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shrna (short hairpin ribonucleic acid) for inhibiting expression of human oral cancer cell PRPS2 (phosphoribosyl pyrophosphate synthetase subunit ii) as well as construction and application of carrier of shrna

A technology for cancer cells and human oral cavity, which is applied in the fields of molecular biology and biomedicine to achieve the effect of inhibiting proliferation and overcoming the short duration of action.

Inactive Publication Date: 2013-10-02
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no report about using the PRPS2 gene as a target gene for the treatment of oral cancer, and there is no report of a eukaryotic expression vector containing both the hTERT promoter sequence and the PRPS2 shRNA interference sequence as a drug for the treatment of oral cancer

Method used

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  • shrna (short hairpin ribonucleic acid) for inhibiting expression of human oral cancer cell PRPS2 (phosphoribosyl pyrophosphate synthetase subunit ii) as well as construction and application of carrier of shrna
  • shrna (short hairpin ribonucleic acid) for inhibiting expression of human oral cancer cell PRPS2 (phosphoribosyl pyrophosphate synthetase subunit ii) as well as construction and application of carrier of shrna
  • shrna (short hairpin ribonucleic acid) for inhibiting expression of human oral cancer cell PRPS2 (phosphoribosyl pyrophosphate synthetase subunit ii) as well as construction and application of carrier of shrna

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Experimental program
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Effect test

Embodiment 1

[0031] Design of the hTERT promoter sequence of the present invention

[0032] The promoter region is the binding region of RNA polymerase, its sequence directly determines the structure, and the structure is directly related to the efficiency of transcription. Using DNAMAN analysis software, the 334bp hTERT promoter sequence (SEQ ID NO: 4) designed and used in the present invention was compared with the 4 hTERT promoter sequences reported in the literature, respectively [Horikawa I, Cable PL, Afshari C, Barrett JC. Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene. Cancer Res. 1999 Feb 15;59(4):826-830.] The 59bp hTERT promoter sequence reported (SEQ ID NO: 5), Literature [Takakura M, Kyo S, Kanaya T, Hirano H, Takeda J, Yutsudo M, Inoue M. Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer...

Embodiment 2

[0034] Design of shRNA that can specifically reduce PRPS2 gene expression in human oral cancer cells

[0035] Search the mRNA base sequence of the human PRPS2 gene in the NCBI database (GeneBank number: NM_001039091.2), and use the siRNA finder software (http: / / www.ambion.com / techlib / misc / siRNA_finder.html) to select the target Sequence, the target sequence is the 422nd to 440th positions of human PRPS2 mRNA, as shown in SEQ ID NO: 2, 5'- AUACGCCCGA CAAGAUAAA -3'. The DNA sequence that can be transcribed to generate shRNA designed according to the target sequence is: SEQ ID NO: 3: 5'- CCATACGCCC GACAAGATAA ACTCGAGTTT ATCTTGTCGG GCGTATGG -3', and the shRNA sequence generated after transcription is: SEQ ID NO: 1: 5' - CCAUACGCCC GACAAGAUAA ACUCGAGUUU AUCUUGUCGG GCGUAUGG -3'.

Embodiment 3

[0037] Construction of recombinant vector pGGN-hTERT-PRPS2 that can specifically reduce the expression of PRPS2 gene in human oral cancer cells

[0038] (1) The design scheme for recombining SEQ ID NO: 3 into pGPU6 / GFP / Neo siRNA eukaryotic expression vector is: EcoRI- Target sense sequence - Hairpin loop - Target antisense sequence - Terminator - BamHI, the specific operation steps are as follows:

[0039] Operation Step 1: Use a DNA synthesizer to synthesize the DNA shown in SEQ ID NO: 3, and add EcoRI and BamHI restriction sites at both ends. The structural model diagram is shown in figure 2 .

[0040] The second step of operation: annealing the above-mentioned artificially synthesized single-stranded DNA sequence to form a double-stranded structure. The annealing reaction system is as follows: each 2mL of forward and reverse DNA oligonucleotides is mixed with 46ml of annealing buffer, at 95°C for 4min, at 70°C for 10min, and slowly cool the annealed oligonucleotides t...

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Abstract

The invention relates to the field of biological medicines, and in particular relates to an shRNA (short hairpin ribonucleic acid) for inhibiting expression of human oral cancer cell PRPS2 (phosphoribosyl pyrophosphate synthetase subunit II) as well as construction and application of carrier of the shRNA. The shRNA is shown in SEQ ID NO.1, a target sequence of the shRNA is as whon in SEQ ID NO:2, and is transcribed and generated through a DNA (deoxyribonucleic acid) template sequence shown in SEQ ID NO.3; the template sequence and a linear carrier are recombined, and are recombined with an hTERT (human telomerase reverse transcriptase) promoter sequence to obtain an shRNA eukaryotic expression plasmid for inhibiting expression of human oral cancer cell PRPS2. Verification indicates that the eukaryotic expression plasmid carrier can be used for stably and constantly reducing the expression level of mRNA (messenger ribonucleic acid) and protein of the PRPS2, and causing occurrence of cell prolifation inhibition, cell cycle repression and apoptosis, so that the defects of short acting duration, high toxicity and the like of in vitro chemically synthesized siRNA (small interfering ribonucleic acid) can be overcome.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, in particular to the construction and application of shRNA for inhibiting the expression of human oral cancer cell PRPS2 and its carrier. Background technique [0002] As a kind of anti-metabolism drug, 5-fluorouracil has been widely used in the treatment of various malignant tumors. Its mechanism of action is to cause cell death by interfering with DNA synthesis and mRNA translation. However, due to its non-specificity, 5-fluorouracil has strong toxic side effects on patients, which may lead to coronary artery spasm, coronary artery thrombosis, myocarditis, and sudden cardiac death [Sorrentino MF, Kim J, Foderaro AE, Truesdell AG. 5-fluorouracil induced cardiotoxicity: review of the literature[J]. Cardiol J. 2012;19(5):453-458.]. Therefore, searching for new effective anti-metabolite drugs is an urgent problem to be solved in tumor treatment. hTERT is only expressed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66A61K48/00A61P35/00A61P1/02
Inventor 陈显久吴彬师磊李冰解军
Owner SHANXI MEDICAL UNIV
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