SiRNA for inhibiting human Rabj gene expression and its application

A gene and small molecule interference technology used in biotechnology and medicine to solve problems such as unsatisfactory results

Inactive Publication Date: 2007-04-18
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] In recent years, although RNA interference has made some progress in the treatment of certain tumors and viral infections in clinical trials and applications, the results obtained are not yet very satisfactory.

Method used

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  • SiRNA for inhibiting human Rabj gene expression and its application
  • SiRNA for inhibiting human Rabj gene expression and its application
  • SiRNA for inhibiting human Rabj gene expression and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Analysis of the expression of RabJ at the mRNA and protein levels after RabJ siRNA interferes with the expression of RabJ

[0082] Separately si-RabJ1 (SEQ ID NO: 1), si-RabJ2 (SEQ ID NO: 2), mutation negative control Scon si-RabJ1 (SEQ ID NO: 3) and Scon si-RabJ2 (SEQ ID NO: 4) HeLa, MCF-7, and SW480 cells were transiently transfected with the transfection reagent lipofectAMINE (Invitrogen), and the cells were harvested 48 hours later for detection.

[0083] In this example, the inhibition of si-RabJ1 and si-RabJ2 on the expression of the oncogene-like molecule RabJ gene was detected from two levels of mRNA and protein levels.

[0084] (a) Detection of mRNA by RT-PCR and quantitative analysis of PCR products

[0085] (1) RT-PCR: Total RNA was extracted according to the TRIzol Total RNA Extraction Kit. The quality of RNA was identified by electrophoresis and quantified by UV spectrophotometer. Take 2.5 μg of total RNA, add 50 μl of reverse transcription re...

Embodiment 2

[0098] Example 2: Inhibition of tumor cell growth after RabJ siRNA interferes with RabJ expression

[0099] This example uses the [ 3 H]-thymine incorporation method.

[0100] HeLa, MCF-7, SW480 cells transfected with RabJ siRNA described in Example 1 at 5×10 4 Cells / well were seeded in 24-well culture plates (Falcon Company), and each cell was seeded in three wells. After culturing for 6 hours, continue culturing in serum-free medium for 24-48 hours, and in the last 4 hours, add 0.5 μCi (1Ci=37GBq) of [ 3 H]-thymine (Amersham). Then, washed three times with PBS, the cells were dissolved in PBS containing 1% Triton X-100, collected with glass fiber filter membrane, and the radioactivity was measured with a liquid scintillation instrument. Or after 24 hours of serum starvation, add normal medium containing 10% FCS, continue culturing for 24 hours, and add [ 3H]-thymidine followed by radiometry.

[0101] The results showed that RabJ siRNA could significantly inhibit the gr...

Embodiment 3

[0102] Example 3: After RabJ siRNA interferes with the expression of RabJ, it inhibits the cell cycle progression of tumor cells

[0103] This embodiment adopts the detection method of propidium iodide (PI) labeled DNA content.

[0104] HeLa, MCF-7, SW480 cells (5×10 cells) transfected with RabJ siRNA described in Example 1 6 ) was digested with 0.25% trypsin, washed twice with PBS, slowly added with 70% ice ethanol, and fixed overnight at 4°C. Centrifuge at 1,000g to collect the cells, wash once with PBS containing 1% BSA, 10% FCS and resuspend the cells. Add 50 μg / ml PI and 100 mg / ml RNase A, label at 37°C for 30 min, monitor on a flow cytometer (FACS, Becton Dickinson Company) and analyze with CELLQUEST software, and the distribution of cells in each cycle is expressed as a percentage.

[0105] The results showed that RabJ siRNA could significantly inhibit the cell cycle progression of HeLa, MCF-7, and SW480 tumor cells in vitro, while the negative control siRNA oligonucl...

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Abstract

This invention relates to an interfering RNA nucleotide sequence aiming directly at oncogene-like molecule RabJ. Exactly to say, this invention relates to a interfering RNA nucleotide sequence which aims directly at the expression and function of oncogene-like molecule RabJ, the interfering RNA nucleotide sequence possesses gene expression and function of target anti- tumour cell RabJ, having the function of restraining tumour cell to grow and oncogenicity of tumour cell, and having the function of promoting apoptosis, thus contributing to cure tumor. This invention also relates to medicine Combination containing this interfering RNA nucleotide sequence, and discloses the method of using this medicine of interfering RNA for disease therapy, especially it is used to cure hyper-expressed malignant tumor.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine. Specifically, the present invention relates to an interfering RNA nucleotide sequence aimed at the expression and function of the oncogene RabJ, the interfering RNA nucleotide sequence has targeted anti-tumor cell RabJ gene expression and function, inhibits tumor cell growth, Inhibit the tumorigenicity of tumor cells and the function of promoting tumor cell apoptosis, thus playing a role in the treatment of tumors. The present invention also relates to a pharmaceutical composition containing the interfering RNA nucleotide sequence, and discloses the application of the interfering RNA drug in tumor treatment, especially the application in the treatment of malignant tumors overexpressing RabJ. Background technique [0002] Small G proteins refer to single-subunit G proteins 1-5 with a molecular weight of about 20-30 kDa. At present, with the completion of the Human Genome Project, more ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C07H21/00A61K48/00A61P35/00C12N15/113
Inventor 陈涛涌李楠万涛曹雪涛
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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