【摘要】 目的：探讨CCR4-NOT复合物亚基2基因（CNOT2）来源的环状RNAcircRNA.0007127在凋亡通路中的作用。创新点：本研究发现，circRNA.0007127与K-562细胞凋亡相关，可能是一个预测K-562细胞凋亡的分子标志物。并首次证实了细胞凋亡中circRNA.0007127/miR-513a-5p/CASP8的调控轴。方法：使用miRanda、TargetScan和RNAhybrid软件对circRNA.0007127的靶微小RNA(miRNA)进行预测，筛选具有凋亡相关基因结合位点的miRNA。在H2O2诱导的K-562细胞中，通过定量聚合酶链反应、流式细胞术、线粒体膜电位、免疫荧光、蛋白印迹和胱天蛋白酶8(CASP8)蛋白活性实验来对circRNA.0007127及其下游靶点miR-513a-5p进行体外验证。circ RNA.0007127–mi R-513a-5p以及CASP8–miR-513a-5p的相互作用分别通过荧光素酶报告基因检测得到验证。结果：沉默circRNA.0007127通过抑制CASP8通路激活降低K-562细胞凋亡。与对照组相比，CASP8表达减少2倍，CASP8蛋白43-kD片段显著减少（P<0.05）。荧光素酶报告实验表明，circRNA.0007127可以结合miR-513a-5p或CASP8，差异极显著（P<0.001）。miR-513a-5p过表达抑制K-562细胞中CASP8基因表达水平（4倍）和CASP8蛋白43-kD片段水平（P<0.01）。挽救实验表明，circRNA.0007127siRNA和miR-513a-5p抑制剂共转染增加CASP8基因表达和凋亡率，提示miR-513a-5p抑制剂是circRNA.0007127的拮抗分子。结论：CircRNA.0007127通过miR-513a-5p/CASP8轴调控K-562细胞凋亡，可作为K-562细胞新的分子靶点。更多还原

【Abstract】 Background: Circular RNAs（circR NAs） 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 subunit2（CNOT2）, which was found to regulate tumor cell apoptosis through caspase pathway. Methods: Potential circR NA.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 circR NA.0007127 and its downstream target, microR NA（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 CASP8in a human myeloid leukemia cell model（75% change） and the level of a 43-kD fragment of CASP8 protein（P small-interfering RNA（siRNA） and the miR-≤0.01）. The rescue experiment showed that cotransfection with circRNA.0007127513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127siRNA 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.更多还原