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Small interference ribonucleic acid molecule for epidermal growth facor gene and its use

A technology of epidermal growth factor and ribonucleic acid, which is applied in the field of preparing drugs that can effectively kill tumor cells, and can solve problems such as lack of treatment methods

Inactive Publication Date: 2005-05-18
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Metastasis and chemotherapy resistance are the biggest problems in the treatment of colon cancer. At present, there is still a lack of effective treatment methods and drugs. Finding effective treatment drugs and treatment methods is a top priority

Method used

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  • Small interference ribonucleic acid molecule for epidermal growth facor gene and its use
  • Small interference ribonucleic acid molecule for epidermal growth facor gene and its use
  • Small interference ribonucleic acid molecule for epidermal growth facor gene and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Synthesis of siRNA

[0040] The synthesis of siRNA can be entrusted to commercial companies that openly carry out synthesis business, such as Dharmacon in the United States, specifically through www.dharmacon.com It is known that all siRNA molecules are deprotected at the 2-position, desalted, purified, and annealed to form double strands, and then dissolved in diethyl pyrocarbonate-treated distilled water.

[0041] The preparation method of the small interfering RNA molecule (siRNA) provided by the present invention can also adopt a conventional solid-phase chemical synthesis method.

[0042] Taking the small interfering RNA molecule (SiRNA) of SEQ ID NO: 1 as an example: the whole chemical synthesis can be roughly divided into four processes (1) synthesis of oligoribonucleic acid; (2) deprotection; (3) purification and separation; 4) Desalting and annealing aseptic disinfection.

[0043] Concrete preparation operation is as follows:

[0044] (1), synthes...

Embodiment 2

[0051] Example 2 Cell culture and transfection of siRNA

[0052] 1. Cell culture Human colon cancer LS-174T cells have high expression of Cripto gene. The cells were cultured in RPMI1640 medium (containing 100 U / ml of penicillin and streptomycin) in vitro with 10% fetal bovine serum, and the medium was changed every day.

[0053] 2. SiRNA transfection: SiRNA was transferred into tumor cells by means of Oligofectamine from Invitrogen (www.invitrogen.com), and the specific steps were carried out according to the instructions. The brief description is as follows, 1.0×10 5 Tumor cells were inoculated overnight on 24-well culture plates, transferred into siRNA the next day, and then cultured for different periods of time, the cells were collected to detect the level of cripto mRNA in the cells.

[0054] 3. Detection of the expression level of cripto mRNA in LS-174T cells: the detection of the expression level of cripto mRNA in tumor cells was carried out by real-time PCR technolo...

Embodiment 3

[0055] Example 3 Inhibitory effect of siRNA on tumor cell cripto mRNA

[0056] 1. Selection of SiRNA: In the present invention, a total of 12 SiRNA molecules targeting the coding region of cripto mRNA were selected, that is, 12 SiRNA molecules listed in SEQ ID NO: 1-12. The attack site is between 45-1414 of the start codon, and the G / C of the 19 complementary double-stranded nucleotide sequences in each SiRNA molecule is between 42%-68%.

[0057] 2. The effect of 12 SiRNAs on the expression of cripto mRNA in LS-174T cells:

[0058] A, the effect of 12 SiRNAs with a concentration of 100nM on the cripto mRNA of SW480 cells:

[0059] After 100nM of these 120 SiRNAs acted on LS-174T cells for 48 hours, when observing their effects on cripto mRNA levels, all 12 SiRNAs had a certain inhibitory effect on cripto RNA. No. C1 (SEQ ID NO: 1) and No. C12 (SEQ ID NO: 12) have the most obvious inhibitory effects on cripto mRNA, reaching 86% and 84%, respectively. See results figure 1 , ...

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Abstract

The present invention provides one small interference ribonucleic acid (SiRNA) as the active component for antitumor medicine to kill tumor cell and for epidermal growth factor Cripto gene mRNA. Its nucleotide sequence has one negative chain and one nucleotide mutant without homology to known human gene. The SiRNA can inhibit the expression of human colon cancer LS-174T cell Cripto gene effectively via intracellular RISC, and is favorable to treating colon cancer and other malignant tumor with up expressing Cripto gene. Therefore, the SiRNA may be used in medicine for treating malignant tumor and relevant disease with up expressing Cripto gene.

Description

technical field [0001] The invention relates to the field of nucleic acid technology, in particular to a small interfering ribonucleic acid molecule (SiRNA) directed at the mRNA of the epidermal growth factor (Cripto) gene and its application in the preparation of drugs for effectively killing tumor cells. Background technique [0002] Malignant tumor is a major disease that endangers human life and health, but so far there is still no effective treatment for it. Scientists have long been trying to develop new drugs that can effectively treat malignant tumors. [0003] According to the statistics of the World Health Organization, there are about 10 million new malignant tumor patients in the world every year, and 6 to 7 million people die from tumors. In my country, there are about 2 million new cases each year, and about 1.4 million deaths (calculated according to a population of 1.3 billion). This trend will continue as the effects of aging populations, smoking, urbaniza...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00C07H21/00
Inventor 丁佳逸范钰郑树
Owner ZHEJIANG UNIV
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