Marker for grading and prognosis of malignant tumors

A tumor marker and tumor technology, applied in the field of biomedicine, can solve problems such as insufficient effective control of tumor progression

Inactive Publication Date: 2020-04-24
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although targeted inhibition of kinase activity by small-molecule drugs has achieved good efficacy in the treatment of various cancers, resistance to therapeutic kinase inhibitors caused by nonsense mutations in the catalytic site of kinases is a rare problem in clinical treatment. This is a difficult problem. Therefore, single-targeted small molecule inhibition of AURKB kinase activity is not enough to effectively control tumor progression. Finding a way to regulate AURKB function independent of kinase activity is of great significance in multi-target AURKB therapy for tumors

Method used

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  • Marker for grading and prognosis of malignant tumors
  • Marker for grading and prognosis of malignant tumors
  • Marker for grading and prognosis of malignant tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the detection of AURKB protein cleavage product and the identification of cleavage site

[0040] 1) Identify the amino acid segments specifically recognized by the AURKB antibody against the N-terminus and C-terminus of the AURKB protein:

[0041] AURKB mutants with different N-terminal and C-terminal segments with GFP tags were selected ( figure 1 C) were transfected into human embryonic kidney 293T cells for expression. Cells were collected 24 hours after transfection, and the cells were lysed to extract proteins for western blotting. The primary antibody against the N-terminus of AURKB protein and the primary antibody against the C-terminus of AURKB protein were used to detect the expression of different segments of AURKB protein. The results show that the primary antibody against the N-terminus of the AURKB protein can specifically recognize the 1-75 amino acid segment, and the primary antibody against the C-terminus of the AURKB protein can specific...

Embodiment 2

[0068] Embodiment 2: AURKB protein cleavage inhibitor hinders the proliferation and tumorigenic ability of KSHV-related tumor cells

[0069] In order to confirm the biological significance of AURKB protein cleavage, targeted protease inhibitors PMSF and Aprotinin were used to inhibit AURKB protein cleavage, and the effect of AURKB protein cleavage inhibition on the tumorigenic ability of KSHV-positive cells was studied by cell proliferation and colony formation experiments. The results showed that inhibition of AURKB protein cleavage significantly hindered the proliferation and tumorigenesis of KSHV-positive tumor cells, but had no effect on KSHV-negative tumor cells ( Figure 5 ).

[0070] Specific steps are as follows:

[0071] 1) Cell proliferation experiment steps:

[0072] a.6.25x10 5 BJAB and BCBL-1 cells were seeded in 25cm 2 FLASK culture flask,

[0073] Add 10ml RPMI1640 medium;

[0074] b. Add PMSF (dissolved in ethanol) and Aprotinin protease inhibitor to the ...

Embodiment 3

[0083] Example 3: AURKB protein C-terminal cleavage large fragment product promotes the tumorigenic ability in vivo and in vitro of KSHV positive cells

[0084] For the cleavage site of AURKB protein determined in Example 1, in order to clarify the effect of AURKB protein cleavage products, especially N-terminal cleavage products, on tumor cell growth and tumorigenic ability. Construct pLVX-YFP-AURKB-WT, D76A, 77-344 or 1-250 lentiviral vectors and package lentiviruses, infect KMM cells and BCBL-1-Luc cells respectively, and obtain stable transfected cell lines by flow cytometry . The results of tumor formation experiments in vivo and in vitro showed that AURKB-77-344 could significantly promote cell proliferation in vivo and in vitro ( Figure 6 A, B) and tumorigenic ability ( Figure 6 C shows).

[0085] 1) Suspension cell tumor formation-agar colony formation experimental steps:

[0086] a. 4% agar preparation: Weigh 0.8g of low-melting point agar powder and dissolve it...

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Abstract

The invention belongs to the field of biomedicine, and relates to a novel marker-AURKB protein cleavage product for predicting tumor grading and prognosis effects, wherein the novel marker-AURKB protein cleavage product is an AURKB protein cleavage product, and the cutting site is one or a plurality of sites selected from of D75, D76, K85, D186, D238, D255 and D289. The invention also provides a detection method and an evaluation standard with the AURKB protein cleavage product as a tumor marker. The AURKB cleavage product can be used as a tumor grading and prognosis potential marker, and thetargeting inhibitor of the protein cleavage site of the AURKB cleavage product can be used for anti-tumor treatment.

Description

technical field [0001] The invention belongs to the field of biomedical technology, and relates to the development and application of tumor markers, in particular to the detection method and evaluation standard of AURKB protein cleavage products as tumor markers, as well as the protein cleavage sites on AURKB and their application in the development of targeted drugs potential applications in . Background technique [0002] Indefinite proliferation and division of tumor cells mainly depend on mitosis-related processes. Targeting proteins involved in mitosis is currently a relatively successful anti-tumor strategy. [0003] AURKB protein kinase belongs to the serine / threonine protein kinase family and is an important regulatory protein involved in chromosome separation and cytoplasmic separation in cell mitosis. Abnormal expression level of AURKB protein and abnormal kinase activity often lead to chromosome instability and multinucleation. Overexpression of AURKB has been ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12G01N33/574G01N33/573
CPCC12Y207/11C12N9/12G01N33/57407G01N33/57423G01N33/57446G01N33/57449G01N33/57438G01N33/57415G01N33/573G01N2333/912
Inventor 蔡启良朱青丁玲朱彩霞
Owner FUDAN UNIV
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