microRNAs 224和21对人胶质瘤干细胞存活的影响及相关的分子机制

摘要

目的：探讨microRNAs 224和21对人胶质瘤干细胞存活的影响及相关的分子机制。方法 qPCR检测恶性胶质瘤样品、人GBM干细胞、人工建立的GBM干细胞系和人体组织中microRNAs的失调表达情况。 GBM神经球干细胞系、GBM干细胞系（0822、0308和A172）、人工建立的细胞系U373及永生化人星形胶质细胞分别转染miR－21，miR－224模拟物或抑制剂，然后通过膜联蛋白Ⅴ染色及Caspase 3／7活性检测细胞凋亡，活细胞计数检测细胞生长情况。利用TargetScan 生物学软件预测miR－21和miR－224的靶基因，并利用荧光素酶报告检测microRNAs对Bim基因的靶向作用。 Western blot 检测细胞转染miR－21或 miR－224模拟物或抑制剂后对Caspase 3，Caspase 9及Bim蛋白表达的影响。结果 qPCR检测结果表明，GSC、人肿瘤组织和GBM神经球干细胞中，miR－21和miR－224显著上调表达（ P＜0．05）。细胞凋亡和细胞生长检测结果表明，miR－224和miR－21能调控GSC细胞凋亡和生长。靶基因预测分析Caspase 3，Caspase 9和Bim 3’－UTR序列为miR－224和miR－21潜在的靶基因，荧光素酶实验进一步证实Caspase 3，Caspase 9和Bim 3’－UTR序列为miR－224和miR－21的直接靶点。进一步的实验证明，miR－224和miR－21通过直接靶向Caspases和Bim调控细胞生长和凋亡。结论上述结果表明miR－224和miR－21是GSC的凋亡抗性的重要生理驱动因子，其为胶质瘤的治疗提供了新的靶标。%Objective To explore the effect of microRNAs 224 and 21 on human glioma stem cells survival and the possible molecular mechanisms.Methods qPCR was used to detect the dysregulated expression of microRNAs in malignant glioma samples, human GBM stem cells, artificially established GBM stem cell lines and human tissues.Caspase 3/7 assay, Annexin V apoptosis/fluorescence assay were performed to determine the effect of miR-21 or miR-224 mimics and inhibitor on cell apoptosis.Living cells count was used to assess miR-21 or miR-224 mimics and inhibitor on cell growth.TargetScan was used to explore potential targets of miR-21 and miR-224, and dual luciferase reporter assay was used to identify whether the 3’UTR of Caspase 3, Caspase 9 and Bim mRNA was a binding target of miR-21 or miR-224.Western blot was used to detect the expression of Caspase 3, Caspase 9 and Bim protein after transfection of miR-21 or miR-224 mimics or inhibitors.Results miR-21 and miR-224 are strongly upregulated in GSC samples, multiple GBM human tumor specimens, and GBM neurosphere stem cell lines ( P<0.05 ) .Caspase 3/7 assay and Annexin V apoptosis/fluorescence assay results showed that miR-224 and miR-21 regulated GSC apoptosis.Living cells count results demonstrated that miR-224 and miR-21 regulated GSC growth.miR-224 and miR-21 regulate pro-apoptotic gene expression by directly targeting Caspase 3, Caspase 9, and Bim 3’-UTRs. Conclusion These results indicate that miR-224 and miR-21 are important physiologic drivers of GSC resistance to apoptosis, providing new points of therapeutic leverage against these treatment-resistant cells.