Novel hypoxia signal regulation molecule and application thereof

A technology of hypoxia-inducible factor and signal, applied in the field of biomedicine, can solve the problems of hypoxia, uncontrolled growth, insufficient oxygen in the area of ​​liver cancer tissue, etc.

Active Publication Date: 2020-06-12
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the main characteristics that distinguish liver cancer cells from normal cells is that their growth is out of control. The rapid gr

Method used

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  • Novel hypoxia signal regulation molecule and application thereof
  • Novel hypoxia signal regulation molecule and application thereof
  • Novel hypoxia signal regulation molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0183] Example 1, OTUD6B inhibits the metastasis of liver cancer cells

[0184] 1. OTUD6B inhibits the migration of liver cancer cells in vitro

[0185] 1. Construction of MHCC-LM3 cells knocking down OTUD6B and Bel-7402 cells knocking down OTUD6B.

[0186] 2. Western Blot detection of knockdown efficiency and expression of cell migration-related protein markers (E-Cadhenrin, Snail) (anti-E-Cadhenrin antibody and anti-Snail antibody are both products of Santa Cruz Company).

[0187] See the test results figure 1 . The results showed that both targeting shRNA sequences had high knockdown efficiency.

[0188] 3. Cell migration experiments were performed using Transwell assay.

[0189] see results figure 1 . The results showed that the downregulation of OTUD6B significantly promoted the migratory ability of MHCC-LM3 cells compared with the non-targeted knockdown sequence; the migratory ability of Bel-7402 cells was also significantly enhanced after knockdown of OTUD6B.

[...

Embodiment 2

[0205] Example 2, OTUD6B is a hypoxia signal regulation molecule

[0206] 1. RNA-Seq analysis

[0207] RNA was extracted from OTUD6B-knockdown MHCC-LM3 cells and MHCC-LM3 cells, and transcriptome sequencing was performed to analyze the expression of differential genes.

[0208] Some sequencing results are shown in Table 5. The results showed that among the genes upregulated in MHCC-LM3 cells knocking down OTUD6B, many genes were related to cell migration. Further bioinformatics analysis found that 80% of the genes (such as DCN, NOS2DUSP6, etc.) could be enriched in the HIF signaling pathway .

[0209] table 5

[0210]

[0211]

[0212] Note: N means no, Y means yes.

[0213] 2. OTUD6B inhibits the expression of HIF downstream target genes

[0214] Vascular endothelial growth factor (VEGF) is a target gene of HIF. It binds to the ligand VEGFR to activate the downstream signal pathway regulating angiogenesis and promote the metastasis of cancer cells. MMP2 gene and L...

Embodiment 3

[0233] Example 3, OTUD6B affects the migration of liver cancer cells by regulating the VHL-HIF pathway

[0234] 1. OTUD6B stabilizes VHL protein levels

[0235] OTUD6B can regulate the protein levels of HIF-1α and HIF-2α without significant changes in their mRNA expression levels. This result suggests that the regulation of HIF by OTUD6B may be through protein post-translational modification pathways. Therefore, it is speculated that OTUD6B regulates its stability by regulating its ubiquitination process. If OTUD6B is the ubiquitination modification of HIF, it should play a positive regulatory role in its stability, but after knocking down OTUD6B, the protein levels of HIF-1α and HIF-2α are up-regulated, so it is speculated that OTUD6B may regulate HIF-1α The stability of the E3 ubiquitin ligase of HIF-2α and HIF-2α in turn regulates its protein level.

[0236] VHL is a tumor suppressor protein, and its main function is to degrade HIF-1α and HIF-2α by ubiquitination as E3. ...

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Abstract

The invention discloses a novel hypoxia signal regulation molecule and application thereof. The hypoxia signal regulation molecule is deubiquitination enzyme OTUD6B protein. Experiments prove that thedeubiquitination enzyme OTUD6B can inhibit liver cancer cell metastasis, and the expression level of the deubiquitinase OTUD6B has significant correlation with liver cancer cell metastasis and recurrence: the lower the expression quantity of the deubiquitinase OTUD6B is, the shorter the total lifetime of liver cancer patients is, and the higher the tumor recurrence rate is; the deubiquitination enzyme OTUD6B can be used for inhibiting generation of blood vessels; the deubiquitination enzyme OTUD6B can be used for lowering the protein levels of HIF-1 alpha and HIF-2 alpha and inhibiting the expression of a plurality of HIF target genes; the deubiquitination enzyme OTUD6B can increase the protein stability of VHL, and the migration ability of the VHL depends on the VHL protein. Therefore, the deubiquitinase OTUD6B is an anoxia signal regulation molecule and can be used for preventing and/or treating hepatocellular carcinoma and other anoxia-related diseases. The invention has importantapplication value.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a novel hypoxia signal regulating molecule and application thereof. Background technique [0002] Liver cancer mainly includes two types: hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). HCC is the most common type of primary liver cancer, accounting for between 85% and 95% of all liver cancers. Cirrhosis (chronic liver damage caused by inflammation and fibrosis) and viral infections (mainly hepatitis B virus) are important factors in the development of HCC. Long-term alcohol abuse and other metabolic syndromes caused by diabetes and obesity are also common factors that induce HCC. Studies have shown that smoking and intake of aflatoxin B1 can also promote the occurrence and development of HCC; infection with adenovirus 2 can also promote the occurrence of HCC, especially in patients without liver cirrhosis. [0003] Hepatocellular carcinoma metas...

Claims

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Application Information

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IPC IPC(8): A61K38/48A61K45/00A61K48/00A61P35/00A61P35/04
CPCA61K38/4813A61K45/00A61K48/005A61P35/00A61P35/04C12Y304/19012
Inventor 张令强崔春萍刘新鑫张晓莉彭志强
Owner ACADEMY OF MILITARY MEDICAL SCI
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