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

使用Ion GeneStudio S5对慢性丙型肝炎病毒感染肝癌患者的42对肿瘤和邻近非肿瘤组织进行CYGB启动子甲基化评价。该区域33个CpG位点的甲基化频率在肿瘤组织中显著高于非肿瘤组织（P = 1.02E-8）。肿瘤组织和非肿瘤组织的甲基化指数平均值分别为43.8%和20.5%。肿瘤组织中CYGB mRNA和蛋白的表达明显低于非肿瘤组织（P < 0.05）。在体外，HCC细胞（HepG2, Huh7, SNU-387， HLE）和众所周知的肌成纤维细胞LX-2细胞中发现高甲基化频率和RNA和蛋白质水平上没有CYGB表达。相比之下，在人肝星状细胞（包括原代和细胞系）中几乎没有甲基化，对应于CYGB阳性表达。用1、3、5和10 μ m 5-aza-2′-脱氧胞苷（DAC）处理4株HCC细胞系，恢复CYGB的表达。有趣的是，DAC处理时间和剂量依赖性地恢复了SNU-387、HLE和Huh7中CYGB mRNA和蛋白水平的表达，以及HepG2细胞中CYGB mRNA水平的表达，而DAC不诱导LX-2中CYGB的表达。值得注意的是，在SNU-387中诱导CYGB表达后，去除DAC导致CYGB在mRNA和蛋白水平上的表达下降。DAC是常规的去甲基化药物，可诱导其他基因启动子的去甲基化。现在我们正在用dCas9和gRNA进行CYGB的靶向去甲基化。

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.