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2021 年度 実施状況報告書

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

研究課題

研究課題/領域番号 21K07921
研究機関大阪市立大学

研究代表者

HOANG HAI  大阪市立大学, 大学院医学研究科, 研究員 (60623246)

研究分担者 LE THITHANHTHUY  大阪市立大学, 大学院医学研究科, 特任講師 (10572175)
河田 則文  大阪市立大学, 大学院医学研究科, 教授 (30271191)
研究期間 (年度) 2021-04-01 – 2024-03-31
キーワードDNA methylation / CYGB promoter / Liver cancer
研究実績の概要

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 (-60%, 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.
Luciferase assay data demonstrated that the CYGB promoter region spanning from -994 to -639 relative to translation start site exhibited a significant decrease in luciferase activity.

現在までの達成度 (区分)
現在までの達成度 (区分)

2: おおむね順調に進展している

理由

Our institutional laboratories supported a lot.

今後の研究の推進方策

1- Create site-directed mutagenesis at other CpG sites. Examine the activity of Cygb promoter: original and site-directed mutagenesis promoters.
2- gRNA Cloning into pPlatTET-gRNA2. Transfection of non-expressing CYGB HCC cells for DNA demethylation.

次年度使用額が生じた理由

Due to COVID-19 situation last year, the incurring amount will be used next fiscal year. This is planned to use in two main experiments:
1- Create site-directed mutagenesis at other CpG sites. Examine the activity of Cygb promoter: original and site-directed mutagenesis promoters.
2- gRNA Cloning into pPlatTET-gRNA2. Transfection of non-expressing CYGB HCC cells for DNA demethylation.

  • 研究成果

    (1件)

すべて 2021

すべて 学会発表 (1件) (うち国際学会 1件)

  • [学会発表] Investigation of CYGB promoter methylation as a biomarker for hepatocellular carcinoma2021

    • 著者名/発表者名
      Hai H, Thuy LTTT, Tamori A, Kubo S, Takemura S, Tanaka S, Hagihara A, Kawamura E, Uchida-Kobayashi S, Enomoto M and Kawada N
    • 学会等名
      AASLD The Liver Meeting
    • 国際学会

URL: 

公開日: 2022-12-28  

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