Cytoglobin (CYGB)-overexpression and targeted demethylation of CYGB promoter impair liver and pancreatic tumor growth

应用Ion GeneStudio S5对42对慢性丙型肝炎病毒感染的肝癌患者的肿瘤及癌旁组织进行了CYGB启动子甲基化检测。在该区域33个CpG位点上，肿瘤组织的甲基化频率显著高于非肿瘤组织(P=1.02E-8)。肿瘤组织和非肿瘤组织甲基化指数均值分别为43.8%和20.5%。检测了部分肝细胞癌组织标本，发现肿瘤组织中细胞色素B基因的mRNA和蛋白表达显著低于非肿瘤组织(P&lt；0.05)。在体外，在肝癌细胞(HepG2、Huh7、SNU-387、HLE)和众所周知的肌成纤维细胞LX-2细胞中发现了较高的甲基化频率，而在RNA和蛋白水平上没有表达。相比之下，人肝星状细胞(原代和细胞系)中几乎没有与CygB阳性表达相对应的甲基化。经1、3、5和10um 5-aza-2‘-deoxcytidine(DAC)处理的四种肝癌细胞株的CygB表达被恢复。有趣的是，DAC处理时间和剂量依赖地恢复了SNU-387、HLE和HuH7细胞中CygB的mRNA和蛋白表达，以及HepG2细胞中的mRNA表达，而DAC不能诱导LX-2细胞中的Cygb表达。值得注意的是，在SNU-387细胞中诱导了CygB的表达后，去掉DAC可以在mRNA和蛋白质水平上导致CygB表达的倒退。DAC是传统的去甲基化药物，它可以诱导其他基因启动子的去甲基化。现在，我们正在通过dCas9和gRNA对Cygb进行靶向去甲基化。

Forty-two pairs of tumor and adjacent non-tumor tissues from liver cancer patients with chronic hepatitis C virus infection were evaluated for CYGB promoter methylation using Ion GeneStudio S5. Methylation frequency in tumors is significantly higher than that in non-tumor tissues at all 33 CpG sites (P = 1.02E-8) in this region. The mean of methylation index in tumor and non-tumor tissues were 43.8% and 20.5%, respectively. A subset of HCC samples was examined, CYGB mRNA and protein expression in tumor is significantly lower than that in non-tumor tissues (P < 0.05).In vitro, high methylation frequency and no CYGB expression at RNA and protein levels were found in HCC cells (HepG2, Huh7, SNU-387, HLE) and also well-known myofibroblast LX-2 cells. In contrast, almost no methylation in human hepatic stellate cells (both primary and cell line) that correspond to positive CYGB expression.Restoration of CYGB expression was performed in four HCC cell lines treated with 1, 3, 5, and 10 uM 5-aza-2′- deoxycytidine (DAC). Interestingly, DAC treatment time- and dose-dependently restored CYGB expression at both mRNA and protein levels in SNU-387, HLE and Huh7, and at mRNA level in HepG2 cells while DAC did not induce CYGB expression in LX-2. Notably, after inducing CYGB expression in SNU-387, removal of DAC resulted in regressing of CYGB expression at both mRNA and protein levels.DAC is conventional demethylation drug, it may induce demethylation of other gene promoters. Now we are doing targeted demethylation of CYGB by dCas9 and gRNA.