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Angptl4 as a marker for hypoxia detection and its application

A technology for detection of markers and uses, applied in the field of oncology and detection, can solve the problems of lack of methods for serum detection of hypoxia, inconvenient detection, short half-life of HIF-1α protein, etc.

Active Publication Date: 2015-10-28
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the perspective of scientific research and experimentation, the half-life of HIF-1α protein is very short under normoxia, making it inconvenient to detect
[0007] In summary, there is still a lack of satisfactory methods for detecting hypoxia in this field, and even less methods for serum detection of hypoxia

Method used

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  • Angptl4 as a marker for hypoxia detection and its application
  • Angptl4 as a marker for hypoxia detection and its application
  • Angptl4 as a marker for hypoxia detection and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0147] Hypoxia can significantly up-regulate the expression of ANGPTL4

[0148] Liver cancer cell lines SMMC-7721, Huh7, and MHCC-97L cells were cultured under hypoxic conditions for 24 hours, and protein and total RNA were collected for Western blot and quantitative PCR. Upregulation ( figure 1 a, b).

[0149] The hypoxia inducer DFO can simulate the hypoxic environment under normoxia. After DFO treated SMMC-7721 and MHCC-97L cells for 24 hours, it was found that DFO stabilized HIF-1α protein and also up-regulated the expression of ANGPTL4 ( figure 1 c MHCC-97L on the left, SMMC-7721 cells on the right).

[0150] Further, the expression of HIF-1α and ANGPTL4 was detected by immunofluorescence in the nude mouse tumor tissue. The results showed that with the prolongation of tumor formation time, the tumor volume increased and the degree of intratumoral hypoxia increased. HIF-1α and ANGPTL4 The expressions of both were up-regulated and co-expressed in the hypoxic area ( figu...

Embodiment 2

[0153] Hypoxia up-regulates the expression of ANGPTL4 through HIF-1α protein

[0154] In order to determine the effect of hypoxia on ANGPTL4 in liver cancer cells, in this example, siRNA technology (see Table 1 for the sequence) was used to further study the transcription factors that play a key role in this process.

[0155] The experimental results showed that in the liver cancer cells MHCC-97L, after interfering with HIF-1α with siRNA, the level of ANGPTL4 was no longer significantly up-regulated even if the cells were cultured under hypoxic conditions; after interfering with HIF-2α, the cells were cultured under hypoxia , ANGPTL4 protein was still significantly up-regulated ( figure 2 a).

[0156] In order to confirm the reliability of the result, the same experiment was carried out in the liver cancer cells SMMC-7721, and the results showed that the results obtained in two different liver cancer cells were consistent ( figure 2 b).

[0157] 2-Methoxyestradiol (2ME2) ...

Embodiment 3

[0161] ANGPTL4 is a target gene of HIF-1α

[0162] The above experimental results have shown that the expression of HIF-1α and ANGPTL4 are closely related, and hypoxia may regulate the expression of ANGPTL4 through HIF-1α. In this example, a chromatin immunoprecipitation (ChIp) experiment was used for further verification. Methods as below:

[0163]SMMC-7721 and MHCC-97L cells were cultured for 24 hours under hypoxic or normoxic conditions. DNA-protein cross-linking was then performed in PBS containing 1% formaldehyde for 10 min at room temperature. Subsequently, the anti-hypoxia-inducible factor-1α (HIF-1α) monoclonal antibody (purchased from Sigma) or control IgG antibody (purchased from Sigma) was used to carry out precipitation reaction according to the instructions of the chromatin immunoprecipitation kit (Sigma). The precipitated DNA was purified and used as a template for PCR detection.

[0164] If HIF-1α binds to the promoter region of ANGPTL4, when the precipitate...

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Abstract

The invention relates to an ANGPTL4 as a marker of hypoxia detection and an application thereof. Specifically, the invention provides the use of angiopoietin-like protein 4 (ANGPTL4 protein) in the preparation of a diagnostic reagent or a kit of hypoxia detection. The invention further provides a corresponding hypoxia detection kit.

Description

technical field [0001] The present invention relates to the fields of oncology and detection. More specifically, the present invention relates to a serum marker ANGPTL4 that can be used for hypoxia detection and its application. Background technique [0002] ANGPTL4 gene (initially the gene was named pp1158 by the inventor's laboratory) [Zhu Hongxin et al., Chinese Journal of Oncology (2002) 24 (2): 123-125] is to use this functional screening technology platform to clone from the human placenta cDNA library. The full-length cDNA of this gene is 1943bp, the open reading frame contains 1218bp, encodes 406 amino acids, and the estimated molecular weight is 45.2kDa. There is a hydrophobic signal peptide and a coiled-coil domain at the N-terminus, and a Fibrinogen-like domain at the C-terminus. And in 2000, the cDNA sequence of the gene (the original gene name was pp1158) was registered on GenBank (accession number is AF202636). PGAR [Mol.Cell.Biol., Jul 2000; 20:5343-5349] c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/533G01N33/535G01N33/68
Inventor 李锦军李红葛超顾健人
Owner SHANGHAI INST OF ONCOLOGY
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