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Special short hairpin ribonucleic acid (shRNA) for reducing human IQGAP1 gene expression and application thereof

A gene expression and species-specific technology, applied in the field of molecular genetics and biomedicine, to overcome the effects of short action time and inhibition of invasion

Inactive Publication Date: 2012-06-27
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no report on the IQGAP1 gene as a target gene for the treatment of esophageal cancer

Method used

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  • Special short hairpin ribonucleic acid (shRNA) for reducing human IQGAP1 gene expression and application thereof
  • Special short hairpin ribonucleic acid (shRNA) for reducing human IQGAP1 gene expression and application thereof
  • Special short hairpin ribonucleic acid (shRNA) for reducing human IQGAP1 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Expression of IQGAP1 in Esophageal Carcinoma and Adjacent Tissues

[0025] Tissue chips were purchased from Shanghai Xinchao Biotechnology Co., Ltd. The whole tissue microarray included 75 cases of esophageal squamous cell carcinoma and matched adjacent normal tissues, with a total of 150 points.

[0026] Immunohistochemical staining steps:

[0027] 1) Paraffin-embedded sections of tissue specimens, routine dewaxing: bake the slices at 70°C for 2 hours, and treat with xylene twice, 10 minutes each time. 2) Hydration: 100%, 85%, 70% ethanol for 3 minutes each. 3) Wash twice with PBS buffer, 3 minutes each time. 4) 3%H 2 o 2The solution was treated at room temperature for 10 min to remove endogenous peroxidase. 5) Wash with double distilled water 3 times, 3 minutes each time. 6) Soak in 0.01 M citrate buffer (pH 6.0) in a 92-98°C water bath for 25 minutes to restore the antigen, and cool down to room temperature naturally. 7) Wash twice with PBS buffer, 3 minutes ...

Embodiment 2

[0032] Design of shRNA that can reduce the expression of human IQGAP1 gene

[0033] According to the mRNA base sequence of the human IQGAP1 gene (GeneBank number NM003870) in the NCBI database, the target sequence was selected with the help of the siRNA tool software (siDirect version 2.0) provided by siDirect on the Internet. Position, as shown in SEQ ID NO.2, namely 5'-GUUAUGGUUGGAUGAAAUUCA-3',

[0034] The corresponding shRNA sequence designed according to the target sequence is:

[0035] SEQ ID NO.1: 5'-GUUAUGGUUGGAUGAAAUUCACUCGAGUGAAUUUCAUCCAACCAUAAC-3'

[0036] The DNA sequence encoding the shRNA is:

[0037] SEQ ID NO.3: 5'-GTTATGGTTGGATGAAATTCACTCGAGTGAATTTCATCCAACCATAAC-3'

[0038] 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:

[0039] SEQ ID NO.4: the sense strand is 5'-GUUAUGGUUGGAUGAAAUUCA-3',

[0040] SEQ ID NO.5: The antisense strand is 5'-UGAAUUUCAUCCAACCAUAAC-3...

Embodiment 3

[0042] Construction of shRNA interference vector capable of reducing human IQGAP1 gene expression

[0043] According to the above sequence, Shanghai Jikai Co., Ltd. synthesized the DNA shown in SEQ ID NO: 3 by chemical synthesis, and added BamHI and Hind III restriction 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 GV102 plasmid (provided by Shanghai Jikai Co., Ltd.), the digested product was purified using a DNA purification kit, and 2 μL of each was ligated with T4 DNA ligase. Transform the recombinant GV102 vector into an agarose plate containing ampicillin, pi...

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Abstract

The invention relates to the field of molecular genetics and biological medicaments, in particular to a special short hairpin ribonucleic acid (shRNA) for reducing human IQGAP1 gene expression and application thereof. A special hairpin type interfering RNA for reducing human IQGAP1 gene expression is indicated in SEQIDNO.1, the corresponding target sequences are the nineteen hundred and first to nineteen hundred and twenty-first of IQGAP1MrRNA and are indicated in SEQIDNO.2, and the shRNA can be shorn in vivo or in vitro to form siRNA as indicated in SEQIDNO.4 and SEQIDNO.5. A deoxyribose nucleic acid (DNA) oligonucleotide chain encoding the shRNA and indicated in SEQIDNO.3 is further provided, and a plasmid of the DNA oligonucleotide chain as indicated in the SEQIDNO.3 is contained. Besides, the fact is proven that after a drug containing IQGAP1-shRNA interfering vector is utilized to instantly transfect human esophageal carcinoma EC9706 cells, transcription level of mRNA of IQGAP1 gene in the cells is remarkably reduced, protein expression level is remarkably reduced, invasion capability of the esophageal carcinoma cells is remarkably reduced, the shortcoming that the action time of siRNA which is chemosynthetic in vitro is transient is overcome, and novel ways are provided for development of novel antitumor drugs.

Description

technical field [0001] The invention relates to the technical fields of molecular genetics and biomedicine, in particular to shRNA that specifically reduces the expression of human IQGAP1 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 suppression,...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N15/11A61K48/00A61P35/00
Inventor 王晓霞陈显久牛勃解军路娜程牛亮
Owner SHANXI MEDICAL UNIV
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