Mutant ubiquitination-specific protease 33 gene and its application

A specific, protease technology, applied in the field of molecular biology, which can solve the problems of inability to deubiquitinate Robo receptors in time, low expression, tumor cell invasion and migration, etc.

Active Publication Date: 2019-05-17
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In normal cells, USP33 can deubiquitinate the ubiquitinated Robo receptor, thereby stabilizing Robo, and affect the Actin cytoskeleton structure in the cytoplasm through the Slit2-Robo signaling pathway, thereby inhibiting cell migration; but in tumor cells, Due to the low expression of USP33, the ubiquitinated Robo receptors cannot be deubiquitinated in time and are degraded, and the Slit2-Robo signaling pathway cannot function normally, resulting in the invasion and migration of tumor cells

Method used

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  • Mutant ubiquitination-specific protease 33 gene and its application
  • Mutant ubiquitination-specific protease 33 gene and its application
  • Mutant ubiquitination-specific protease 33 gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 KDM5B is highly expressed in colorectal cancer cells and down-regulates the expression of USP33

[0021] Routine immunohistochemical analysis was performed on 273 clinical cases of colorectal cancer tissue samples. The results showed that KDM5B was significantly highly expressed in colorectal cancer tissues, while USP33 was significantly less expressed, as shown in figure 1 shown.

[0022] Conventional cell culture techniques were used to culture human colorectal cancer cell lines HCT-16, SW480, Caco-2 and normal human colon cells NCM460, and harvest the cells when the cells were growing well. Various cell lysates were obtained by conventional cell protein preparation technology, and the expression levels of KDM5B and USP33 in the above cells were detected by conventional Western-blot technology. The results showed that the expression of KDM5B and USP33 in colon cancer cells was negatively correlated, as shown in figure 2 shown. Antibodies used in Western-...

Embodiment 2

[0023] Example 2 KDM5B binds to the USP33 gene promoter region

[0024] To clarify whether KDM5B binds to the USP33 gene promoter region, a qChIP protocol was used. details as follows:

[0025] (1) Collect cultured colon cancer cells at room temperature, centrifuge at 2000 g for 5 min to collect 4×10 7 Cells were resuspended in 4°C pre-cooled cell culture medium, transferred to a 15ml centrifuge tube, and placed in ice for 10 min.

[0026] (2) Use 1% formaldehyde solution for cross-linking reaction, the purpose is to stabilize the combination of protein factors and DNA, and stop with 0.125M glycine after the cross-linking reaction.

[0027] (3) Centrifuge to obtain the cell pellet, first use 10ml of L1 solution (50 mMHepes KOH, pH7.5, 140 mMNaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) to lyse, 4°C, 10 min. Centrifuge after L1 lysis, 3000rpm, 10min, 4°C.

[0028] (4) The obtained cell pellet was further lysed with 10ml of L2 solution (0.2 M NaCl, 1 mM EDT...

Embodiment 3

[0041] Example 3 KDM5B down-regulates the transcription of USP33 gene

[0042] To confirm the effect of KDM5B on USP33 gene transcription, a luciferase reporter assay was used, as follows:

[0043] (1) Construction of the luciferase assay reporter vector. Get the upstream 3kb sequence of USP33 from the UCSC website (http: / / genome.ucsc.edu / ), design the upstream and downstream primers (see attached table) according to the genome sequence, get USP33-3kb, 2kb, 1kb promoter sequences respectively, and insert KpnI and XhoI restriction sites in the pGL4.15 vector were used to construct pGL4.15-USP33-3kb, pGL4.15-USP33-2kb and pGL4.15-USP33-1kb, respectively. The KDM5B expression vector was constructed, primers were designed according to the KDM5B gene (NM_006618.3), and then cloned into the pcDNA3.1-HA vector NheI and

[0044]

[0045] (2) Cell transfection test. Using conventional cell transfection techniques, the above constructed pcDNA3.1-KDM5B and pGL4.15-USP33-3kb, or pGL...

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Abstract

The invention discloses a mutated ubiquitin specific protease 33 gene and application of the mutated ubiquitin specific protease 33 (USP33) gene to production of a medicine for treating colorectal cancer. A research finds out that KDM5B has high expression in a colorectal cancer clinical sample and a colorectal cancer cell line and promotes the proliferation of colorectal cancer cells through inhibiting USP33. A KDM5B inhibitor or a monoclonal antibody of the KDM5B can be used for remarkably inhibiting the proliferation of the colorectal cancer cells. A further research proves that the KDM5B is combined with a USP33 gene promotor region, and the inhibition of the expression to the USP33 gene is realized through combining an upstream specific regulation and control sequence GCACA / C or G / TGTGC. The mutated ubiquitin specific protease 33 gene obtained through a site-directed mutation PCR (Polymerase Chain Reaction) technology can be used for resisting the inhibition effect of the KDM58B, so that the growth of the colorectal cancer cell line is inhibited.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a mutated ubiquitination-specific protease 33 gene and its application in the production of medicines for treating colorectal cancer. Background technique [0002] Colorectal cancer has become one of the three most common tumors in the world. According to the data released by the World Health Organization (WHO), colorectal cancer is the third most prone tumor in men, second only to lung cancer and gastric cancer; it is the second most prone tumor in women, second only to breast cancer. At present, there are more than 1 million new cases of colorectal cancer every year in the world. In my country, the mortality rate of colorectal cancer is second only to lung cancer, gastric cancer, liver cancer, and esophageal cancer, ranking fifth, seriously endangering the health of residents and causing a heavy burden on society and family economy. Among them, death caused by can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N15/85A61K38/48A61P35/00
CPCA61K38/48C12N9/6421C12N15/85C12N2800/107
Inventor 王海军宋娜赵铁锁冯志伟钟加滕陈秋月齐金博邹亚文
Owner XINXIANG MEDICAL UNIV
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