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Novel anti-tumor amiRNA (artificial micro Ribonucleic Acid) sequence and application thereof

A technology of anti-tumor and anti-tumor drugs, applied in the direction of anti-tumor drugs, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effects of inhibiting growth, inhibiting tubule formation, and promoting apoptosis

Inactive Publication Date: 2012-09-19
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the highly anticipated amiRNA has only just begun. Although there are a few successful cases of using amiRNA to regulate the expression of target genes, there are no reports of using amiRNA to target hTERT to achieve high-efficiency and low-toxicity treatment of tumor cells ( Dickins et al., 2005)

Method used

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  • Novel anti-tumor amiRNA (artificial micro Ribonucleic Acid) sequence and application thereof
  • Novel anti-tumor amiRNA (artificial micro Ribonucleic Acid) sequence and application thereof
  • Novel anti-tumor amiRNA (artificial micro Ribonucleic Acid) sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Design and synthesis of amiRNA-hTERT.

[0038] The cDNA sequence number (NM_198253) of the human telomerase reverse transcriptase gene hTERT was found from GenBank, and amiRNA design was carried out in combination with the design principles of commonly used siRNA and miRNA mimics and the structural characteristics of natural miRNA: ① First, use the siRNA rule for positioning, Then the pre-selected action sites are transformed into miRNA mimics, and finally the structure is optimized according to the structural characteristics of the natural miRNA to obtain the candidate amiRNA; ② Select the translated region (ORF) and 3' untranslated region (3' -UTR) is designed for the target region; ③The percentage content of GC is controlled at 35%-55%; ④The evaluation level is set to the highest level of 5 stars; ⑤The specificity of the sequence is verified by BLAST. As a result, 2 amiRNAs (amiRNA-hTERT 1, amiRNA-hTERT 2) were obtained, and amiRNA-hTERT 3 was obtained by structural ...

Embodiment 2

[0041] In vitro cell experiments were used to evaluate the anti-tumor effect of amiRNA-hTERT.

[0042] 1. Determination of cell culture and amiRNA-hTERT transfection efficiency

[0043] In this example, 4 strains of cells were selected according to the purpose of the experiment (all were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences / Cell Resource Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences), which were normal cells with low expression of telomerase (HUVEC), telomerase-negative tumor cells (U2-OS) and tumor cells with high telomerase expression (Hela and NCI-H446). All cells were grown in RPMI1640 medium (Invitrogen) containing 10% fetal bovine serum (Gibico), 100 U / ml ampicillin, and 100 ug / ml streptomycin sulfate (Sigma), at 37°C and 5% CO 2 Continuous cultivation was carried out under the environmental conditions of saturated humidity. The day before transfection, ce...

Embodiment 3

[0070] Animal experiments evaluated the anti-tumor effect of amiRNA-hTERT.

[0071] NCI-H446 cells were inoculated into a 15cm-diameter culture dish (Corning Company) at 1.5E+7 cells / dish, and were transfected with amiRNA-hTERT 1, amiRNA-hTERT 2 and NC with Lipo2000 20 hours after plate placement, and transfected The concentration was 50nM, and the transfection scheme was carried out according to the instructions of lipo2000. Digest and count cells 24 hours after transfection, collect cells by centrifugation at 800rpm x 5min, resuspend cells in pre-cooled PBS and collect by centrifugation, resuspend cells in PBS and adjust cell density to 0.5E+8 cells / ml, 100ul / piece The amount of cells was inoculated into the left forelimb axilla of 4-week-old BABL / c nude mice (NLARSH, Shanghai Branch of National Rodent Laboratory Animal Seed Center (NLARSH) Shanghai Slack Experimental Animal Co., Ltd.), with 6 nude mice in each group. The length a and width b of the tumor were measured ever...

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Abstract

The invention discloses a novel anti-tumor amiRNA (artificial micro Ribonucleic Acid) sequence, which comprises any one or more than one of the following sequences: amiRNA-hTERT (human Telomerase Reverse Transcriptase) 1: positive-sense strand 5'-TGACCAAATGTGCCCTGTA-3'; anti-sense strand 5'-TACAGGGCACACCTTTGGTCA-3'; amiRNA-hTERT 2: positive-sense strand 5'-CAGAGCCACTCACCTTCAA-3'; anti-sense strand 5'-TTGAAGGTGAGACTGGCTCTG-3'; amiRNA-hTERT 3: positive-sense strand 5'-CAGAGCCAGTCTCACCTTCAA-3'; and anti-sense strand 5'-TTGAAGGTGAGACTGGCTCTG-3'. According to the novel anti-tumor amiRNA sequence, growth, proliferation and migration of tumor can be inhibited, so that tumor cells enter an aging state to promote the apoptosis of the tumor cells.

Description

technical field [0001] The present invention relates to a novel anti-tumor amiRNA sequence and its application. Background technique [0002] Telomere is a short repetitive DNA sequence 5'-GGTTAG-3' that exists at the end of linear chromosomes in eukaryotic cells. Together with telomere binding proteins, it forms a special "cap" structure and maintains the integrity of chromosomes and genomes Properties and stability (Rlackburn, E.H. Nature, 350, 569-573; 1991.). In normal somatic cells, telomeres shorten a little each time the cell divides due to the "Okazaki fragment" effect during DNA replication. Once telomeres are depleted, chromosomes are susceptible to mutations that lead to apoptosis or other cancerous changes. Therefore, there is a clear relationship between telomeres and cellular aging. Its length reflects the replication history and potential of cells, and is called the "mitotic clock" of cell lifespan (Kim Sh, S.H., et al. Oncogene 21, 503-511; 2002.). When t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 许瑞安唐明青
Owner HUAQIAO UNIVERSITY
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