Estrogen-induced epigenetic silencing of <i>FTH1</i> and <i>TFRC</i> genes reduces liver cancer cell growth and survival

Estrogen (E2) regulates hundreds of genes involved in cell metabolism and disrupts iron homoeostasis in various cell types. Herein, we addressed whether E2-induced epigenetic modifications are involved in modulating the expression of iron-regulatory genes. Epigenetic status of FTH1 and TFRC genes was assessed in E2-treated cancer cells. E2-induced DNA methylation was associated with decreased FTH1 and TFRC expression in Hep-G2 and Huh7 cells, but not in AGS or MCF7 cells. Demethylation with 5-Aza-2-deoxycytidine upregulated the expression of both these genes in Hep-G2 cells. The expression of DNMT3B, PRMT5, and H4R3me2s increased in E2-treated cells. Chromatin immunoprecipitation showed that E2 treatment recruited PRMT5 and H4R3me2s on FTH1 but not on TFRC. Knockdown of PRMT5, DNMT3B, and Estrogen-receptor alpha rescued FTH1 from E2-induced silencing. However, knockdown of DNMT3B alone blocked the inhibitory effects of E2 on TFRC. Analysis of human liver tissues in publicly available datasets showed that FTH1 and TFRC are highly expressed in primary liver tumours, but a lower expression is associated with better survival. Interestingly, we showed that the silencing of FTH1 and/or TFRC inhibited carcinogenesis in Hep-G2 cells. For the first time, our findings uncovered the novel signalling pathway involved in the protective effects of E2 against liver cancer.雌激素（E2）可调控数百个参与细胞代谢的基因，并在多种细胞类型中破坏铁稳态。本研究旨在探讨雌激素诱导的表观遗传修饰是否参与调控铁调节基因的表达。研究检测了经E2处理的癌细胞中铁蛋白重链1（FTH1）与转铁蛋白受体（TFRC）基因的表观遗传状态。结果显示，在Hep-G2和Huh7细胞中，E2诱导的DNA甲基化与FTH1、TFRC的表达下调相关，但在AGS与MCF7细胞中无此关联。采用5-氮杂-2'-脱氧胞苷（5-Aza-2-deoxycytidine）进行去甲基化处理，可上调Hep-G2细胞中这两个基因的表达。经E2处理的细胞中，DNA甲基转移酶3B（DNMT3B）、蛋白质精氨酸甲基转移酶5（PRMT5）以及组蛋白H4精氨酸3位点二甲基化（H4R3me2s）的表达水平显著升高。染色质免疫沉淀实验证实，E2处理可将PRMT5与H4R3me2s招募至FTH1基因位点，但无法招募至TFRC基因位点。敲低PRMT5、DNMT3B以及雌激素受体α（Estrogen-receptor alpha）可逆转E2诱导的FTH1基因沉默；但仅敲低DNMT3B即可阻断E2对TFRC的抑制作用。对公共数据集中人肝组织的分析显示，FTH1与TFRC在原发性肝肿瘤中呈高表达，而其低表达与患者更佳的生存预后相关。有趣的是，本研究证实敲低FTH1和/或TFRC可抑制Hep-G2细胞的致癌能力。本研究首次揭示了介导E2对抗肝癌保护作用的全新信号通路。