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Structure of siRNA for target survivin gene and antineoplastic usage

An anti-tumor drug and its application technology, applied in the structure of siRNA and its anti-tumor field, can solve problems such as loss of resistance, achieve the effects of reducing drug resistance, inducing tumor cell apoptosis, and inhibiting tumor cell division

Inactive Publication Date: 2007-09-26
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the C84A mutation occurs in the only BIR region, survivin will lose its resistance to the apoptosis induced by the chemotherapeutic drug Taxol

Method used

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  • Structure of siRNA for target survivin gene and antineoplastic usage
  • Structure of siRNA for target survivin gene and antineoplastic usage
  • Structure of siRNA for target survivin gene and antineoplastic usage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] siRNA design and synthesis

[0028] Instruments OligoPilot II DNA / RNA Synthesizer (Pharmacia, Sweden), HP-1100 High Performance Liquid Chromatography (Hewlett-Packard, USA), DU 2640 UV Spectrophotometer (Backman, USA).

[0029] Materials and Reagents Synthesis column (6.3mL), molecular sieve (3A Sigma), 30HL carrier (lot number 256723), RNA Pac PA100 analysis column (Dionex, USA), 5 kinds of nucleotide monomers (A, U, C, G, dT), acetonitrile, dichloroacetic acid, tetrazole, N2 methylimidazole, except the manufacturers indicated, the rest are products of Pharmacia, Sweden.

[0030] Design of siRNA According to the sequence of the open reading frame (ORF) of the survivin gene, design small molecule interference RNA CPUsiRNA2 and 1 pair of negative control siRNA, compare their sequences in the human EST database, and confirm that there are no other gene sequences identical to them. CPUsiRNA2 sense strand: 5’-GAAUUUGAGGAAACUGCGAdTdT-3’, antisense strand: 5’-UCGCAGUUUCCUAAC...

Embodiment 2

[0035] Antitumor Pharmacodynamics Test

[0036] Materials and methods:

[0037] Tumor cell lines A549, HepG2 and HL60 were preserved by our laboratory, transfection reagent Lipofectamin TM 2000 was purchased from Invitrogen Company, Taq DNA polymerase, dNTP, DNA Marker and protein Marker were purchased from Takara Company. Rabbit anti-human antibody of Survivin protein, HRP-goat anti-rabbit IgG antibody, and DAB chromogenic kit were purchased from Wuhan Boster Company.

[0038] Cell culture Tumor cell lines A549, HepG2 and HL60 were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C, 5% CO 2 cultured in an incubator.

[0039] MTT method will 2.5×10 6 / L cells were inoculated in a 96-well plate, 180 μl per well, 3 wells in parallel for each group, and a blank and negative drug control were set up. After 44 hours, discard the supernatant, add 20 μL of freshly prepared 5g / L MTT to each well, incubate for 4 hours, discard the supernatant, add 150 μL DMSO to ...

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Abstract

The invention discloses a micro-molecular interference RNA (siRNA) structure and tumour-proof utility of target dying inhibiting gene surviving, which is characterized by the following: designing double-chain siRNA of functional conserving area of ORF to inhibit tumour such as lung carcinoma, liver cancer and leukemia cell; connecting positive-meaning chain of CPUsiRNA2 and mRNA of surviving; interfering the interpretation after transcription; inducing cell to dye for tumour cell; inhibiting cell from splitting; reducing the drug tolerance of tumour cell.

Description

technical field [0001] The present invention relates to the structure and application of small molecule interfering RNA (siRNA). Specifically, it relates to the structure of the siRNA of the apoptosis suppressing gene survivin and its anti-tumor application. Background technique [0002] Tumor molecular biology studies have shown that tumors are diseases caused by gene abnormalities. The pathogenic mechanism includes the activation of oncogenes, the inactivation of tumor suppressor genes and the abnormality of cell cycle. Further clarification of the molecular mechanism of tumor pathogenesis provides a basis for tumor gene therapy. Reliable theoretical basis. RNA interference technology is a biotechnology that has emerged in recent years. Using small molecule interference RNA to interfere with the expression of specific genes is an important part of gene therapy. [0003] The apoptosis suppressor gene survivin is an important tumor-related gene discovered in 1997, which wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/02A61K31/7088A61K48/00A61P35/00A61P35/02
Inventor 奚涛陆云华王淑珍邢滢滢
Owner CHINA PHARM UNIV
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