siRNA dual-interference composition for inhibiting growth and metastasis of tumors and application thereof

A composition and double-interference technology, applied in the application field of tumor therapy drugs, can solve problems such as not having advantages

Inactive Publication Date: 2015-04-22
葛银林 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of single inhibition of targeted gene expression has its limitations. For example, the inhibition of VEGF-C is mainly aimed at the treatment of lymphatic metastasis of tumors, although it has been reported that VEGF-C can inhibit apoptosis and promote survival of tumors. However, the effect of inhibiting VEGF-C on promoting tumor cell apoptosis and inhibiting growth is still not superior to the inhibition of survivin

Method used

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  • siRNA dual-interference composition for inhibiting growth and metastasis of tumors and application thereof
  • siRNA dual-interference composition for inhibiting growth and metastasis of tumors and application thereof
  • siRNA dual-interference composition for inhibiting growth and metastasis of tumors and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Design and synthesis of siRNA targeting mouse VEGF-C and survivin

[0035] The siRNA sequence targeting VEGF-C described in the present invention (hereinafter referred to as siVC),

[0036] The sense strand is: 5'-GCAAGACGUUGUUUGAAAUtt-3' (SEQ ID NO.1);

[0037] The antisense strand is: 5'-AUUUCAAACAACGUCUUGCtt-3' (SEQ ID No.2);

[0038] The siRNA sequence targeting survivin (hereinafter referred to as siSV) in the present invention,

[0039] The sense strand is 5'-GGAAUUGGAAGGCUGGGAAtt-3' (SEQ ID NO.3);

[0040] The antisense strand is 5'-UUCCCAGCCUUCCAAUUCCtt-3' (SEQ ID No.4).

[0041] The above two siRNA sequences were obtained by the following methods: First, the full-length sequence of mouse VEGF-C mRNA (GenBank accession No.NM_009506) and the full-length sequence of mouse survivin mRNA (GenBank accession No. .NM_009689), and then input them into Invitrogen's online design software for targeted siRNA design and BLAST homologous gene sequence comparis...

Embodiment 2

[0047] Embodiment 2: According to the effect of inhibiting tumor cell proliferation and migration after the transfection of the mixture of different ratios of the siRNA molecules.

[0048] A siRNA double interference composition, including oligomeric nucleic acid VEGF-C-siRNA molecule and oligomeric nucleic acid survivin-siRNA molecule; the nucleotide sequence of the oligomeric nucleic acid VEGF-C-siRNA molecule, the sense strand is SEQ ID NO .1, the antisense strand is SEQ ID No.2; the nucleotide sequence of the oligonucleotide survivin-siRNA molecule, the sense strand is SEQ ID NO.3, and the antisense strand is SEQ ID No.4. The oligonucleotide VEGF-C-siRNA molecule and the oligonucleotide survivin-siRNA molecule are mixed according to the mass ratio of the substances.

[0049] The steps of the MTT method are as follows: 4T1 cells in the logarithmic growth phase were digested with 0.25% trypsin containing 0.02% EDTA, blown into a single cell suspension with DMEM solution cont...

Embodiment 3

[0056] Example 3. RT-PCR method was used to detect the effect of siRNA transfection on the mRNA expression of VEGF-C and survivin in tumor cells.

[0057] The Hifectin II eukaryotic cell transfection reagent from Beijing Pulilai Company was used for transfection, and all operations were performed according to the operating procedures provided by Beijing Pulilai Company. Mouse breast cancer 4T1 cells were inoculated into 6-well plates (1×10 5 Cells / well), cultured with DMEM medium containing 10% fetal bovine serum (purchased from Hyclone) for 16-24 hours, sucked out the old medium, and replaced it with 1500 μL of new DMEM medium without serum and antibiotics. The oligonucleotide powders (siVC, siSV and siNC) were respectively dissolved in RNase-free sterile water to prepare an oligonucleotide solution with a final concentration of 20 μmol / L.

[0058]Dilute 10 μL oligonucleotide solution (siVC, siSV and siNC above) and 5 μL Hifectin I in 250 μL serum-free culture medium (DMEM),...

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Abstract

The invention relates to the field of tumor treatment medicine application, and particularly relates to a siRNA dual-interference composition for inhibiting tumor growth and metastasis of breast cancer and application thereof. The siRNA dual-interference composition is prepared by mixing an oligonucleotide VEGF-C-siRNA molecule and an oligonucleotide survivin-siRNA molecule in a mass ratio of (0-2):(2-0), wherein the nucleotide sequence of the oligonucleotide VEGF-C-siRNA molecule has a positive-sense strand of SEQ ID NO.1 and a negative-sense strand of SEQ ID NO.2; and the nucleotide sequence of the oligonucleotide survivin-siRNA molecule has a positive-sense strand of SEQ ID NO.3 and a negative-sense strand of SEQ ID NO.4. The result of an experiment, which is used for inoculating a mouse mammary carcinoma cell line 4T1 in an underarm fat pad of a Balb/c mouse and transfecting a transplanted tumor by using a chemically synthesized and modified oligonucleotide molecule, indicates that the composition of VEGF-C-siRNA and survivin-siRNA can be used for effectively inhibiting growth and pulmonary metastasis of the tumor cells. The chemically synthesized oligonucleotide serving as a novel siRNA anti-tumor drug cannot be easily decomposed, has a relatively high effect half-life, and can be used in in-vitro experiments and in-vivo treatment.

Description

technical field [0001] The invention relates to the application field of tumor therapy drugs, in particular to an siRNA double interference composition for inhibiting growth and metastasis of breast cancer tumors and its application. Background technique [0002] Breast cancer is one of the most common malignant tumors in women. In recent years, the incidence of breast cancer in women in developing countries, including China, is showing an obvious upward trend. According to statistics, the incidence of female breast cancer in my country in 2008 was about 47.64 / 100,000, ranking second in the incidence of female malignant tumors, second only to lung cancer. The current policy of breast cancer treatment is mainly surgery, radiotherapy and chemotherapy. Thanks to the popularization of breast cancer prevention knowledge and the improvement of early diagnosis technology, the growth trend of breast cancer mortality has been eased in recent years. However, advanced breast cancer a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P35/00
Inventor 葛银林刘永超薛美兰张金玉郑征
Owner 葛银林
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