shRNA for specifically reducing human Aurora-A gene expression and application thereof

A gene expression and species-specific technology, applied in the field of molecular genetics and biomedicine, can solve the problems of poor prognosis, patient death, tumor recurrence, etc., and achieve the effect of overcoming the short-term effect

Inactive Publication Date: 2012-09-26
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although surgical resection and postoperative radiotherapy and chemotherapy have greatly improved the survival rate of patients, the overall prognosis is still not optimistic, and the main cause of patient death is tumor recurrence and metastasis.

Method used

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  • shRNA for specifically reducing human Aurora-A gene expression and application thereof
  • shRNA for specifically reducing human Aurora-A gene expression and application thereof
  • shRNA for specifically reducing human Aurora-A gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Design of shRNA that can reduce the expression of human Aurora-A gene

[0025] According to the mRNA base sequence of the human Aurora-A gene (GeneBank number NM_003600.2) in the NCBI database, the target sequence was selected with the help of siRNA tool software (siDirect version 2.0) provided by siDirect on the Internet, and the target sequence was human Aurora-A The 995th to 1015th positions of the mRNA, as shown in SEQ ID NO.2, that is, 5'-GCACCACUUGGAACAGUUUAU-3',

[0026] The corresponding shRNA sequence designed according to the target sequence is:

[0027] SEQ ID NO.1: 5'-GCACCACUUGGAACAGUUUAUUUCAAGAGAAUAAACUGUUCCAAGUGGUGC-3'

[0028] The DNA sequence encoding the shRNA is:

[0029] SEQ ID NO.3: 5'-GCACCACTTGGAACAGTTTATTTCAAGAGAATAAACTGTTCCAAGTGGTGC-3'

[0030] The shRNA sequence can be cleaved in vivo or in vitro to form siRNA sense strand and antisense strand as shown in SEQ ID NO.4 and SEQ ID NO.5:

[0031] SEQ ID NO.4: the sense strand is 5'-GCACCACUUGGA...

Embodiment 2

[0034] Construction of shRNA interference vector capable of reducing human Aurora-A gene expression

[0035] According to the above sequence, Shanghai Gemma Co., Ltd. synthesized the DNA shown in SEQ ID NO: 3 by chemical synthesis, and added BamHI and BbsI enzyme cutting sites at both ends. Dissolve the DNA oligonucleotide in sterile, nuclease-free water to a final concentration of 3 mg / mL. The annealing reaction is to mix each 1mL of forward and reverse DNA oligonucleotides with 48ml of annealing buffer (10mM Tris, pH 7.5-8.0, 50mM NaCl, 1mM EDTA), incubate at 90°C for 4min, and incubate at 70°C. Incubate for 10 min, and slowly cool the annealed oligonucleotides to 10°C. The annealed product was double digested with the empty pGPU6 / GFP / Neo plasmid (provided by Shanghai Gemma Co., Ltd.), and the digested product was purified using a DNA purification kit, and 2 μL of each was ligated with T4 DNA ligase. Transform the recombinant pGPU6 / GFP / Neo vector into an agarose plate cont...

Embodiment 3

[0038] Preparation of drug containing human Aurora-A-shRNA interference carrier

[0039] Dilute 8 μg of plasmid in 500 μL of medium-free medium and mix gently. Dilute 8 μL of Lipofectamine 2 000 (liposome-mediated method) into 500 μL serum-free medium, mix well, and incubate at room temperature for 5 min. Mix the diluted liposomes with the diluted plasmid DNA and incubate at room temperature for 20min.

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Abstract

The invention relates to the field of molecular genetics and biomedicines, in particular to a shRNA for specifically reducing human Aurora-A gene expression and application of the shRNA. The invention provides hairpin interference RNA (ribonucleic acid) for specifically reducing human Aurora-A gene expression, shown by SEQIDNO. 1; an aimed target sequence is the 995th-1015th bit of human Aurora-AmRNA, shown by SEQIDNO. 2; the shRNA can be sheared in vivo or in vitro to form siRNA shown by SEQIDNO. 4 and SEQIDNO. 5; the invention further provides a DNA (deoxyribonucleic acid) oligonucleotide chain for coading the shRNA, shown by SEQIDNO. 3, and a plasmid including the DNA oligonucleotide chain shown by SEQIDNO. 3. According to the invention, after medicines of Aurora-A-shRNA interference carriers transfect human esophageal cancer cells EC9706, an mRNA transcriptional level and a protein expression level of Aurora-A genes in cells are significantly reduced so as to significantly reduce invasiveness of esophageal cancer cells and prompt apoptosis of esophageal cancer cells irradiated and induced by UV (ultra violet), thereby overcoming the defect that the siRNA chemically synthetized in vitro has short acting time, and providing a new way for developing novel antitumor drugs.

Description

technical field [0001] The invention relates to the technical fields of molecular genetics and biomedicine, in particular to a shRNA that specifically reduces the expression of human Aurora-A gene and its application. Background technique [0002] RNA interference technology (RNAi) refers to the phenomenon that double-stranded RNA specifically induces the degradation of its homologous sequence mRNA molecules, resulting in the inhibition of corresponding gene expression. After the small double-stranded RNA (small interfering RNA, siRNA) produced by artificial chemical synthesis or intracellular transcription enters the cell, it can bind to the complementary target gene mRNA, thereby mediating specific cutting enzymes to degrade the mRNA to achieve reduction The purpose of gene expression of interest. [0003] At present, although siRNA chemically synthesized in vitro can reduce the expression of target genes, its effect is relatively short-lived. For long-term gene suppress...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 王晓霞裴毅李晓钟路娜陈显久牛勃
Owner SHANXI MEDICAL UNIV
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