siRNA molecule for inhibiting survivin gene expression and application of siRNA molecule

A gene expression and molecular technology, applied in the field of biomedicine, can solve the problems of tumor treatment and no radical cure.

Inactive Publication Date: 2015-10-28
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Malignant tumors are a serious threat to human life and health, and its mortality rate is second only to cardiovascular and cerebrovascular diseases; although various treatments continue to emerge, no truly effective cure has been found so far, and the treatment of tumors has long plagued the medical community. world-wide problem

Method used

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  • siRNA molecule for inhibiting survivin gene expression and application of siRNA molecule
  • siRNA molecule for inhibiting survivin gene expression and application of siRNA molecule
  • siRNA molecule for inhibiting survivin gene expression and application of siRNA molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Detection experiment of chemically synthesized siRNA on survivin gene silencing effect

[0028] 1. Main instruments, reagents and materials.

[0029] 1.1 Instruments: Nucleic acid synthesizer (GE Company), PCR instrument (ABI Company); real-time quantitative PCR instrument (Bio-Rad); cell incubator (Thermo), etc.

[0030] 1.2 Materials and reagents: RNAiMAX TM (invitrogen), DMEM medium (Gibco),

[0031] TurboCapture mRNA kit (QIAGEN), SensiMix TM one-Step Kit (Quantace), etc. Other biochemical reagents were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0032] 1.3PCR primers (synthesized by Biomaike Biotechnology Co., Ltd.):

[0033] Survivin forward primer: 5'-AGAACTGGCCCTTCTTGGAGG-3'

[0034] survivin reverse primer: 5'-CTTTTTATGTTCCCTCTATGGGGTC-3'

[0035] GAPDH forward primer; 5'-GAAGGTGAAGGTCGGAGTC-3'

[0036] GAPDH reverse primer: 5'-GAAGATGGTGATGGGATTTC-3'

[0037] 2. Chemical synthesis of siRNA

[0038] Accordi...

Embodiment 2

[0048] Example 2: Effect of survivin gene silencing on tumor cell proliferation

[0049] Cervical cancer cells Hela, breast cancer cells MCF-7 and liver cancer cells HepG2 cultured in 3.1 of Example 1 were divided into 5×10 3 Each cell / well was inoculated into a 96-well cell culture plate, and the three kinds of cells were cultured separately and tested separately at 37°C and 5% CO 2 Cells were cultured in the incubator for 24 hours, in DMEM medium containing 10% FBS without double antibody, according to RNAiMAX TM According to the instruction manual for transfection, the siRNA synthesized in step 2 of Example 1 was added at 10, 20, 40 nM / well, and after incubation at 37°C for 48 hours, 20 μL of MTT (5 mg / mL) was added to each well, incubated at 37°C for 4 hours, and then sucked out. Add 200 μL DMSO to the culture medium to dissolve formazan crystals, and measure the absorbance at 540 nm.

[0050] The results of the inhibitory effect of siRNA on tumor cell proliferation dete...

Embodiment 3

[0054] Example 3: Western Blot detection of siRNA inhibition of survivin expression

[0055] MDA-MB-231 cells, Hela cells, A549 cells and HepG2 cells were divided into 1.5×10 5 cells / well were respectively inoculated into 6-well cell culture plates, and the four kinds of cells were cultured separately, and the inhibition test was carried out respectively, at 37°C, 5% CO 2 Cells were cultured in the incubator for 24 hours, in DMEM medium containing 10% FBS without double antibody, according to RNAiMAX TM Instructions for transfection, the siRNA synthesized in Step 2 of Example 1 was added at 20nM / well, after incubation at 37°C for 48 hours, the medium was aspirated, washed twice with PBS, 100μL of cell lysate was added, and lysed in an ice bath for 10min , Scrape the cells with a cell scraper, transfer to a 1.5ml centrifuge tube, centrifuge at 10000rpm for 5min, take the supernatant to measure the protein concentration. Take 30 μg of protein from each group for SDS-PAGE, tran...

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Abstract

The invention discloses a siRNA sequence for inhibiting survivin gene expression. The siRNA sequence has the broad spectrum siRNA anti-tumor effect, and the excellent inhibiting effect on breast cancer, lung cancer, intestinal cancer, liver cancer, leukemia, stomach cancer, cervical cancer, human oral epidermoid carcinoma and the like in vitro, particularly on breast cancer, lung cancer and cervical cancer cells. When the siRNA double-strand sequence enters an organism through a certain manner, the expression of disease related protein can be efficiently and specifically inhibited, so that related disease genes are silent, the target gene expression can be effectively knocked out, and the treating purpose can be achieved.

Description

technical field [0001] The invention discloses a double-strand siRNA molecule inhibiting the expression of survivin gene, and also provides the medical application of the siRNA molecule, which belongs to the technical field of biomedicine. Background technique [0002] Malignant tumors are a serious threat to human life and health, and its mortality rate is second only to cardiovascular and cerebrovascular diseases; although various treatments continue to emerge, no truly effective cure has been found so far, and the treatment of tumors has long plagued the medical community. worldwide problems. Evasion of apoptosis is a hallmark of all tumors, allowing tumor cells to survive abnormal growth stimuli and resist chemotherapy and radiotherapy. The anti-apoptotic protein survivin has become a new and most potential therapeutic target to inhibit the growth of tumor cells by interacting with apoptosis signals. [0003] The phenomenon of RNA interference (RNAi) is an evolutionari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63A61K31/713A61P35/00A61P35/02
Inventor 谢晶孙亚厅李剑光滕乐生孟庆繁逯家辉刘艳权宇彤张洋
Owner JILIN UNIV
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