Medicinal use of e3 ligase fbw7 in delaying aging and related diseases

A technology of ligase and aging, which is applied in the field of medical application of E3 ligase FBW7 in delaying aging and related diseases, and can solve the problem that the inhibitory effect of FBW7 is not completely clear

Active Publication Date: 2020-12-15
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In mammalian mice, loss of FBW7 function activates stem cell expansion; FBW7 is mutated in a variety of human tumors, but the inhibitory role of FBW7 in cell proliferation is not fully understood

Method used

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  • Medicinal use of e3 ligase fbw7 in delaying aging and related diseases
  • Medicinal use of e3 ligase fbw7 in delaying aging and related diseases
  • Medicinal use of e3 ligase fbw7 in delaying aging and related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Inhibition of the protective effect of FBW7 on telomere-binding protein TPP1 under stress conditions

[0023] Experimental objects: human alveolar basal adenocarcinoma cell A549 and human embryonic kidney cell 293T.

[0024] Experimental method: A549 cells were transfected (transfection reagent was lipofectemin2000) with siRNA against FBW7, MKRN1, CHIP, FBX4, hDM2, RNF8, SIAH2 and control to inhibit their expression, and hydrogen peroxide (100 μM , 16 hours), bleomycin (BLM, 200 μM, 16 hours) and X-ray irradiation (0 Gy and 6 Gy) treatment, using real-time quantitative PCR to detect the interference of FBW7, MKRN1, CHIP, FBX4, hDM2, RNF8 and SIAH2 Efficiency, the expression of TPP1 and internal reference GAPDH was detected by immunoblotting; the plasmids overexpressing FBW7, FBW7γ and TPP1 with HA tag were transfected into 293T cells by lipofectemin2000, and the cells were subjected to CHX after 48 hours of transfection (respectively treated 3, 6 and 9 hours)...

Embodiment 2

[0026] Example 2 Protective effect of inhibiting FBW7 on telomere DNA damage under stress conditions

[0027] Experimental object: human cervical cancer cell Hela.

[0028] Experimental method: Hela cells were transfected with siRNA against FBW7, MKRN1, CHIP, FBX4, hDM2, RNF8, SIAH2 and the control (the transfection reagent was lipofectemin2000) to inhibit their expression, and the cells were given hydrogen peroxide (100 μM and 200μM for 4 hours), bleomycin (BLM, 200μM for 16 hours) and X-ray irradiation (5Gy) treatment, using immunofluorescence (primary antibody is 53BP1) combined with in situ hybridization (Cy3-labeled telomere probe ) (IF-FISH) to detect telomere DNA damage; Hela cells were transfected with siRNA against FBW7 and TPP1, and after 48 hours of transfection, the cells were irradiated with X-rays (0Gy and 5Gy), and then cultured for 1 hour and then used Telomere DNA damage was detected by IF-FISH (supra), and mRNA expression of TPP1 and FBW7 was detected by qua...

Embodiment 3

[0030] Example 3 Effects of FBW7 on telomere length, telomere DNA damage and cell senescence

[0031] Experimental objects: human cervical cancer cells Hela and human fibrosarcoma cells HT1080.

[0032] Experimental method: Transfect Hela or HT1080 cells with plasmids expressing human FBW7α, β and γ subunits, and detect the expression of the three subunits by immunoblotting 48 hours after transfection; detect the opposite ends of the three subunits by chromatin immunoprecipitation The role of telomeric DNA damage; using in situ hybridization and flow combined in situ hybridization (Flow-FISH) to detect the length of telomeric DNA; the cells in the metaphase after the cells were treated with colchicine were detected by FISH. granule signal; use flow cytometry to detect the distribution of cell cycle, and use western blot to detect the expression of senescence-related proteins p16, p53 and p21; use immunofluorescence to detect the expression of HP1γ and p21; use siRNA to transfe...

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Abstract

Disclosed is a use of E3 ligase FBW7 in preparing a drug for delaying aging and for preventing and treating aging-related diseases by means of inhibiting telomere damage, wherein the amino acid sequence of the FBW7 is as shown in SEQ. No.2. Knockout or RNA interference inhibiting the gene expression of the FBW7, a gene mutant expressing the FBW7 competitively inhibiting the FBW7 protein, or the overexpression of TPP1 overcoming the degradation caused by the FBW7 can protect the chromosome telomere from damage caused by stress, such as radiation, bleomycin and hydrogen peroxide, prevent the degradation of TPP1 in the basic state or in the stress state, result in the effect of extending telomere length, increase the number of tissue stem cells, improve the pulmonary respiratory function in the basic state, and prevent the occurrence of lung tissue aging and pulmonary fibrosis in the stress state, and same can be used in preparing a drug for preventing and treating telomere dysfunction to provide ideas and means for anti-aging.

Description

technical field [0001] The invention belongs to biotechnology and relates to the medical application of inhibiting the expression of FBW7 protein, in particular to the medical application of E3 ligase FBW7 in delaying aging and related diseases. The use of protecting its substrate TPP1 by inhibiting FBW7 protein in the preparation of drugs for delaying aging and preventing and treating aging-related diseases, especially related to anti-aging and preventing and treating related diseases by inhibiting TPP1 homeostasis abnormality, excessive degradation, and telomere damage and shortening. application in medicines. Background technique [0002] Aging-related chronic diseases such as pulmonary fibrosis and skin fiber damage are caused by telomere DNA damage and cell aging. Progressive shortening of chromosomal telomere damage is a well-recognized mechanism leading to cellular senescence. Stress, DNA breaks caused by chemicals such as radiation exposure and antibiotics, and oxi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/53A61P39/06A61P11/00A61P9/00A61P1/16A61P13/12A61P19/08A61P7/06A61P37/04A61P15/00A61P19/02A61P19/10A61P25/00A61P3/10A61P35/00
CPCA61K38/53
Inventor 刘俊平王丽辉李果陈如萍
Owner HANGZHOU NORMAL UNIVERSITY
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