Method for inhibiting tumor growth by DDX24 helicase point mutation and application of DDX24 helicase
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A DDX24, point mutation technology, applied in the field of biomedicine, can solve the problems of lack of systemic cells or living animal research
Active Publication Date: 2020-06-09
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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[0003] The current TCGA clinical database shows that DDX24 is overexpressed in head and neck squamous cell carcinoma, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, skin melanoma, lymphoma, adenothelioma, thymoma and other tumors, suggesting that DDX24 has Cancer-promoting function, but lack of systematic cell or live animal studies
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Embodiment 1
[0023] 1. A method for amplifying wild and point mutation genes of DDX24, screening monoclonal cell lines, and constructing point mutation models, wherein the DDX24 mutation site includes K11E or E271K, comprising the following steps:
[0024] Search the gene library to obtain the nucleotide and amino acid sequences of the wild-type human DDX24; design and screen primer pairs in the online primer design of the NCBI database to amplify the target genes of the wild-type and point mutant DDX24 of human origin. Primer pairs such as figure 1 shown.
[0025] Then construct the plasmid containing the target gene respectively. In order to achieve dynamic observation research at the cell level, the target carrier pmCherry-N1 of Clontech was selected. mCherry is a red fluorescent protein behind the carboxyl terminal of the target gene DDX24, and its maximum absorption / emission peaks are located at 587 nm and 610 nm, respectively. Therefore, the expression of the target gene DDX24 can ...
Embodiment 2
[0038] 1. Proliferation experiments of wild and mutant monoclonal cells of DDX24:
[0039] The cell suspension (100 μL / well) seeded in a 96-well plate, the number of cells is 800. The plates were pre-incubated for 24 hours in an incubator (37°C, 5% CO2). Add 10 μL of CCK-8 solution to each well of the culture plate, place it in an incubator and incubate for 2 hours, and measure the absorbance at 450 nm with a microplate reader.
[0040] The results showed that CHO-WT-DDX24 cells proliferated faster, followed by CHO-Vector cells, while cells with DDX24 point mutation showed slower proliferation.
[0041] 2. Tumor-bearing experiment in nude mice
[0042] The animal strain used in this example is Balb / cnu nude mice. Female nude mice aged 5-6 weeks were randomly divided into four groups, including CHO-Vector control group, CHO-WT-DDX24, CHO-MT-E271K-DDX24 , CHO-MT-K11E-DDX24 experimental group, 10 rats in each group.
[0043] Wild-type and point-mutated monoclonal cell lines w...
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Abstract
The invention discloses a newly discovered cancer promoting effect of DDX24 helicase, a method for realizing a cancer inhibition effect through point mutation and application of the DDX24 helicase. The contents of the invention comprise: 1, high expression of wild type DDX24 has significant correlation with tumor growth, and the growth of tumors can be promoted through high expression of the wildtype DDX24; 2, different from the mechanism of the pathogenic gene DDX24 for familial vascular malformation, DDX24 point mutation including K11E or E271K obviously inhibits the growth of tumors; 3, based on the new function and induced point mutation of DDX24 provided in the invention, a novel targeted therapy method can be provided for treatment of latent and refractory tumors, and a new thoughtcan also be provided for preparation of tumor-related vaccines; and 4, application of 1-3 described above is provided. The point mutation can be used for treating solid tumors including, but not limited to, squamous cell carcinoma of the head and the neck, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, skin melanoma, lymphoma, adenoma, thoracic adenoma, lung cancer, colorectal cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, nasopharyngeal cancer, bone cancer andmalignant myoma.
Description
technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for promoting cancer by wild-type DDX24 and suppressing tumor after point mutation and its application. Background technique [0002] The DDX24 gene is located in the 32 region of the long arm of chromosome 14 and is widely distributed in various tissues of the human body. It was previously considered that DDX24 is a member of the DEAD-box family, the largest member of the non-cyclic superfamily 2 helicase (Fairman-Williams, Guenther et al, 2010, Curr Opin Struct Biol. 20(3): 313-324), with ATP Dependent RNA helicase activity and RNA-dependent ATPase activity are involved in translation initiation and post-translational modification to regulate cellular physiological activities (Jarmoskaite, Russell, 2011, 2010 John Wiley&Sons, Ltd. 2: 135-152). Another article pointed out that DDX24 may be involved in the formation of visceral vascular malformations (Pang, Hu et al...
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