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Fusaric acid decreases p53 expression by altering promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells
Epigenetics ( IF 3.7 ) Pub Date : 2020-07-07 , DOI: 10.1080/15592294.2020.1788324
Terisha Ghazi 1 , Savania Nagiah 1 , Anil A Chuturgoon 1
Affiliation  

ABSTRACT

Fusaric acid (FA) is a food-borne mycotoxin that mediates toxicity with limited information on its epigenetic properties. p53 is a tumour suppressor protein that regulates cell cycle arrest and apoptotic cell death. The expression of p53 is regulated transcriptionally by promoter methylation and post-transcriptionally by N-6-methyladenosine (m6A) RNA methylation. We investigated the effect of FA on p53 expression and its epigenetic regulation via promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells. HepG2 cells were treated with FA [0, 25, 50, 104, and 150 µg/ml; 24 h] and thereafter, DNA, RNA, and protein was isolated. Promoter methylation and expression of p53 was measured using qPCR and Western blot. RNA immuno-precipitation was used to determine m6A-p53 levels. The expression of m6A methyltransferases (METTL3 and METTL14), demethylases (FTO and ALKBH5), and readers (YTHDF1-3 and YTHDC2) were measured using qPCR. FA induced p53 promoter hypermethylation (p < 0.0001) and decreased p53 expression (p < 0.0001). FA decreased m6A-p53 levels (p < 0.0001) by decreasing METTL3 (p < 0.0001) and METTL14 (p < 0.0001); and suppressed expression of YTHDF1 (p < 0.0001), YTHDF3 (p < 0.0001), and YTHDC2 (p < 0.0001) that ultimately reduced p53 translation (p < 0.0001). Taken together, the data shows that FA epigenetically decreased p53 expression by altering its promoter methylation and m6A RNA methylation in HepG2 cells. This study reveals a mechanism for p53 regulation by FA and provides insight into future therapeutic interventions.



中文翻译:

Fusaric acid 通过改变人肝细胞癌 (HepG2) 细胞中的启动子甲基化和 m6A RNA 甲基化来降低 p53 表达

摘要

Fusaric acid (FA) 是一种食源性真菌毒素,可介导毒性,但其表观遗传特性的信息有限。p53 是一种肿瘤抑制蛋白,可调节细胞周期停滞和细胞凋亡。p53 的表达受启动子甲基化的转录调控和 N-6-甲基腺苷 (m6A) RNA 甲基化的转录后调控。我们通过启动子甲基化和 m6A RNA 甲基化研究了 FA 对 p53 表达的影响及其在人肝细胞癌 (HepG2) 细胞中的表观遗传调控。HepG2 细胞用 FA [0、25、50、104 和 150 µg/ml;24 小时],然后分离 DNA、RNA 和蛋白质。使用 qPCR 和蛋白质印迹测量 p53 的启动子甲基化和表达。RNA免疫沉淀用于测定m6A- p53水平。使用 qPCR 测量m6A 甲基转移酶(METTL3METTL14)、去甲基化酶(FTOALKBH5)和读数器(YTHDF1-3YTHDC2)的表达。FA 诱导p53启动子超甲基化 ( p < 0.0001) 并降低p53表达 ( p < 0.0001)。FA降低m6A- p53的水平(p <0.0001)通过降低METTL3p <0.0001)和METTL14p <0.0001); 并抑制YTHDF1 的表达(p < 0.0001)、YTHDF3 ( p < 0.0001) 和YTHDC2 ( p < 0.0001) 最终减少了 p53 的翻译 ( p < 0.0001)。综上所述,数据表明 FA 通过改变 HepG2 细胞中的启动子甲基化和 m6A RNA 甲基化,在表观遗传上降低了 p53 的表达。这项研究揭示了 FA 调节 p53 的机制,并为未来的治疗干预提供了见解。

更新日期:2020-07-07
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