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Deoxyribozyme for promoting tumor cell apoptosis

A technology of tumor cell apoptosis and deoxyribozyme, which is applied in anti-tumor drugs, gene therapy, DNA/RNA fragments, etc., can solve the problems of cell apoptosis and tumor cell overgrowth, and achieve the promotion of apoptosis and low synthesis cost Effect

Inactive Publication Date: 2010-12-15
孙仑泉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that many tumors have dysregulation of apoptosis, accompanied by high expression of anti-apoptotic genes, leading to excessive growth of tumor cells

Method used

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  • Deoxyribozyme for promoting tumor cell apoptosis
  • Deoxyribozyme for promoting tumor cell apoptosis
  • Deoxyribozyme for promoting tumor cell apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Analysis and screening targeting bcl-2 and bcl-xL mRNA deoxyribozyme cleavage sites

[0023] Bcl-2 and Bcl-xL are highly expressed in many tumors, and inhibiting the expression of Bcl-2 or Bcl-xL can promote tumor cell apoptosis.

[0024]In this example, the cDNA clone of Bcl-2 used for deoxyribozyme design comprises 31 bp of 5'UTR, 720 bp of ORF, and 2.2 kb of 3'UTR sequence. By scanning bcl-2 mRNA, 141 potential AU and GU cleavage sites were identified. Take 9 nucleotides at the 5' end of these cleavage sites (excluding the R at the cleavage site R*Y) and 9 nucleotides at the 3' end (including the Y at the cleavage site R*Y) For the sequence, the enzyme-substrate complex thermodynamic stability analysis (-ΔG°kcal / mol) was performed on the sequence, and the hybridization free energy of the sequence at each site was obtained. Further analysis obtained Tm values ​​to ensure that the DNAzyme does not form intramolecular structures. Through the above analysis...

Embodiment 2

[0030] Example 2: Inhibitory effect of deoxyribozymes on tumor cell bcl-2 expression

[0031] Bcl-2 and Bcl-xL are highly expressed in many tumors. In order to verify the activity of DNAzymes screened in vitro in cells, we selected DNAzymes targeting Bcl-2 with higher activity in vitro, and used the transfection reagent TMP to transfect different DNAzymes at a concentration of 2mM DNAzymes. tumor cells. After 24 hours, the cellular proteins were extracted for Western Blot analysis. figure 2 The results showed that the deoxyribozymes DT895, DT899, DT908, DT910 and DT912 targeting Bcl-2 could all inhibit the expression of Bcl-2 in prostate cancer cell PC3 and bladder cancer cell T24.

Embodiment 3

[0032] Example 3: Inhibitory effect of deoxyribozymes on tumor cell bcl-xL expression

[0033] The same analysis results for deoxyribozymes targeting Bcl-xL showed ( image 3 ), DT888, DT884, DT883, DT882 showed a strong inhibitory effect in prostate cancer cell PC3. Further, DT882 was selected for analysis in bladder cancer cell line T24, lung cancer cell line A549, and colon cancer cell line HCT116. The results showed that DT882 could effectively inhibit the expression of Bcl-xL in these cells.

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Abstract

The invention discloses deoxyribozyme for promoting tumor cell apoptosis, which comprises a deoxynucleotide sequence 5'-GGCTAGCTACAACGA-3', a first binding sequence and a second binding sequence, wherein the deoxynucleotide sequence 5'-GGCTAGCTACAACGA-3' has catalyzing function; and the first binding sequence and the second binding sequence are respectively connected with the 5' end and the 3'end of the catalyzing function sequence, respectively comprise 7 to 12 nucleotides and are complemented to the 3'end and 5' end sequences of an R*Y cutting site of an apoptosis inhibiting gene mRNA or pre-mRNA in a bc1-2family. The deoxyribozyme of the invention reduces the expression of the apoptosis inhibiting gene by specifically recognizing and cutting the apoptosis inhibiting gene mRNA or pre-mRNA, thereby promoting the tumor cell apoptosis and inhibiting the growth of tumors.

Description

technical field [0001] The invention relates to a class of deoxyribozyme targeting tumor cell apoptosis gene family, in particular to a class of deoxyribozyme capable of inhibiting bcl family gene expression and promoting tumor cell apoptosis. Background technique [0002] Apoptosis is an important physiological process for body development, emergency response and removal of mutant cells. Abnormalities in this process can lead to many pathological changes in the body, including tumors, viral infections, and neurological diseases. There are two main pathways of cell apoptosis, one is to activate intracellular caspase by extracellular signal, and the other is to activate caspase by releasing caspase activating factor from mitochondria. These activated caspases can degrade important intracellular proteins and cause apoptosis. The regulation of apoptosis involves many gene products, including ICE, Apaf-1, Bcl-2, Fas / APO-1, c-myc, p53, ATM, etc. [0003] bcl-2 is an apoptosis ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 孙仑泉
Owner 孙仑泉
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