Xiajing Li1, Yiyu Zhang2, Ning Wang3,4, Zhaohu Yuan5,6, Xiaojie Chen5,6, Qicong Chen3,4, Hui Deng5,6, Xinxin Tong5,6, Honglin Chen3, Yuyou Duan7, Yaming Wei8,9. 1. School of Medicine, South China University of Technology, Guangzhou 510000, China. 2. Department of Blood Transfusion, Shenzhen Longhua Central Hospital, Shenzhen 518000, China. 3. Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510000, China. 4. School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou 510000, China. 5. Department of Blood Transfusion, the Second Affiliation Hospital of South China University of Technology, Guangzhou 510000, China. 6. Guangdong Engineering Research Center of Precise Transfusion, Guangzhou 510000, China. 7. Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510000, China. eywym@scut.edu.cn, yuyouduan@scut.edu.cn. 8. Department of Blood Transfusion, the Second Affiliation Hospital of South China University of Technology, Guangzhou 510000, China. eywym@scut.edu.cn. 9. Guangdong Engineering Research Center of Precise Transfusion, Guangzhou 510000, China. eywym@scut.edu.cn.
Abstract
BACKGROUND: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs with multiple biological functions. CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2 (CNOT2), which was found to regulate tumor cell apoptosis through caspase pathway. METHODS: Potential circRNA.0007127 target microRNAs (miRNAs) were analyzed by miRanda, TargetScan, and RNAhybrid software, and the miRNAs with binding sites for apoptosis-related genes were screened. The roles of circRNA.0007127 and its downstream target, microRNA (miR)-513a-5p, were validated by quantitative real-time polymerase chain reaction (qPCR), flow cytometry, mitochondrial membrane potential, immunofluorescence, western blot, and caspase-8 (CASP8) protein activity in vitro in H2O2-induced K-562 cells. The circRNA.0007127‒miR-513a-5p and CASP8‒miR-513a-5p interactions were verified by luciferase reporter assays. RESULTS: Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells. Compared with the control group, the expression of CASP8 was reduced by 50% and the 43-kD fragment of CASP8 protein was significantly reduced (P≤0.05). The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8, with extremely significant differences (P≤0.001). The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model (75% change) and the level of a 43-kD fragment of CASP8 protein (P≤0.01). The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA (siRNA) and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist. CONCLUSIONS: CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis, which can serve as a novel powerful molecular target for K-562 cells.
BACKGROUND: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs with multiple biological functions. CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2 (CNOT2), which was found to regulate tumor cell apoptosis through caspase pathway. METHODS: Potential circRNA.0007127 target microRNAs (miRNAs) were analyzed by miRanda, TargetScan, and RNAhybrid software, and the miRNAs with binding sites for apoptosis-related genes were screened. The roles of circRNA.0007127 and its downstream target, microRNA (miR)-513a-5p, were validated by quantitative real-time polymerase chain reaction (qPCR), flow cytometry, mitochondrial membrane potential, immunofluorescence, western blot, and caspase-8 (CASP8) protein activity in vitro in H2O2-induced K-562 cells. The circRNA.0007127‒miR-513a-5p and CASP8‒miR-513a-5p interactions were verified by luciferase reporter assays. RESULTS: Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells. Compared with the control group, the expression of CASP8 was reduced by 50% and the 43-kD fragment of CASP8 protein was significantly reduced (P≤0.05). The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8, with extremely significant differences (P≤0.001). The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model (75% change) and the level of a 43-kD fragment of CASP8 protein (P≤0.01). The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA (siRNA) and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist. CONCLUSIONS: CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis, which can serve as a novel powerful molecular target for K-562 cells.
Authors: Alfonso Rodriguez-Gil; Olesja Ritter; Juliane Hornung; Hilda Stekman; Marcus Krüger; Thomas Braun; Elisabeth Kremmer; Michael Kracht; M Lienhard Schmitz Journal: Mol Biol Cell Date: 2016-04-27 Impact factor: 4.138