Application of TRAF6 inhibitor in preparation of melanoma drug

A melanoma and inhibitor technology, applied in the field of biomedicine, to achieve the effect of promoting immune function, improving effect and reducing immune tolerance

Pending Publication Date: 2021-09-21
SUN YAT SEN UNIV SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are also reports disclosing the research progress on the relationship between RAF6 and tumors (Li Miao, Zhou Lijun. Research progress on the relationship between TRAF6 and tumors [J]. Biotechnology Bulletin, 2017,33(6):24-31.), however, the current The role of TRAF6 in tumor cell PD-L1 expression regulation and immunosuppression has not been reported yet

Method used

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  • Application of TRAF6 inhibitor in preparation of melanoma drug
  • Application of TRAF6 inhibitor in preparation of melanoma drug
  • Application of TRAF6 inhibitor in preparation of melanoma drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 GEO database analysis shows that TRAF6 is a molecule that potentially regulates PD-L1

[0028]1. Search for PD-L1 in the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), and the number GSE129968 is the screening data of high-throughput genetic libraries related to PD-L1. This analysis is to combine Human genome-wide CRISPR / Cas9 knockout (Genome-Scale CRISPR Knock-Out, GeCKO) lentiviral library was introduced into lung cancer cells, screened with 1 μg of puromycin for two weeks, then incubated with APC-PD-L1 flow antibody for flow cytometry Sorting, enrichment of PD-L1 high and PD-L1 low Cell. Then extract unsorted cell control group, PD-L1 low Cell experiment group, PD-L1 high The genomic DNA of the cell experimental group, two rounds of PCR amplification of the genomic DNA containing sgRNA, and finally, illumina sequencing was performed to screen out the genes corresponding to the significantly enriched sgRNA in the experimental group, so as to obtain th...

Embodiment 2

[0031] Example 2 TRAF6 can positively regulate PD-L1

[0032] 1. Construct a melanoma cell line that knocks down or overexpresses TRAF6 by means of lentiviral packaging infection. First, 2×10 6 HEK 293T cells were plated in a 6cm cell culture dish, after adding 5mL DMEM (containing 10% FBS and 1% P / S) complete medium, placed in 5% CO 2 , cultured in a cell culture incubator at 37°C. After 18-24 hours, the HEK 293T cells reached a confluence of 60%-70%, and the cell culture medium was replaced with 3 mL of 5% FBS DMEM complete medium. Add 475 μL of HBS solution into a 1.5 mL centrifuge tube, then add 5 μg of packaging plasmid, 5 μg of target plasmid (human TRAF6 empty control plasmid, TRAF6 gene cDNA lentiviral expression plasmid, human TRAF6 knockdown shRNA empty control plasmid, human TRAF6 knockdown shRNA lentiviral expression plasmids #1 and #2) were added sequentially, and finally 25μL 2.5mol / L CaCl was added dropwise 2 solution at room temperature in the dark for 20 mi...

Embodiment 3

[0037] Example 3 TRAF6 inhibitors can enhance CD8 by down-regulating PD-L1 expression + T cell activity (in vitro)

[0038] 1. The above results have proved that TRAF6 can positively regulate PD-L1, so inhibiting TRAF6 is expected to reduce endogenous PD-L1 and thus weaken tumor immune escape. Since TRAF6 also plays an important role in innate immunity and acquired immunity, it is necessary to select a suitable TRAF6 inhibitor to exert tumor killing effect without significantly affecting the normal immunity of the body. According to literature reports, parthenolide or quinine can effectively inhibit the activity of TRAF6 to inhibit the proliferation of cancer cells, and the proteasome inhibitor MG132 can slow down the proliferation of tumor cells by inhibiting the expression of TRAF6. In addition, the reversible proteasome inhibitor bortezomib can inhibit the activity of proteasome 26S subunit, reduce the degradation of NF-κB inhibitor IκB, promote the combination of IκB and ...

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Abstract

The invention discloses an application of a TRAF6 inhibitor in preparation of a melanoma drug. Researches show that mRNA and protein level of PD-L1 in a melanoma cell line with knockdown or overexpression of TRAF6 are subjected to consistent change, that is, TRAF6 can positively regulate PD-L1. By inhibiting the expression of TRAF6, the expression of PD-L1 on the surfaces of tumor cells can be inhibited, the immune escape phenomenon of combination of PD-L1 and PD-1 on the surfaces of T cells can be reduced, the normal immune function of the T cells can be promoted, the body immunity can be enhanced, the tumor progress can be reduced, and the immune tolerance can be reduced. Furthermore, when the TRAF6 inhibitor is applied as a PD-L1 regulator to a mouse melanoma lung metastasis model, the activity of lung infiltration T cells can be remarkably enhanced, and melanoma can be effectively killed.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and more specifically, relates to the application of TRAF6 inhibitors in the preparation of melanoma drugs. Background technique [0002] Melanoma, usually referring to malignant melanoma, is a highly malignant tumor derived from melanocytes, referred to as melanoma for short, mostly occurs in the skin, and can also be found in mucous membranes and internal organs, accounting for about 3% of all tumors. Cutaneous melanoma has the characteristics of strong invasion, easy transfer, and easy recurrence, ranking first in the cause of death of skin tumors. In my country, the most common clinical types of skin melanoma are acral type and mucosal type, and the incidence rate is increasing rapidly at an annual rate of 3-5%. The surgical effect of early melanoma is better, but 60-70% of patients with advanced skin melanoma, the 5-year survival rate is only 5%, and the degree of malignancy is extremel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K45/00A61K38/05A61P35/00
CPCA61K38/1793A61K45/00A61K38/05A61P35/00
Inventor 程芳杨欣蕊陈红波刘晓燕王凌璐奚丽芳韩雨航
Owner SUN YAT SEN UNIV SHENZHEN
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