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Application of chromatin remodeled protein 4A (CHMP4A) in enhancing stability and transcriptional activity of hypoxia-inducible factor 1alpha (HIF-1alpha)

A hypoxia-inducible factor and chromatin technology, applied in the fields of application, recombinant DNA technology, genetic engineering, etc.

Inactive Publication Date: 2011-02-16
SINOGENOMAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on its relationship with cellular hypoxia and its effect on the protein stability and transcriptional activity of hypoxia-inducible factor 1α

Method used

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  • Application of chromatin remodeled protein 4A (CHMP4A) in enhancing stability and transcriptional activity of hypoxia-inducible factor 1alpha (HIF-1alpha)
  • Application of chromatin remodeled protein 4A (CHMP4A) in enhancing stability and transcriptional activity of hypoxia-inducible factor 1alpha (HIF-1alpha)
  • Application of chromatin remodeled protein 4A (CHMP4A) in enhancing stability and transcriptional activity of hypoxia-inducible factor 1alpha (HIF-1alpha)

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Experimental program
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Embodiment 1

[0039] The construction of embodiment 1 reporter gene plasmid (pHRE-LUC)

[0040] The HRE sequence of the promoter region of three genes consisting of erythropoietin (Erythropoietin, EPO), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor (VEGF) 3×HRE sequences were synthesized by Shanghai Sangon. Sense strand (5'-AGCTGCCCTA CGTGCTGTCT CATGCATACG TGGGCTCCAA CAGGTGACTA CGTGCTGCCT AG-3', containing sticky-end sequence of HindIII) and antisense strand (5'-TCGACTAGGC AGCACGTAGT CACCTGTTGG AGCCCACGTA TGCATGAGAC AGCACGTAGG GC-3', containing sticky-end sequence of Xho I ) annealed into a double strand and connected to the pLUC-MCS empty vector cut by HindIII and Xho I, thereby obtaining the reporter gene plasmid pHRE-LUC (such as figure 1 shown).

Embodiment 2HI

[0041] Construction of embodiment 2 HIF-1α eukaryotic expression plasmid

[0042] According to the full-length HIF-1α sequence (GenBank NM_001530), the upstream primer (5-GAAGACATCG CGGGGACC-3) and the downstream primer (5-GGGGTACCGAAAAAAGCTCAGTTAACTTGATC-3) were PCR-amplified from the human normal fetal liver cDNA library to a full-length 2507 bases , connected to the T-easy carrier after gel recovery. Then use Not I single enzyme digestion and connect to pcDNA3.1 / myc-His(-)B (pCDB) with isoenzyme cutting site, thereby obtain HIF-1α eukaryotic expression plasmid pCDB-HIF-1α( figure 2 ). The size of the plasmid conforms to the calculated value ( image 3 ).

Embodiment 3

[0043] The detection of embodiment 3 dual-luciferase reporter system

[0044] Hela cells were routinely subcultured in DMEM medium (HyClone, SH30022.01B) containing 10% fetal bovine serum (HyClone, SH30084.03) at 37°C, 5% CO 2 cultured in an incubator. According to 1.1×10 6 The density of cells / ml is inoculated in 96-well plates, 100 μl per well, and cultured in an incubator for 18 hours to 24 hours, so that the cell density reaches 40% to 60%, ready for transfection. According to the instructions, 50ng of pHRE-LUC, 5ng of pRL-TK-LUC and 50ng of the gene to be screened or the control plasmid were co-transfected into Hela cells using VigoFect cationic transfection reagent (Vigolas Biotechnology (Beijing) Co., Ltd.). Cells transfected with pcDNA3.1 / myc-His(-)B empty vector were used as blank control. Cells transfected with pCDB-HIF-1α and ING4 gene (ING4 connected in PCDB vector, as a positive control, similar to HIF-1a) were used as positive controls for activating the expre...

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Abstract

The invention relates to an application of a chromatin remodeled protein 4A (CHMP4A) in promoting growth of tumour cells, in particular to an application of CHMP4A in enhancing the stability and transcriptional activity of a hypoxia- inducible factor 1alpha(HIF-1alpha) and an application thereof in screening a lead compound capable of inhibiting growth of tumour cells. In the invention, a cis-reporter gene screening system is adopted to find that CHMP4A can obviously enhance the stability and transcriptional activity of the hypoxia-inducible factor 1alpha in a Hela cell.

Description

technical field [0001] The present invention relates to the application of chromatin remodeling protein 4A (CHMP4A) in promoting the growth of tumor cells, especially the application of CHMP4A in enhancing the stability and transcriptional activity of hypoxia-inducible factor 1α (HIF-1α); Application of lead compounds in tumor cell growth. Background technique [0002] Tumor hypoxia is a common phenomenon in solid tumors. Hypoxia can trigger a series of emergency protective responses in the body, making tumor cells adapt to the hypoxic microenvironment. Hypoxia inducible factor 1 (hypoxia inducible factor 1, HIF-1) is the transcription factor most closely related to hypoxia found so far. , function, regulation and the relation to the hypoxia signal transduct of hypoxia inducible factor 1. Progress Of Physiology science, 2001, 32(1): 62-64). In recent years, the key regulatory role of HIF-1α in tumor occurrence, invasion, survival, and drug resistance has attracted increasi...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12N15/12C12N15/85
Inventor 石太平董云巧李静高鹏邓唯唯雄英马大龙
Owner SINOGENOMAX
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