Image J (NIH) and normalized by G6PD.(E) Schematic representation of the reporter construct containing the luciferase-coding sequence fused to the BNIP3L/Nix 3′UTR. Two predicted targeting sites for miR-302/367 cluster were designated as BS1 and BS2. See also Figure S3.(F) BNIP3L/Nix 3′UTR luciferase reporter assay. 293T cells were transfected with the reporter plasmid pGL3-BNIP3L/Nix carrying mutations BS1 or BS2 or both, together with pMIGR1 (vector control) or pMIGR1_miR-302/367 and then cultured for 72 hr before luciferase activity assay. pCMV-LacZ was included in each transfection as an internal control to normalize luciferase activity. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).

Mentions: Because transforming growth factor-β1 (TGF-β1) is involved in apoptotic pathways in several types of cells (Lee and Bae, 2002; Schuster and Krieglstein, 2002), we thus asked whether miR-302/367 cluster regulates apoptosis via TGF-β1 signaling. To address this question, we treated two groups of hESCs (control-KRAB versus TALE1-KRAB) with or without TGF-β1 or SB431542 (a chemical inhibitor of TGF-β1 receptor). Our data showed that TGF-β1 or SB431542 had little effect on apoptosis in hESCs expressing TALE1-KRAB or control-KRAB (Figure S2). To dissect the molecular mechanisms by which miR-302/367 cluster dually regulates cell cycle and apoptosis in hESCs, we examined the expression of 21 cell cycle regulators by qPCR and found that knockdown of the endogenous miR-302/367 cluster by TALE1-KRAB inhibits the expression of BMI-1, CDK2, CCND1, CCND2, CDK6 (Figure 4A; Table S1). It has been shown that these molecules play important roles in regulation of G0/G1- to S-phase transition (Abdelalim, 2013). Thus, it is likely that the endogenous miR-302/367 cluster controls hESC cell cycle progression through the regulation of these key cell cycle regulators.


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)

Target Genes of miR-302/367 Cluster Associated with Cell Cycle and Apoptosis in hESCs(A) qPCR analysis of potential targets of miR-302/367 cluster regulating cell cycle in hESCs. Data are represented as mean ± SD of technical replicates (n = 3). See also Figure S2 and Table S1.(B) Screening of apoptosis-related target genes of the miR-302/367 cluster. Patterns of gene expression were compared between the hESCs expressing control-KRAB or TALE1-KRAB using Apoptosis PCR Array. See also Table S2.(C) qPCR analysis of BNIP3L and BCL-xL gene expression in control-KRAB- and TALE1-KRAB-expressing hESCs. Data are represented as mean ± SD of technical replicates (n = 3).(D) Western blot analysis of BINP3L/Nix and BCL-xL in control-KRAB- and TALE1-KRAB-expressing hESCs. BINP3L/Nix and BCL-xL were detected by western blot analysis with each specific antibody. Relative expression of the BINP3L/Nix and BCL-xL was quantified by Image J (NIH) and normalized by G6PD.(E) Schematic representation of the reporter construct containing the luciferase-coding sequence fused to the BNIP3L/Nix 3′UTR. Two predicted targeting sites for miR-302/367 cluster were designated as BS1 and BS2. See also Figure S3.(F) BNIP3L/Nix 3′UTR luciferase reporter assay. 293T cells were transfected with the reporter plasmid pGL3-BNIP3L/Nix carrying mutations BS1 or BS2 or both, together with pMIGR1 (vector control) or pMIGR1_miR-302/367 and then cultured for 72 hr before luciferase activity assay. pCMV-LacZ was included in each transfection as an internal control to normalize luciferase activity. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
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fig4: Target Genes of miR-302/367 Cluster Associated with Cell Cycle and Apoptosis in hESCs(A) qPCR analysis of potential targets of miR-302/367 cluster regulating cell cycle in hESCs. Data are represented as mean ± SD of technical replicates (n = 3). See also Figure S2 and Table S1.(B) Screening of apoptosis-related target genes of the miR-302/367 cluster. Patterns of gene expression were compared between the hESCs expressing control-KRAB or TALE1-KRAB using Apoptosis PCR Array. See also Table S2.(C) qPCR analysis of BNIP3L and BCL-xL gene expression in control-KRAB- and TALE1-KRAB-expressing hESCs. Data are represented as mean ± SD of technical replicates (n = 3).(D) Western blot analysis of BINP3L/Nix and BCL-xL in control-KRAB- and TALE1-KRAB-expressing hESCs. BINP3L/Nix and BCL-xL were detected by western blot analysis with each specific antibody. Relative expression of the BINP3L/Nix and BCL-xL was quantified by Image J (NIH) and normalized by G6PD.(E) Schematic representation of the reporter construct containing the luciferase-coding sequence fused to the BNIP3L/Nix 3′UTR. Two predicted targeting sites for miR-302/367 cluster were designated as BS1 and BS2. See also Figure S3.(F) BNIP3L/Nix 3′UTR luciferase reporter assay. 293T cells were transfected with the reporter plasmid pGL3-BNIP3L/Nix carrying mutations BS1 or BS2 or both, together with pMIGR1 (vector control) or pMIGR1_miR-302/367 and then cultured for 72 hr before luciferase activity assay. pCMV-LacZ was included in each transfection as an internal control to normalize luciferase activity. Data are represented as mean ± SD of three independent experiments (∗∗p < 0.01).
Mentions: Because transforming growth factor-β1 (TGF-β1) is involved in apoptotic pathways in several types of cells (Lee and Bae, 2002; Schuster and Krieglstein, 2002), we thus asked whether miR-302/367 cluster regulates apoptosis via TGF-β1 signaling. To address this question, we treated two groups of hESCs (control-KRAB versus TALE1-KRAB) with or without TGF-β1 or SB431542 (a chemical inhibitor of TGF-β1 receptor). Our data showed that TGF-β1 or SB431542 had little effect on apoptosis in hESCs expressing TALE1-KRAB or control-KRAB (Figure S2). To dissect the molecular mechanisms by which miR-302/367 cluster dually regulates cell cycle and apoptosis in hESCs, we examined the expression of 21 cell cycle regulators by qPCR and found that knockdown of the endogenous miR-302/367 cluster by TALE1-KRAB inhibits the expression of BMI-1, CDK2, CCND1, CCND2, CDK6 (Figure 4A; Table S1). It has been shown that these molecules play important roles in regulation of G0/G1- to S-phase transition (Abdelalim, 2013). Thus, it is likely that the endogenous miR-302/367 cluster controls hESC cell cycle progression through the regulation of these key cell cycle regulators.

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