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Application of ZNF498 protein expression quantity inhibiting substance in preparing cancer preventing and treating products

A technology of expression quantity and protein, applied in the field of biomedicine

Active Publication Date: 2018-03-20
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The p53 gene is so far found to be the most relevant gene to human tumors. Mutations of the p53 gene have been found in more than 50% of human tumor tissues, and most of the tumors without p53 gene mutations have defects in the regulation mechanism of the p53 gene

Method used

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  • Application of ZNF498 protein expression quantity inhibiting substance in preparing cancer preventing and treating products
  • Application of ZNF498 protein expression quantity inhibiting substance in preparing cancer preventing and treating products
  • Application of ZNF498 protein expression quantity inhibiting substance in preparing cancer preventing and treating products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1, the construction of plasmid, the preparation of oligonucleotide and the acquisition of HepG2 shRNA-ZNF498 cell

[0094] 1. Construction of Myc-ZNF498 plasmid

[0095] The small fragment between the recognition sequences of the restriction endonucleases EcoRI and XhoI of the pCMV-Myc plasmid was replaced with the DNA molecule shown in sequence 2 in the sequence listing, and the resulting recombinant plasmid was the Myc-ZNF498 plasmid.

[0096] The Myc-ZNF498 plasmid expresses the ZNF498 protein shown in Sequence 1 in the Sequence Listing.

[0097] 2. Construction of Flag-p53 plasmid

[0098] The small fragment between the recognition sequences of the restriction endonucleases EcoRI and BamHI of the pCMV-Flag plasmid is replaced with the DNA molecule shown in sequence 4 in the sequence table, and the resulting recombinant plasmid is the Flag-p53 plasmid.

[0099] The Flag-p53 plasmid expresses the p53 protein shown in sequence 3 in the sequence listing.

...

Embodiment 2

[0120] Example 2, ZNF498 protein can inhibit endogenous p53 protein activity

[0121] Experiment 1. ZNF498 protein can inhibit the transcriptional activity of endogenous p53 protein

[0122] The experiment was repeated three times to take the average value, and the steps for each repetition were as follows:

[0123] 1. Convert p53 + / + HepG2 cells were seeded in 12 wells of a 24-well plate containing 0.5 mL of DMEM medium (4.0 × 10 4 cells), and then placed at 37°C, 5% CO 2 Cultivate in an incubator, and when the fusion rate reaches 70-90%, they are randomly divided into four groups, and each group is set with three replicate wells, and the following treatments are carried out:

[0124] The first group: add 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.4μg pCMV-Myc plasmid to each well, and co-transfect for 36h.

[0125] The second group: add 20ng pG13L plasmid, 0.2ng pRL-TK plasmid, 0.3μg pCMV-Myc plasmid and 0.1μg Myc-ZNF498 plasmid to each well, and co-transfect for 36h...

Embodiment 3

[0150] Embodiment 3, inhibit the expression of ZNF498 protein and increase the expression level of Puma protein

[0151] Experiment 1: After inhibiting the expression of ZNF498 protein, the expression levels of Puma protein and Gadd45 protein were increased

[0152] The experiment was repeated three times to take the average value, and the steps for each repetition were as follows:

[0153] 1. Convert p53 + / + HepG2 cells were seeded in 3 wells of a 24-well plate containing 0.5 mL DMEM medium (4.0 × 10 4 cells), and then placed at 37°C, 5% CO 2 Cultivate in an incubator, and when the fusion rate reaches 70-90%, proceed as follows:

[0154] The first well: add 0.5 μg siRNA-con, and co-transfect for 36 hours;

[0155] The second well: add 0.5 μg siRNAa, and co-transfect for 36 hours;

[0156] The third well: add 0.5 μg siRNAb, and co-transfect for 36 hours.

[0157] 2. After completing step 1, extract the total protein of each cell, and perform Western Blot with Puma antibo...

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Abstract

The invention discloses application of ZNF498 protein expression quantity inhibiting substance in preparing cancer preventing and treating products. Experiments prove that ZNF498 protein can be combined with p53 protein to inhibit transcriptional activity of the p53 protein, reduce an expression level of Puma protein and lower an expression quantity of puma gene. Inhibiting expression of the ZNF498 protein can improve the transcriptional activity of the p53 protein, promote expression of the Puma protein and Gadd45 protein, up regulate the expression quantity of the puma gene, improve a phosphorylation level of 46th site serine, promote apoptosis of p53 positive cancer cells and inhibit proliferation of cancer cells and growth of cancer. Thereofore, inhibiting expression of the ZNF498 protein has important application value in promoting apoptosis of the cancer cells, inhibiting proliferation of the cancer cells and inhibiting growth of the cancer, and inhibiting the expression of the ZNF498 protein can prevent and treat cancer.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of a substance inhibiting the expression of ZNF498 protein in the preparation of products for preventing and treating cancer. Background technique [0002] The KRAB zinc finger protein (KRAB-containing zinc finger protein, KZNF) family is the largest transcription factor / transcription regulator family in mammals, and its protein structure is mainly composed of the conserved KRAB (Krüppel -associated box) domain, the middle and the C-terminus contain multiple C2H2-type zinc finger structures that continuously recognize continuous DNA sequences. Among them, the KRAB domain recruits a variety of transcriptional repressors by combining with KAP-1 (KRAB-associated protein-1), forming a transcriptional repression complex, and the DNA and / or other transcriptional factors in the C-terminal zinc finger region and the target gene regulatory region After binding, the transcriptio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K48/00A61K31/7105A61P35/00G01N33/68
CPCA61K31/7105A61K45/00G01N33/68
Inventor 田春艳王建令吉明张秀园原艳芝
Owner ACADEMY OF MILITARY MEDICAL SCI
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