INSERM U1256, NGERE-Nutrition, Genetics, and Environmental Risk Exposure, University of Lorraine, 54500 Nancy, France.
Service de Génopathies, Centre de Biologie Pathologie Génétique, Centre Hospitalier Régional et Universitaire de Lille, CEDEX, 59037 Lille, France.
Nutrients. 2022 Apr 30;14(9):1887. doi: 10.3390/nu14091887.
Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that , a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.
先前，在五种不同的脑癌细胞系中，以肿瘤球体的形式体外培养癌症干细胞，发现其生长依赖于蛋氨酸。由于早期的研究表明，叶酸依赖性线粒体乙醛脱氢酶基因在神经胶质瘤干细胞中上调，因此我们在本研究中使用 CRISPR-cas9 技术对其进行了无效化处理。我们在这里报告，这种无效化在 U251 神经胶质瘤细胞中是有效的，并且通过对两个独立的靶向外显子 I 或外显子 III 的敲除靶点的基因组测序，无法检测到 cas9 脱靶位点。在 U251 细胞中敲除基因使 U251 细胞中癌症干细胞的生长对蛋氨酸不再依赖。此外，与野生型细胞相比，在敲除细胞中可以检测到更高水平的 ROS（活性氧自由基）。我们的证据将敲除细胞中过量的 ROS 水平与总细胞 NADPH 的减少联系起来。我们的证据还表明，敲除肿瘤球体生长较慢的原因可能与其部分分化有关。
