2020 Fiscal Year Research-status Report
Cytoglobin overexpression inhibits liver fibrosis and cancer development via anti-oxidant function
| Project/Area Number |
19K08428
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| Research Institution | Osaka City University |
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Principal Investigator |
LE THITHANHTHUY 大阪市立大学, 大学院医学研究科, 特任講師 (10572175)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | Cytoglobin / Hepatic stellate cells / Anti-fibrosis / Liver cancer |
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| Outline of Annual Research Achievements |
We have examined therapeutic effects of 6-His tagged recombinant human Cytoglobin (rhCYGB) protein against mouse liver injuries, fibrosis and human HSC activation. In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes via clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of Col1a1 production and HSC activation. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis and oxidative cell damage in TAA- or DDC-administered mice. The injected His-CYGB predominantly localised to HSCs, suggesting specific targeting effects. His-CYGB exhibited no toxicity in humanised liver chimeric PXB mice.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
RNA-Seq analysis of His-CYGB-treated HHSteC samples in comparison with controls revealed the down-regulation of the fibrosis-related genes. Besides that, we also found IFN-stimulated genes such as the genes encoding IFN-inducible proteins IFI27, IFI6, and IFI44L, ISG15, IRF7 and IRF9, and OAS2 were upregulated, suggesting the involvement of IFNb signaling during His-CYGB treatment. His-CYGB treated HHSteC increased the levels of P-TBK1, and secreted IFN-β protein. Simultaneous with IFN-β secretion, STAT1 phosphorylation was observed, secreted IFNb 8 h after His-CYGB challenge. In opposite, the JAK1-specific inhibitor momelotinib attenuated the reduction in COL1A1 production in both His-CYGB- and rhIFN-β treated HHSteCs.
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| Strategy for Future Research Activity |
We have pointed that IFN- β is one of down-stream targets of Cygb in protecting liver injuries. Beside that, we overexpression of Cygb in human HSCs and cancer cells. Immunoprecipitation experiments using Pull-Down Poly-His Protein:Protein Interaction Kit, Sulfo-SBED Biotin Label Transfer Kit, and Dynabeads His-Tag Isolation and pulldown (Invitrogen) to triple confirm the binding targets. We have found some potential CYGB targets including Flotilin-1, Annexin A2, ATPb5. We are now explore the role of each target. For rhCYGB protein production and application: we will perform the shortening full-length of rhCYGB protein to be shorten peptides and explore the important portion of CYGB for its effect.
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| Causes of Carryover |
We need to shorten full-length of rhCYGB protein and explore the CYGB targets. We need to: design primers; PCR amplification of DNA fragments; Double digestion vectors; Ligation reaction; Transformation into DH5α, propagation the plasmids; Verify sequencing; Choose the corrected vectors and transformation into Ecoli BL21-AI; Produce rhCYGB short-peptides; Purify the peptides; In vitro application. Silencing or overexpression of each potential CYGB targets: Flotilin-1, Annexin A2, ATPb5. Examine the interaction between these targets with rhCYGB in vitro and in vivo.
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[Journal Article] Early Change in the Plasma Levels of Circulating Soluble Immune Checkpoint Proteins in Patients with Unresectable Hepatocellular Carcinoma Treated by Lenvatinib or Transcatheter Arterial Chemoembolization2020
Author(s)
2.Odagiri N, Hai H, Thuy LTT, Dong MP, Suoh M, Kotani K, Hagihara A, Uchida-Kobayashi S, Tamori A, Enomoto M, Kawada N
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Journal Title
Cancers (Basel)
Volume: 12
Pages: 2045
DOI
Peer Reviewed / Open Access
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[Journal Article] Hepatocellular carcinoma incidence with tenofovir versus entecavir in chronic hepatitis B: a systematic review and meta-analysis.2020
Author(s)
3.Cheng-Hao Tseng, Yao-Chun Hsu, Tzu-Haw Chen, Fanpu Ji, I-Sung Chen, Ying-Nan Tsai, Hoang Hai, Le Thi Thanh Thuy, Tetsuya Hosaka, Hitomi Sezaki, John A Borghi, Ramsey Cheung, Masaru Enomoto, Mindie H Nguyen
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Journal Title
Lancet Gastroenterol Hepatol
Volume: 5
Pages: 1039-1052
DOI
Peer Reviewed / Open Access
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[Presentation] Hepatocellular carcinoma incidence with tenofovir vs entecavir in chronic hepatitis b: a systematic review and meta-analysis2020
Author(s)
Tseng, CH; Hsu, YC; Chen, TH; Ji, FP; Chen, IS; Tsai, YN; Hai, H; Le Thuy, TT; Hosaka, T; Sezaki, H; Borghi, JA; Cheung, R; Enomoto, M; Nguyen, MH.
Organizer
Annual Meeting of American Association for the Study of Liver Diseases (AASLD)
Int'l Joint Research
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[Presentation] Early change in the plasma levels of circulating soluble immune checkpoint proteins in patients with unresectable hepatocellular carcinoma treated by lenvatinib or transcatheter arterial chemoembolization.2020
Author(s)
Odagiri N, Enomoto M, Hai H, Le Thuy TT, Dong MP, Suoh M, Kotani K, Hagihara A, Uchida S, Tamori A, Kawada N.
Organizer
Annual Meeting of American Association for the Study of Liver Diseases (AASLD)
Int'l Joint Research
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[Presentation] Cytoglobin inhibits pancreatic cancer growth in vitro and in vivo2020
Author(s)
2.Dinh Viet Hoang, Le Thi Thanh Thuy, Hoang Hai, Vu Ngoc Hieu, Ninh Quoc Dat, Truong Huu Hoang, Dong Minh Phuong, Ngo Vinh Hanh, Yoshihiro Ikura, Kenjiro Kimura, and Norifumi Kawada
Organizer
107th General Meeting of the Japanese Society of Gastroenterology
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