Application of transcription factor ZEB1 for preparing medicine capable of accelerating breast cancer chemosensitivity

A technology of transcription factor and chemotherapeutic drug, applied in the field of tumor drug preparation, can solve the problems of easy occurrence of chemoresistance, large toxic and side effects, and restriction of clinical application, and achieves the effect of solving the problem of chemoresistance and promoting chemosensitivity.

Active Publication Date: 2016-07-13
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its clinical application has been greatly restricted due to its high

Method used

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  • Application of transcription factor ZEB1 for preparing medicine capable of accelerating breast cancer chemosensitivity
  • Application of transcription factor ZEB1 for preparing medicine capable of accelerating breast cancer chemosensitivity
  • Application of transcription factor ZEB1 for preparing medicine capable of accelerating breast cancer chemosensitivity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] ZEB1 is closely related to chemotherapy resistance in breast cancer

[0055] (1) Tissue samples of 233 breast cancer cases receiving standard neoadjuvant chemotherapy were collected, and the efficacy of chemotherapy was evaluated using the WHO chemotherapy efficacy evaluation criteria. Among them, 69 were drug-resistant cases and 164 were non-drug-resistant cases. chemical staining;

[0056] (2) Baking slices: first place the tissue slices in a 65°C oven and bake for 2 hours, then take them out and cool to room temperature;

[0057] (3) Prepare the reagents required for dewaxing in the fume hood, put the tissue sections in xylene I for 10 min, xylene II for 10 min, absolute ethanol for I5 min, absolute ethanol II for 5 min, 95% ethanol for 10 min, and 80% ethanol for 10 min, then Wash with TBST on a shaker, 5min, twice, 100rpm;

[0058] (4) Antigen retrieval and 3% H 2 o 2 Remove endogenous hydrogen peroxide;

[0059] (5) Seal the tissue with 5% goat serum at room ...

Embodiment 2

[0067] Construction of stable cell lines of breast cancer overexpressing ZEB1

[0068] (1) Cloning the full-length CDS of the human ZEB1 gene by means of molecular biology;

[0069] (2) PCR amplification and recovery of cloned products;

[0070] (3) Ligate the recovered product to the vector plasmid pLV-EF1-MCS-IRES-Bsd, identify by double enzyme digestion, and sequence to ensure the correctness of the constructed target vector;

[0071] (4) Utilize the Letivirus system to package the target plasmid successfully constructed above, and measure the virus titer;

[0072] (5) The packaged virus was used to transfect human breast cancer cell MDA-MB-231. After 48 hours of transfection, 10 μM BSD was added for stable strain selection, and finally a stable cell line with ZEB1 overexpression was obtained through screening.

[0073] The overexpression efficiency of ZEB1 was verified by Western blot method ( figure 2 ), the successful construction of this stable cell line provides an...

Embodiment 3

[0075] Preparation of Le-shZEB1

[0076] (1) Design and synthesize ZEB1shRNA sequence online;

[0077] (2) After annealing the ZEB1shRNA sequence (95°C, 5 minutes), ligate it to the vector plasmid pLV-H1-EF1α-puro, and double-enzyme digest to determine whether the target plasmid (BamH1, Sac1) is successfully constructed;

[0078] (3) Use the Letivirus system to package the target plasmid successfully constructed above, and measure the virus titer to construct Le-shZEB1:

[0079] a. Dilute 293T cells at 1.0×10 per well 6 The amount of cells was placed in a 6-well plate, and the cells were transfected when the cell density reached about 90%;

[0080] b. Take a sterile 1.5ml centrifuge tube, add 7.5μl Lipofectamine2000 and 250μl Opti-MEM into it, mix gently, and let stand at room temperature for 5min;

[0081] c. Take another sterile 1.5ml centrifuge tube, add 1.5μg construct pLV vector (purpose plasmid), 1.5μg packaging plasmid (each packaging plasmid 0.5μg, Gag-pol+Rev+VSV-G...

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Abstract

The invention discloses application of a transcription factor ZEB1 for preparing a medicine capable of accelerating breast cancer chemosensitivity. The transcription factor ZEB1 can mediate the chemotherapy resistance of the breast cancer, and the breast cancer chemosensitivity can be improved through the targeted intervention of the ZEB1. On one hand, the ZEB1 can accelerate the breast cancer chemotherapy resistance through a way of accelerating DNA (Deoxyribose Nucleic Acid) damage repair, and the generation of the chemotherapy resistance is realized through the regulation and the control on ATM (Ataxia Telangiectasia-Mutated Gene) by the ZEB1. On the other hand, the ZEB1, P300 and/ or PCAF (P300/CBP-Associated Factor) can form a compound, wherein the compound performs a regulation and control function on the ATM. Meanwhile, a ZEB1-shRNA lentivirus gene knock-down system is used for verifying the function of Le-shZEB1 in enhancing the anti-tumor curative effect of anti-tumor medicines through breast carcinoma cell lines and an in vivo nude mouse model, and the tolerance problem of the breast cancer on chemotherapy is effectively solved. The invention provides a new target spot and a therapeutic strategy for alleviating the chemotherapy resistance of the breast cancer, increasing the sensitivity of chemotherapy medicines and the like.

Description

technical field [0001] The invention relates to a new application of transcription factor ZEB1 and related genes for improving chemotherapy sensitivity, in particular to the application of transcription factor ZEB1 in the preparation of drugs for improving breast cancer chemotherapy sensitivity, and belongs to the technical field of tumor drug preparation. Background technique [0002] Breast cancer (Breast Cancer) has become one of the most common malignant tumors in women worldwide, and it is also the leading cause of death from tumor-related diseases in women worldwide. Although the 5-year survival rate of early breast cancer has increased to over 85% in some developed countries due to the development of comprehensive treatment technologies such as breast surgery, neoadjuvant therapy and targeted drugs, advanced breast cancer or recurrent and metastatic breast cancer The annual survival rate is only 23%. The occurrence of chemotherapy resistance in breast cancer has beco...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P35/00A61K31/704A61K31/7048
CPCA61K31/704A61K31/7048A61K45/00A61K2300/00
Inventor 杨爽张翔张震向荣
Owner NANKAI UNIV
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