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Method for inducing cell to generate epithelial-mesenchymal transition of cells and method for screening ferroptosis inducing substance

A technology for inducing cell and ferroptosis, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve problems such as poor characteristics and difficult to meet clinical needs, and achieve the effect of improving efficiency

Active Publication Date: 2019-06-25
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now commonly used ferroptosis-inducing molecules such as Erastin and RSL3 are obtained by screening Ras-dependent tumor cells (Dolma et al., 2003, PMID: 12676586; Yang et al., 2008, PMID: 18355723), and they The characteristics of medicinal chemistry are not good, and it is difficult to meet the clinical needs

Method used

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  • Method for inducing cell to generate epithelial-mesenchymal transition of cells and method for screening ferroptosis inducing substance
  • Method for inducing cell to generate epithelial-mesenchymal transition of cells and method for screening ferroptosis inducing substance
  • Method for inducing cell to generate epithelial-mesenchymal transition of cells and method for screening ferroptosis inducing substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] In this example, the tumor cells in the mesenchymal state were established, and the specific scheme was as follows:

[0044] Construction and screening of MCF7-MIB1 cells: The human MIB1 gene coding sequence (Entrez Gene: 57534) was cloned into the SnaBI site of the pSIN-EF2-IRES-Puro expression vector by adding the Flag-tag to the C-terminus by conventional molecular cloning techniques (like figure 1 (a)), then the virus was packaged in HEK293T cells according to the general method, and the virus was collected after 48 and 72 hours. After the virus was collected, MCF7 cells were infected. MCF7 cells were cultured in high-glucose DMEM medium containing 10% FBS, MCF7 cells were plated at 5 × 10 per well the day before infection 5 The cells were seeded on a six-well plate, and the next day, when the cells were about 70%, the virus was infected (2ml virus solution, plus 8μg / ml polybrene), 24 hours later, the fresh medium was replaced, and 48 hours later, puromycin (5μg / m...

Embodiment 2

[0047] In this example, MCF7-MIB1 cells are analyzed for mesenchymal cell-related functions, mainly the analysis of cell migration ability in vitro and in vivo. The specific scheme is as follows:

[0048] The migration ability of MCF7 and MCF7-MIB1 cells was compared by cell scratch assay. That is, use a needle or pipette tip to draw a cell-free area on the surface of the culture dish covered with monolayer cells, and take pictures and record. During subsequent cell culture, cells at the edge of the scratch gradually migrated into the cell-free area. The same area was photographed again after 12 hours, the boundaries of the scratches were determined, and the average distance of cell migration was calculated. The result is as image 3 (a,b).

[0049] The in vivo migration ability of MCF7 and MCF7-MIB1 cells was compared using an embryonic zebrafish model. About 1 million MCF7 and MCF7-MIB1 cells in good condition and digested into single cells were taken, labeled with Dil d...

Embodiment 3

[0051] In this example, MCF7-MIB1 cells are analyzed for mesenchymal cell-related functions, mainly the analysis of the sensitivity of the ferroptosis pathway. The specific scheme is as follows:

[0052] MCF7 and MCF7-MIB1 cells were seeded in a 96-well plate at 10,000 cells per well (100 μl of 10% FBS / high glucose DMEM medium per well), and drug treatment was performed after the cells adhered the next day. The used drugs RSL3, Erastin, Ferrostatin-1 (FER-1) and Ciclopirox ethanolamine (CPX) were purchased from SELLECK Company, and L-Ascorbic acid (ie L-vitamin C) was purchased from SIGMA Company. cell press Figure 4 The drug gradient treatment shown contains three replicate wells for each drug concentration. Figure 4 In the rescue experiments in (a,b), CPX or FER-1 was added simultaneously with RSL3. After 24 hours of drug treatment, the cell viability was detected with CCK8 kit (Keygen Biotech), and the relative survival rate of cells in each group was calculated accordi...

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Abstract

The invention provides a purpose of a reagent in preparation of a kit. The reagent is used for overexpressing a MIB1 gene, and the kit is used in at least one of the following: inducing cell epithelial-mesenchymal transition; obtaining mesenchymal cells; and obtaining cells highly sensitive to a ferroptosis pathway. The inventors find that after overexpressing the MIB1 gene, the cells can efficiently realize epithelial-mesenchymal transition, and the mesenchymal cells can be effectively obtained, The mesenchymal cells are highly sensitive to the ferroptosis pathway and are suitable for high-throughput screening of small molecule compounds and other regulatory molecules that are effective in inducing ferroptosis in mesenchymal cells.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the use of reagents in the preparation of kits, a method for inducing epithelial-mesenchymal transition of cells, recombinant cells and a method for screening ferroptosis-inducing substances. Background technique [0002] Epithelial-mesenchymal transition (EMT) is a common cellular biological phenomenon that plays a variety of roles in different stages of tumorigenesis. For example, the mesenchymal state after EMT can promote tumor cell metastasis, the acquisition of tumor stem cell properties, and the development of drug resistance, all of which are associated with tumor recurrence (Nieto et al., 2016, PMID: 27368099). Most existing tumor chemotherapy and targeted drugs are more effective for epithelial cells, but there is currently no effective treatment for drug-resistant tumor cells in mesenchymal state. Several recent studies have found that the survival of various mese...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N15/85
Inventor 舒晓东裴端卿王海云
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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