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Application of MR-1 in tumor diagnosis and treatment as tumor marker and target molecule

A tumor marker and tumor diagnosis technology, applied in the field of tumor gene therapy, can solve problems such as no related reports, and achieve the effect of inhibiting tumorigenesis and growth

Inactive Publication Date: 2008-06-18
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The research on MR-1 described in the present invention as a new tumor diagnostic marker and therapeutic target molecule has not been reported so far

Method used

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  • Application of MR-1 in tumor diagnosis and treatment as tumor marker and target molecule
  • Application of MR-1 in tumor diagnosis and treatment as tumor marker and target molecule
  • Application of MR-1 in tumor diagnosis and treatment as tumor marker and target molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Expression of MR-1 in tumor cells and normal cells

[0071] 1), Western blotting method to detect the expression level of MR-1 in cells

[0072] Add 120μl of freshly prepared cell lysate (50mM Tris-HCl, pH7.5; 1% NP-40; 150mM NaCl; 1mg / ml aprotinin; 1mg / ml leupeptin: 1mM NaCl) to cells of different cell lines 3 VO 4 ; 1mM NaF), immediately scrape the cells with a cell scraper to mix them well with the lysate, and place them on ice for 30 minutes to 1 hour. Centrifuge at 13,000 rpm at 4 degrees for 15 minutes, collect the supernatant in a new EP tube, and use the standard Bradford method to determine the protein content. Take an equal amount of total protein (20-50 μg) from each sample, add an equal volume of 3-fold loading buffer, boil water and denature for 5 minutes, separate the protein on 10% SDS-PAGE gel electrophoresis, and then transfer the blotted protein to on PVDF membrane. Afterwards, the PVDF membrane was soaked in TBS solution containing 5% (w / v) skimm...

Embodiment 2

[0075] Design and synthesis of MR-1-siRNA

[0076] At present, the most effective siRNA duplex is composed of a sense strand and an antisense strand of 21 nucleotides, with two nucleotides overhanging at the 3' end of each strand, and 19 nucleotides in the middle are paired.

[0077] According to the MR-1 gene sequence provided in GenBank (serial number AF417001), the present invention selects and designs two pairs of siRNA sequences. The target mRNA sequence and siRNA nucleotide sequence corresponding to siRNA are as follows:

[0078] mRNA target sequence 1 (19nt): 5'-ACC GUG UGA AGC AGA UGA A-3'

[0079] Oligonucleotide sequence: 5'-UUC AUC UGC UUC ACA CGG UdTdT-3'

[0080] 3’-dTdTA AGU AGA CGA AGU GUG CCA-5’

[0081] mRNA target sequence 2 (19 nt): 5'-CCU AGG CUAUUG ACU GUU A-3'

[0082] Oligonucleotide sequence: 5'-UAA CAG UCA AUA GCC UAG GdTdT-3'

[0083] 3’-dTdTA UUG UCA GUU AUC GGA UCC-5’

[0084] The mock sequence used in the experime...

Embodiment 3

[0089] Design and vector construction of MR-1 shRNA target sequence and its DNA template

[0090]The ShRNA target sequence is MR-1-siRNA target 2. Synthesize the DNA template of shRNA according to the existing rules. The template includes two complementary strands of sense and antisense. The basic units are: BamHI restriction site, sense strand of target sequence, 9bp loop sequence, Sense strand, 5 consecutive T, HindIII restriction sites, total length 65bp.

[0091] The MR-1 shRNA oligonucleotide sequence is as follows:

[0092] Justice Chain:

[0093] 5'-GATCCCG CCTAGGCTATTGACTGTTA TTCAAGAGA TAACAGTCAATAGCCTAGG TTTTTTGGAAA-3'

[0094] Antisense strand:

[0095] 3'-GGC GGATCCGATAACTGACAAT AAGTTCTCT ATTGTCAGTTATCGGATCC AAAAAACCTTTTCGA-5'mock shRNA oligonucleotide sequence is as follows:

[0096] 5'-GATCCCG TTCTCCGAACGTGTCACGT TTCAAGAGA ACGTGACACGTTCGGAGAA TTTTTTGGAAA-3'

[0097] 3'-GGC AAGAGGCTTGCACAGTGCA AAGTTCTCT TGCACTGTGCAAGCCTCTT AAAAAACCTTTTCGA-5' ...

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Abstract

The invention relates to the application of MR-1 gene which is used as a tumor marker and a target molecular in the preparation of tumor diagnosis reagents and therapeutic drugs. Specifically speaking, the invention is to detect the expression of the novel gene MR-1 in human normal cells and tumor cells and finds that the migration and the growth of the tumor cells are restrained after the highly expressed MR-1 is restrained in the tumor cells through the RNA jamming technology. And that MR-1 is expected to become a novel marker for the tumor diagnosis and a novel target for the clinical treatment.

Description

Technical field: [0001] The present invention relates to a tumor gene therapy technology, specifically, according to the newly discovered target gene MR-1, through corresponding means, inhibit its influence on the growth and movement of tumor cells, and then clarify the role of human MR-1 in tumor diagnosis The feasibility of new markers and new targets for clinical treatment. Background technique: [0002] Molecular diagnosis and treatment of tumors is an important part of molecular medicine and has become a hot spot in tumor research in recent years. With the development of molecular biology, especially the smooth implementation of the Human Genome Project, the analysis of the human genome sequence and the identification of related gene functions, more and more tumor molecular markers and therapeutic targets have been discovered. [0003] Invasion and metastasis are important features of malignant tumors and the main causes of patient death. This complex pathological pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68A61K48/00A61K35/00
Inventor 邵荣光任开环王以光何红伟金海霞边春景
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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