Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1, 32-083, Balice, Poland.
Center for Experimental and Innovative Medicine, The University of Agriculture in Krakow, Redzina 1C, 30 248, Krakow, Poland.
J Neurooncol. 2023 May;163(1):61-69. doi: 10.1007/s11060-023-04304-8. Epub 2023 May 11.
We have previously shown that TRDMT1 methyltransferase is a regulator of chemotherapy-associated responses in glioblastoma cells. Despite the fact that glioblastoma, a common and malignant brain tumor, is widely characterized in terms of genetic and epigenetic markers, there are no data on TRDMT1-related changes in 5-methylcytosine pools in the genome. In the present study, the effect of TRDMT1 gene knockout (KO) on DNA methylome was analyzed.
CRISPR-based approach was used to obtain TRDMT1 KO glioblastoma cells. Total 5-methylcytosine levels in DNA, DNMT1 pools and DNMT activity were studied using ELISA. Reduced representation bisulfite sequencing (RRBS) was considered to comprehensively evaluate DNA methylome in glioblastoma cells with TRDMT1 KO.
TRDMT1 KO cells were characterized by decreased levels of total 5-methylcytosine in DNA and DNMT1, and DNMT activity. RRBS-based methylome analysis revealed statistically significant differences in methylation-relevant DMS-linked genes in control cells compared to TRDMT1 KO cells. TRDMT1 KO-associated changes in DNA methylome may affect the activity of several processes and pathways such as telomere maintenance, cell cycle and longevity regulating pathway, proteostasis, DNA and RNA biology.
TRDMT1 may be suggested as a novel modulator of gene expression by changes in DNA methylome that may affect cancer cell fates during chemotherapy. We postulate that the levels and mutation status of TRDMT1 should be considered as a prognostic marker and carefully monitored during glioblastoma progression.
我们之前已经表明，TRDMT1 甲基转移酶是胶质母细胞瘤细胞中与化疗相关反应的调节剂。尽管胶质母细胞瘤是一种常见且恶性的脑肿瘤，但在基因组中 5-甲基胞嘧啶池方面，尚无关于 TRDMT1 相关变化的资料。在本研究中，分析了 TRDMT1 基因敲除（KO）对 DNA 甲基组的影响。
使用基于 CRISPR 的方法获得 TRDMT1 KO 胶质母细胞瘤细胞。使用 ELISA 研究 DNA 中总 5-甲基胞嘧啶水平、DNMT1 池和 DNMT 活性。考虑使用简化代表性双硫代修饰测序（RRBS）全面评估具有 TRDMT1 KO 的胶质母细胞瘤细胞中的 DNA 甲基组。
TRDMT1 KO 细胞的特征是 DNA 和 DNMT1 以及 DNMT 活性中的总 5-甲基胞嘧啶水平降低。基于 RRBS 的甲基组分析显示，与 TRDMT1 KO 细胞相比，对照细胞中与甲基化相关的 DMS 相关基因存在统计学上的显著差异。TRDMT1 KO 相关的 DNA 甲基组变化可能会影响几个过程和途径的活性，例如端粒维持、细胞周期和长寿调节途径、蛋白质稳态、DNA 和 RNA 生物学。
TRDMT1 可能被认为是通过影响化疗期间癌细胞命运的 DNA 甲基组变化的基因表达的新型调节剂。我们假设 TRDMT1 的水平和突变状态应被视为预后标志物，并在胶质母细胞瘤进展过程中仔细监测。
