2022 Fiscal Year Research-status Report
Cytoglobin (CYGB)-overexpression and targeted demethylation of CYGB promoter impair liver and pancreatic tumor growth
| Project/Area Number |
21K07921
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| Research Institution | Osaka Metropolitan University |
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Principal Investigator |
HOANG HAI 大阪公立大学, 大学院医学研究科, 研究員 (60623246)
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| Co-Investigator(Kenkyū-buntansha) |
LE THITHANHTHUY 大阪公立大学, 大学院医学研究科, 特任講師 (10572175)
河田 則文 大阪公立大学, 大学院医学研究科, 教授 (30271191)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | DNA methylation / CYGB promoter / Liver cancer |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Our institutional laboratories supported a lot.
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| Strategy for Future Research Activity |
gRNA Cloning into pPlatTET-gRNA2. Transfection of non-expressing CYGB HCC cells for DNA demethylation.
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| Causes of Carryover |
Due to COVID-19 situation last year, the incurring amount will be used next fiscal year. This is planned to use in the main experiment:
gRNA Cloning into pPlatTET-gRNA2. Transfection of non-expressing CYGB HCC cells for DNA demethylation.
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