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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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have shown that many tumors have dysregulation of apoptosis, accompanied by

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

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Example Embodiment

[0022] Example 1: Analysis and screening of targeted 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 Bcl-2 cDNA clone used for DNAzyme design contains a 31bp 5'UTR, a 720bp ORF, and a 2.2kb 3'UTR sequence. By scanning bcl-2mRNA, 141 potential AU and GU cutting sites were identified. Take 9 nucleotides at the 5'end of these cutting sites (not including the R at the cutting site R*Y) and 9 nucleotides at the 3'end (including the Y at the cutting site R*Y) For the sequence, the thermodynamic stability analysis of the enzyme-substrate complex (-ΔG°kcal / mol) was performed on the sequence to obtain the hybridization free energy of the sequence at each site. Further analysis obtained the Tm value to ensure that the DNAzyme does not form an intramolecular structure. Through the above analysis, based on -25kcal / ...

Example Embodiment

[0030] Example 2: Inhibition of DNAzyme on the expression of bcl-2 in tumor cells

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

Example Embodiment

[0032] Example 3: Inhibition of DNAzyme on the expression of bcl-xL in tumor cells

[0033] The same analysis of DNAzyme targeting Bcl-xL showed that ( image 3 ), DT888, DT884, DT883, DT882 show strong inhibitory effects in prostate cancer cell PC3. DT882 was further selected for analysis in the 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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