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Application of antisense oligonucleotide ASOYAP1 in inhibition of multiple YAP1 positive cancers

An antisense oligonucleotide and oligonucleotide technology, which is applied in the direction of DNA / RNA fragments, organic active ingredients, recombinant DNA technology, etc., can solve the problems of insufficient research and development of inhibitors, and achieve high knockout efficiency

Pending Publication Date: 2022-04-29
天津市泌尿外科研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Studies on antisense oligonucleotides targeting YAP1 have not been reported, and the development of its inhibitors is still insufficient

Method used

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  • Application of antisense oligonucleotide ASOYAP1 in inhibition of multiple YAP1 positive cancers
  • Application of antisense oligonucleotide ASOYAP1 in inhibition of multiple YAP1 positive cancers
  • Application of antisense oligonucleotide ASOYAP1 in inhibition of multiple YAP1 positive cancers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Embodiment 1 Design and detection of YAP1 antisense oligonucleotide

[0133] 1. Biological identification of ASO sequences and comparison of modification schemes and sites

[0134] Use the BLAST biological serial information primary structure online comparison tool in NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to compare and analyze the mRNA structure information of each transcript of YAP1 mRNA, and record it consensus sequence. Use the "soligo" page (http: / / sfold.wadsworth.org / cgi-bin / index.pl) in the sfold RNA secondary structure online prediction tool to input the consensus sequence of each transcript of YAP1, and select the oligo length as 20nt. Submit and record screening results.

[0135] According to the basic principle of complementary base pairing, we designed 9 (see Table 1) antisense oligonucleotides ASO covering 20 nucleic acids including functional sites Yap1 , and use phosphorothioate full-chain modification as basic modification (Table 2)

[0136...

Embodiment 2

[0164] The antisense oligonucleotide ASO of embodiment 2 methoxyethyl modification YAP1 The mode of action of knocking out YAP1

[0165] The C4-2 cells were cultured in a six-well plate in vitro, and the cells were transfected 24 hours after the cells were plated. methoxyethyl-modified antisense oligonucleotide ASO YAP1 Mix ASO-3-flank5 and liposome transfection reagent evenly and incubate at room temperature for 20min, then add the mixture evenly into the culture medium. makes ASO YAP1 The final concentrations in the medium were 0nM, 50nM, 100nM, 200nM, and the cell culture medium was removed after 48h, and Trizol / RIPA reagent was added to extract the total RNA and total protein of the cells. Another plate was spread, and cells were transfected 24 hours later, and ASO was transfected with liposomes at a final concentration of 100 nM. After 0, 12h, 24h and 48h respectively, the same method was used to extract the total RNA and total protein of the cells. The total RNA wa...

Embodiment 3

[0167] Embodiment 3 detects ASO YAP1 Knockout ability of YAP1 protein level in various cancer cells

[0168] Use ASO-3-flank5 and liposome transfection reagent to transfect breast cancer MCF-7 cells, lung cancer cells A549 cells, cervical cancer cells Hela cells, bladder cancer T24 cells, and liver cancer cells HUH-6. Western blot and RT-QPCR were used to detect the expression levels of YAP1 protein and mRNA 48h after transfection with 100nM ASO.

[0169] The result is as Figure 5 As shown, in breast cancer MCF-7 cells, lung cancer cells A549 cells, cervical cancer cells Hela cells, bladder cancer T24 cells, and liver cancer cells HUH-6, the expression levels of YAP1 mRNA and protein in the experimental group were significantly down-regulated.

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Abstract

The invention discloses an application of an antisense oligonucleotide ASOYAP1 in inhibition of a plurality of YAP1 positive cancers. A bioinformatics on-line tool is used for analyzing sequence information of a YAP1 protein transcriptome, antisense oligonucleotide sites which have the length of 20 basic groups and can be combined are screened, meanwhile, antisense oligonucleotide is modified, and it is proved that the modified antisense oligonucleotide has the high capacity of inhibiting the level of YAP1 mRNA and protein thereof in cells. The invention provides a new means of nucleic acid drug treatment for clinically treating various cancer cells with high YAP1 expression and important YAP1 effects.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to antisense oligonucleotide ASO YAP1 Application in inhibiting various YAP1 positive cancer cells. Background technique [0002] In 2018, there were as many as 18,078,957 new cancer cases in the world, the incidence rate was 236.9 / 100,000, and the world standard population standardized incidence rate was 197.9 / 100,000; the number of cancer deaths was as high as 9,555,027, and the mortality rate was 125.2 / 100,000, the world standard population standardized death rate The rate is 101.1 / 100,000. Among the 185 countries in the world, prostate cancer is the number one cancer in men in 105 countries; lung cancer is the number one cancer in men in 37 countries; liver cancer and colorectal cancer are the number one cancer in 13 and 10 countries respectively. Lung cancer is the leading cause of death among men in 93 countries, prostate cancer in 46 countries, and liver cancer in 20 countries. In ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7105A61P35/00
CPCC12N15/1135A61K31/7105A61P35/00C12N2310/11C12N2310/341C12N2310/315C12N2310/321C12N2310/3525
Inventor 牛远杰尚芝群李阳
Owner 天津市泌尿外科研究所
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