Application of FHL3 (four and a half LIM domains 3) in preparation of medicines for treating tumors

1. FHL3, application technology, applied in the application field of FHL3 in the preparation of drugs for treating tumors, can solve the problems of unclear molecular mechanism, unclear expression of FHL3, etc.

Active Publication Date: 2010-06-23
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the expression of FHL3 in human tumors is still unclear, and the molecular mechanism of whether FHL3 can inhibit the growth of human tumor cells and how to regulate this growth is also unclear

Method used

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  • Application of FHL3 (four and a half LIM domains 3) in preparation of medicines for treating tumors
  • Application of FHL3 (four and a half LIM domains 3) in preparation of medicines for treating tumors
  • Application of FHL3 (four and a half LIM domains 3) in preparation of medicines for treating tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, FHL3 mammalian cell expression vector and FHL3 recombinant adenovirus vector

[0047] (1) Method

[0048] 1. Construction of FHL3 mammalian cell expression vector

[0049] The complete coding sequence of the FHL3 gene was amplified by PCR from a mammary gland cDNA library (purchased from Clontech, USA) according to conventional methods. It is: 94°C for 5min, 94°C for 1min, 60°C for 1min, 72°C for 1min for 30 cycles, and finally 72°C for 7min. Use BamHI and Xba I enzymes (purchased from Biolabs, UK) to digest the PCR product and the corresponding mammalian cell expression vector (pcDNA3, purchased from Invitrogen, USA) at 37°C, and use T4 DNA for the digested PCR product and vector Ligase (purchased from Promega, USA) was ligated at 12-16°C to obtain a recombinant vector (which contains the CMV promoter / enhancer, the complete coding region sequence of the FHL3 gene, the BGH polyA termination signal, and the ampicillin resistance gene) , transformin...

Embodiment 2

[0081] Example 2, FHL3 inhibits the growth of various tumor cells cultured in vitro

[0082] (1) Method

[0083] The influence of FHL3 on the growth of various tumor cells was determined by crystal violet assay, and the steps were as follows: Digest various tumor cells, including breast cancer cell lines (MCF7, ZR75-1) with 0.25% trypsin (purchased from Sigma-Aldrich, USA) , liver cancer cell lines (HepG2, SMMC7721), lung cancer cell lines (H460). After counting the cells, dilute to an appropriate concentration, put 0.5ml of the cell solution into a 24-well plate, so that the number of cells in each well is about 10,000. Transfect these tumor cells with the FHL3 mammalian cell expression vector according to the method described in Example 1 or infect these tumor cells with 20-100 MOI of FHL3 recombinant adenovirus, after cultivating for 1-4 days, remove the medium, and add 0.5 ml to each well 1% glutaraldehyde (fixative solution), stand still for 15-20min, remove glutara...

Embodiment 3

[0086] Example 3, FHL3 inhibits the anchorage-independent growth of various tumor cells

[0087] (1) Method

[0088] The effect of FHL3 on the anchorage-independent growth of tumor cells was determined by soft agar assay. First, transfect tumor cells with the FHL3 mammalian cell expression vector according to the method described in Example 1 or infect tumor cells with 20-100 MOI of FHL3 recombinant adenovirus, including breast cancer cell line (MCF7), liver cancer cell line (HepG2) , lung cancer cell line (H460), and then use 60mm 2 Cell culture dish, add 3ml DMEM medium containing 0.7% agar (purchased from U.S. Invitrogen Company) in the culture dish, add 3ml DMEM medium containing above-mentioned 20,000 tumor cells and 0.25% agar on it after solidification, each group The cells were set up in 3 parallel samples, and the number of colonies formed by cells in each group was counted under an inverted microscope after 3 weeks of culture.

[0089] (2) Results

[0090] ...

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Abstract

The invention relates to an application of FHL3 (four and a half LIM domains 3) in the preparation of medicines for treating tumors, in particular to application of FHL3 in the preparation of medicines for adjusting the expression of oncogenes and / or cancer suppressor genes in mammal cells as well as an application of FHL3 in the preparation of medicines for inhibiting the growth of the tumor cells of the mammals. The invention also relates to an expression vector containing the nucleotide sequence of the code FHL3, application of the expression vector in the preparation of medicines for adjusting the representation of cancer genes and / or cancer-inhibiting genes in cells of mammals, as well as application of the expression vector in the preparation of medicines for inhibiting the growth of the tumor cells of the mammals. The expression vector in the invention can effectively inhibit the growth of various human tumor cells, and can adjust the expression of the oncogenes and / or cancer suppressor genes so as to inhibit the growth of various human tumor cells.

Description

technical field [0001] The present invention relates to a new medical application of four and a half LIM domain proteins, in particular to the application of four and a half LIM domain proteins 3 (FHL3) in the preparation of medicines for treating tumors. Background technique [0002] The four and a half LIM domains protein (Four and a Half LIM domains protein, hereinafter referred to as FHL) family belongs to the LIM superfamily, and its family members are composed of four and a half LIM domains. The LIM domain consists of approximately 50 amino acids, is rich in cysteine, and forms 2 zinc finger structures. The LIM domain mediates many protein-protein interactions and regulates the actions of cytoskeletal proteins, enzymes, and transcription factors. Five members of the FHL family have been found, which are FHL1, FHL2, FHL3, FHL4 and FHL5 / ACT. The expression of FHL members is tissue specific, FHL1, FHL2 and FHL3 are mainly expressed in skeletal muscle and cardiac muscle,...

Claims

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Application Information

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IPC IPC(8): A61K36/17A61K48/00A61P35/00C12N15/63C12N15/86C12N15/861
Inventor 叶棋浓丁丽华秦玺杨晓牛畅程龙严景华黄翠芬
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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