Sajadian Sahar Olsadat, Ehnert Sabrina, Vakilian Haghighat, Koutsouraki Eirini, Damm Georg, Seehofer Daniel, Thasler Wolfgang, Dooley Steven, Baharvand Hossein, Sipos Bence, Nussler Andreas K
Eberhard-Karls University Tübingen, BG Trauma Clinic, SWI, Schnarrenbergstraße 95, 72076 Tübingen, Germany.
Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Clin Epigenetics. 2015 Sep 11;7(1):98. doi: 10.1186/s13148-015-0133-x. eCollection 2015.
Global deregulation of DNA methylation is one of the crucial causes of hepato cellular carcinoma (HCC). It has been reported that the anti-cancer drug 5-azacytidine (5-AZA) mediates the activation of tumor suppressor genes through passive demethylation by inhibiting DNMT1. Recent evidence suggests that active demethylation which is mediated by ten-eleven translocation (TET) proteins may also be an important step to control global methylation. However, there exists a controversial discussion in which TET proteins are involved in the demethylation process in HCC. Therefore, we firstly wanted to identify which of the TETs are involved in demethylation and later to study whether or not 5-AZA could trigger the TET-dependent active demethylation process in HCC. HCC cell lines (Huh-7, HLE, HLF), primary human hepatocytes (hHeps), and tissues from both healthy (55 patients) and HCC patients (55 patients) were included in this study; mRNA levels of isocitrate dehydrogenase (IDH1, 2) and TETs (TET1-3) were studied via qPCR and confirmed by Western blot. The expression of 5hmC/5mC was determined by immunohistochemistry in human HCC tissues and the corresponding adjacent healthy liver. HCC cell lines were stimulated with 5-AZA (0-20 μM) and viability (Resazurin conversion), toxicity (LDH release), proliferation (PCNA), and 5hmC/5mC distribution were assessed. In addition, knockdown experiments on TET proteins in HCC cell lines using short interference RNAs (siRNAs), in the presence and absence of 5-AZA, were performed.
Our data applying qPCR, immunofluorescence, and Western blotting clearly show that TET2 and TET3 but not TET1 were significantly decreased in HCC tissue and different HCC cell lines compared to non-tumor liver tissues and hHeps. In addition, we show here for the first time applying knockdown experiments that 5-AZA is able to trigger an active TET2-dependent demethylation process with concomitant significant changes in 5hmC/5mC in HCC cell lines and hHeps.
Our data clearly show that the expression and activity of TET2 and TET3 proteins but not TET1 are impaired in hepatocellular carcinoma leading to the reduction of 5hmC in HCCs. Furthermore, this study identified a novel function of 5-azacytidine in promoting a TET-mediated generation of 5hmC suggesting that the availability of 5-AZA in cancer cells will have various effects on different epigenetic targets. These findings may open new therapeutic strategies for epigenetic drugs to treat HCC.
DNA甲基化的整体失调是肝细胞癌（HCC）的关键病因之一。据报道，抗癌药物5-氮杂胞苷（5-AZA）通过抑制DNA甲基转移酶1（DNMT1）介导的被动去甲基化来激活肿瘤抑制基因。最近的证据表明，由十一-易位（TET）蛋白介导的主动去甲基化也可能是控制整体甲基化的重要步骤。然而，关于TET蛋白是否参与HCC的去甲基化过程存在争议性讨论。因此，我们首先想确定哪些TET蛋白参与去甲基化，随后研究5-AZA是否能在HCC中触发TET依赖的主动去甲基化过程。本研究纳入了HCC细胞系（Huh-7、HLE、HLF）、原代人肝细胞（hHeps）以及来自健康人群（55例患者）和HCC患者（55例患者）的组织；通过定量聚合酶链反应（qPCR）研究异柠檬酸脱氢酶（IDH1、2）和TET蛋白（TET1 - 3）的mRNA水平，并通过蛋白质免疫印迹法进行确认。通过免疫组织化学法测定人HCC组织及相应的相邻健康肝脏组织中5-羟甲基胞嘧啶（5hmC）/5-甲基胞嘧啶（5mC）的表达。用5-AZA（0 - 20 μM）刺激HCC细胞系，并评估细胞活力（刃天青转化）、毒性（乳酸脱氢酶释放）、增殖（增殖细胞核抗原，PCNA）以及5hmC/5mC分布。此外，在有和没有5-AZA存在的情况下，使用小干扰RNA（siRNAs）对HCC细胞系中的TET蛋白进行敲低实验。
我们应用qPCR、免疫荧光和蛋白质免疫印迹法的数据清楚地表明，与非肿瘤肝脏组织和hHeps相比，HCC组织和不同的HCC细胞系中TET2和TET3而非TET1显著降低。此外，我们首次通过敲低实验表明，5-AZA能够在HCC细胞系和hHeps中触发依赖TET2的主动去甲基化过程，同时5hmC/5mC发生显著变化。
我们的数据清楚地表明，肝细胞癌中TET2和TET3蛋白而非TET1的表达和活性受损，导致HCC中5hmC减少。此外，本研究确定了5-氮杂胞苷在促进TET介导的5hmC生成方面的新功能，表明癌细胞中5-AZA的可用性将对不同的表观遗传靶点产生多种影响。这些发现可能为表观遗传药物治疗HCC开辟新的治疗策略。
