Application of cancer suppressor gene FBXW7 in preparation of drugs used for preventing or treating breast tumors, expression vector and diagnosis medicine

A breast tumor and tumor suppressor gene technology, applied in the field of medicine and biology, can solve the problems of unreported clinical significance of function and expression changes, increased chromosomal instability, and incompletely clear mechanism of breast cancer.

Active Publication Date: 2013-07-10
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutation or deletion of FBXW7 leads to increased chromosomal instability and uncontrolled cell growth
[0005] However, the mechanism of action of FBXW7 in breast cancer is still not completely clear, and the clinical significance of its function and expression changes has not been reported yet.

Method used

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  • Application of cancer suppressor gene FBXW7 in preparation of drugs used for preventing or treating breast tumors, expression vector and diagnosis medicine
  • Application of cancer suppressor gene FBXW7 in preparation of drugs used for preventing or treating breast tumors, expression vector and diagnosis medicine
  • Application of cancer suppressor gene FBXW7 in preparation of drugs used for preventing or treating breast tumors, expression vector and diagnosis medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Meta-analysis of FBXW7 gene in normal breast tissue and breast cancer tissue

[0055] 1. Materials and methods 2 transcriptome databases containing normal and breast cancer samples and 10 breast cancer databases containing clinical data and gene expression data were selected. The samples in these databases have all passed the Affymetrix microarray assay (either HG-U133A or HG U133Plus2.0) or Agilent oligo microarray detection analysis ( figure 1 a and figure 1 b, The expression of FBXW7 mRNA was detected by Affymetrix microarray method. Through the analysis of the two major databases GSE10780 (a) and GSE3844 (b), it was found that the expression level of FBXW7 mRNA in breast tumor tissue was significantly lower than that in normal breast tissue). Analyzed by retrieving relevant data from the GEO website. In total, disease-free survival (DFS) data were available for 1900 of 1935 patient samples in 10 databases.

[0056] 2. Statistical analysis The differenc...

Embodiment 2

[0064] Example 2 Cloning of FBXW7 cDNA and Construction of Expression Vector

[0065] 1. Cloning-specific cDNA synthesis

[0066] The reverse transcription reaction (RT) was performed using the Fermentas First Synthesis System for RT-PCR kit, and 2 μg of total RNA was used in each reaction. The total reaction volume is 20 μl: 2 μl of 10× RT buffer, 5 μl of total RNA, 1 μl of oligodT20 primer, 1 μl of 10 mM dNTP, 25 mM MgCl 2 4 μl, 1 μl of 0.1M DTT, 1 μl of RNaseOUT, 1 μl of SuperScript III reverse transcriptase, and 4 μl of high-purity deionized water. The reverse transcription reaction was performed using an ABI PCR machine, and the specific operating parameters were as follows: 25°C for 10 min; 37°C for 60 min; 75°C for 5 min; cooling to 4°C. Store at -20°C for later use.

[0067] 2. Cloning of FBXW7 cDNA

[0068] The open reading frame of the full-length FBXW7 gene was amplified from the total RNA of normal breast tissue by RT-PCR. The PCR used Fermentas' Mix, and the t...

Embodiment 3

[0077] Example 3 Colony formation assay analysis of tumor suppressor function of FBXW7 gene

[0078] 1. Operation process

[0079] The effect of FBXW7 gene on the growth of breast cancer cells was detected by colony formation assay. 100,000 cells were seeded in 12-well culture plates and cultured overnight, and FBXW7 plasmid was transfected with FUGENE6 reagent, and an empty pcDNA3.1 vector was used as a control. 48h after transfection, the transfected cells were divided into three groups and re-inoculated, and cultured in G418 medium containing 400μg / ml for 10-15d, the formed clones were fixed with methanol, and then stained with gentian violet, and the number of cells was greater than or equal to 50 cells. The clones were counted for analysis.

[0080] 2. Analysis of results

[0081] In BT549 cells, when the FBXW7 gene was exogenously transferred, the ability of tumor cells to form clones was significantly reduced ( Figure 9 ). It indicated that FBXW7 has the function o...

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Abstract

The invention discloses application of a cancer suppressor gene FBXW7 in preparation of drugs used for preventing or treating breast tumors, an expression vector and a diagnosis medicine, belonging to the field of medical biotechnology. Specifically, the invention provides application of the cancer suppressor gene FBXW7 in preparation of drugs used for preventing or treating breast tumors, the expression vector constructed from the gene FBXW7 and a vector pcDNA3.1 and a preparation method thereof, and the breast tumor diagnosis medicine at least including a pair of primers capable of specific amplification of the gene FBXW7 and composed of an upstream primer and a downstream primer. Expression of the cancer suppressor gene FBXW7 is closely related to breast cancer molecular subtyping; the cancer suppressor gene FBXW7 has specific low expression in a breast cancer with high grade malignancy and exerts an inhibitory effect on growth of breast cancer cells. Thus, screening of breast cancers with low expression of the gene has significant meaning to prognostic prediction of the breast cancers; and specific recovery of expression of the cancer suppressor gene in breast cancer cells provides a novel approach for targeted individualized treatment of the breast cancers.

Description

technical field [0001] The invention relates to the field of medicine and biotechnology, in particular to the application of a tumor suppressor gene FBXW7 in the preparation of drugs for preventing, diagnosing and treating breast tumors and an expression vector thereof. Background technique [0002] The basic characteristics of tumor cells are uncontrolled cell growth and blocked differentiation. This uncontrolled cell growth and stunted differentiation are the result of the accumulation of multiple gene defects, mainly manifested in two aspects: one is the activation or overexpression of oncogenes, and the other is the inactivation of tumor suppressor genes. Under normal circumstances, tumor suppressor genes can inhibit the malignant transformation of cells and negatively regulate the proliferation of normal cells. When tumor suppressor genes are inactivated, normal cells proliferate out of control and transform into tumor cells. The research on oncogenes and tumor suppres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/85C12N15/66C12Q1/68A61P35/00A61P15/14
Inventor 魏光伟王允山张鹏举何秀全
Owner SHANDONG UNIV
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