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

细胞珠蛋白 (CYGB) 过度表达和 CYGB 启动子的靶向去甲基化会损害肝脏和胰腺肿瘤的生长

• 批准号: 21K07921
• 负责人: HOANG HAI
• 金额: $ 2.66万
• 依托单位: Osaka Metropolitan University (2022)Osaka City University (2021)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21K07921/
• 关键词: DNA methylation; CYGB promoter; Liver cancer; Cytoglobin; Promoter; Demethylation; liver tumor; pancreatic tumor

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.

使用Ion GeneStudio S5对来自慢性丙型肝炎病毒感染的肝癌患者的42对肿瘤和邻近非肿瘤组织进行CYGB启动子甲基化评价。在该区域的所有33个CpG位点上，肿瘤组织中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的甲基化指数分别为43.8%和20.5%。肝癌组织中CYGB的mRNA和蛋白表达均显著低于非癌组织（P < 0.05），肝癌细胞（HepG 2、Huh 7、SNU-387、HLE）和肌成纤维细胞LX-2中CYGB的mRNA和蛋白表达均不存在，且甲基化频率较高。相反，在对应于CYGB阳性表达的人肝星状细胞（包括原代细胞和细胞系）中几乎没有甲基化。有趣的是，DAC处理时间和剂量依赖性地恢复了SNU-387、HLE和Huh 7中mRNA和蛋白质水平以及HepG 2细胞中mRNA水平的CYGB表达，而DAC没有诱导LX-2中的CYGB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消退。DAC是常规的去甲基化药物，它可能诱导其他基因启动子的去甲基化。现在我们正在通过dCas 9和gRNA对CYGB进行靶向去甲基化。

项目成果

Investigation of CYGB promoter methylation as a biomarker for hepatocellular carcinoma

CYGB启动子甲基化作为肝细胞癌生物标志物的研究

• 发表时间: 2021
• 作者: Hai H;Thuy LTTT;Tamori A;Kubo S;Takemura S;Tanaka S;Hagihara A;Kawamura E;Uchida-Kobayashi S;Enomoto M and Kawada N
• 通讯作者: Enomoto M and Kawada N

Down-regulation of CYGB expression by promoter methylation is associated with hepatocellular carcinoma progression

启动子甲基化下调 CYGB 表达与肝细胞癌进展相关

• 发表时间: 2022
• 作者: MAWATARI Fumihiro;SHIMIZU Tadashi;MIYAAKI Hisamitsu;ARIMA Tetsuhiko;FUKUDA Sachiko;KITA Yoshiko;FUKAHORI Aiko;ITO Hiroyuki;MATSUKI Kei;IKEMATSU Yoshito;RYU Nobutoshi;NAKAO Kazuhiko;Hoang Hai
• 通讯作者: Hoang Hai