Low Level Radiation Research Section, Radiation Biology and Health Sciences Division, Bio-Sciences Group, Bhabha Atomic Research Centre, Trombay, Mumbai, 400 085, India.
Homi Bhabha National Institute, Anushaktinagar, Trombay, Mumbai, 400 094, India.
Mol Cell Biochem. 2022 Jan;477(1):267-281. doi: 10.1007/s11010-021-04265-4. Epub 2021 Oct 27.
DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR) genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1 Gy (low) and 2.0 Gy (high) doses of γ-radiation and proliferated for 48 h and 72 h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM) method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1 Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72 h. At 2.0 Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48 h and 72 h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.
DNA 甲基化是一种表观遗传机制，在基因调控中起着重要作用。本研究评估了 LINE1 重复序列的 DNA 甲基化谱以及人外周血单个核细胞（PBMCs）中 DNA 损伤反应（DDR）和 DNA 修复（DR）基因（PARP1、ATM、BRCA1、MLH1、XPC、RAD23B、APC、TNFα、DNMT3A、MRE11A、MGMT、CDKN2A、MTHFR）启动子甲基化对γ射线的反应。选择 DDR 和 DR 基因（APC、MLH1、PARP1、MRE11A、TNFα、MGMT）的基因表达谱与甲基化水平相关联，以了解它们在基因调控中的作用。从 15 名随机健康供体中采集血液样本，分离 PBMCs，用 0.1Gy（低）和 2.0Gy（高）γ射线照射，并在 48 小时和 72 小时进行增殖。从照射和未照射的 PBMC 中分离基因组 DNA 和总 RNA。用亚硫酸氢盐转化的 DNA 进行甲基化谱测定，并通过甲基化敏感高分辨率熔解（MS-HRM）方法进行扩增。将总 RNA 转化为 cDNA，并使用实时定量 PCR 分析相对表达。我们的结果表明，在 0.1Gy 时，MRE11A 和 TNFα 在 72 小时时甲基化水平显著增加（P<0.05）。在 2.0Gy 时，在 LINE1、MRE11A、PARP1、BRCA1、DNMT3A 和 RAD23B 中观察到甲基化谱在 48 小时和 72 小时时显著增加（P<0.05）。PARP1 的甲基化状态与基因表达呈显著正相关。总之，低剂量和高剂量的 γ 射线对 LINE1、DDR 和 DR 基因的 DNA 甲基化状态有显著影响，这表明它们作为人类 PBMCs 中表观遗传标记的潜在作用，这可以在人类群体中进一步探索。
