Application of TRAF6 inhibitors in the preparation of drugs for the treatment of human malignant melanoma

Abstract

The present invention discloses the use of a TRAF6 inhibitor in the preparation of a drug for treating human malignant melanoma, and specifically relates to the use of a tumor necrosis factor receptor-associated factor 6 (TRAF6) inhibitor combined with retinoic acid in the preparation of a drug for treating human malignant melanoma. The present invention uses RNA interference technology and Western bolt analysis to study the role of TRAF6 in human malignant melanoma and the relationship between TRAF6 and retinoic acid sensitivity. Through the research of the present invention, it can be known that TRAF6 inhibition can increase the sensitivity of human malignant melanoma to retinoic acid, and the development of TRAF6 inhibitors can provide a new strategy for the treatment of human malignant melanoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a tumor necrosis factor receptor-associated factor 6 (TRAF6) inhibitor combined with retinoic acid in the preparation of a drug for treating human malignant melanoma. Background Art

[0002] Human malignant melanoma is a malignant tumor that develops from the malignant transformation of melanocytes in the skin or mucosal epithelium. It ranks third among all skin malignant tumors and has clinical characteristics such as low survival rate, easy recurrence, and high drug resistance. In recent years, a series of treatment strategies such as surgical resection, conventional chemotherapy, and biological therapy have been applied to the treatment of human malignant melanoma, and have achieved certain therapeutic effects. However, this malignant tumor is insensitive to traditional chemotherapy, and drug resistance of malignant melanoma is also the biggest obstacle to the treatment effect, so the survival rate of patients is low. With the in-depth exploration of the molecular mechanism of drug resistance of malignant melanoma, overcoming drug resistance has important research significance for the treatment of malignant melanoma.

[0003] Retinoic acid (RA) is derived from the metabolic intermediate of vitamin A. It can regulate multiple developmental processes and is an anticancer drug with great application prospects. Studies have shown that RA binds to the retinoic acid receptor (RXRs) in the nucleus, triggering the extranuclear non-transcriptional signaling pathway of the kinase cascade reaction, thereby activating the transcription and expression of target genes, and ultimately exerting the activity of inhibiting proliferation and inducing differentiation. In recent years, RA has shown good anti-proliferative and antioxidant activities in the treatment of human malignant melanoma, further inducing its apoptosis. Despite this, RA is insoluble in water, unstable in the presence of light, air, and oxidants, and has side effects such as large local irritation when applied externally, and poor inducing apoptosis, which limits its clinical application. At the same time, many melanoma cells are resistant to the anti-proliferative effects of all trans-retinoic acids. Recently, many molecular mechanism studies have explored the genetic and molecular changes associated with the occurrence and development of melanoma, such as RAR-β2, PIN1 and other genes are closely related to retinoic acid resistance. However, these potential targets have not yet led to the development of effective / curative treatments. Although retinoic acid as a single agent is not a clear treatment for metastatic melanoma, it has good promise in combination with inhibitors of these potential targets.

[0004] Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor protein that mediates extensive protein-protein interactions through the TRAF domain and the RING finger domain with E3 ubiquitin ligase activity. As a regulatory protein, TRAF6 is involved in the activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), interferon regulatory factor pathway, interleukin-1 receptor (IL-1R)-mediated activation of NFκB signaling pathway, etc. Studies have shown that TRAF6 is overexpressed in primary and metastatic melanoma tumors and melanoma cell lines. At the same time, TRAF6 is involved in regulating the invasion and metastasis of melanoma, indicating that TRAF6 may be a potential target for the treatment or chemoprevention of melanoma. Therefore, the potential therapeutic effect of inhibiting TRAF6 or combining it with other methods is worth studying.

[0005] Currently, there are no reports on the role of TRAF6 in retinoic acid treatment of human malignant melanoma. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a tumor necrosis factor receptor-associated factor 6 inhibitor combined with retinoic acid in the preparation of a drug for treating human malignant melanoma.

[0007] The present invention uses RNA interference technology and Western bolt analysis to study the role of TRAF6 in human malignant melanoma and the relationship between TRAF6 and retinoic acid sensitivity. First, knocking down TRAF6 significantly reduced the IC of retinoic acid on Mewo cells. 50 , indicating that inhibition of TRAF6 increased the sensitivity of human malignant melanoma cells to retinoic acid; then the effect of TRAF6 inhibition on cell clone formation of human malignant melanoma cells Mewo under retinoic acid treatment was further investigated; finally, the effect of TRAF6 gene knockdown combined with retinoic acid on pro-apoptotic factors and anti-apoptotic factors was studied.

[0008] The above studies show that TRAF6 inhibition can increase the sensitivity of human malignant melanoma to retinoic acid, and the development of TRAF6 inhibitors can provide a new strategy for the treatment of human malignant melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The results show that knockdown of TRAF6 increases the sensitivity of human malignant melanoma cell lines to retinoic acid treatment. Specifically, MeWo cells transfected with negative control (siNEG) or siRNA targeting TRAF6 (siTRAF6) were treated with different concentrations of retinoic acid.

[0010] Figure 2The results of knocking down TRAF6 inhibited the proliferation of human malignant melanoma cells. A shows the cell clone formation of Mewo cells after transfection with siNEG or siTRAF6 and treatment with 8 μM retinoic acid; B shows the results of quantitative analysis of cell clone formation in different treatment groups by GraphPad Prism 8.2.0. All data are from three independent repeated experiments and are expressed as the mean ± standard deviation of three measurements, ** p<0.01, *** p<0.001.

[0011] Figure 3 The results of the changes in protein levels of apoptosis-related factors in MeWo cells after TRAF6 knockdown before and after retinoic acid (RA) treatment. DETAILED DESCRIPTION

[0012] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art. Example

[0013] 1. Cell Culture

[0014] The human malignant melanoma cell line MeWo was purchased from the American Type Culture Collection (ATCC). The cells were cultured in a 37°C constant temperature cell incubator with 5% CO2 using DMEM high glucose medium (containing 10% FBS, 1% penicillin-streptomycin). The experimental cell culture time did not exceed three months, and mycoplasma detection was performed every three months. Cells were cryopreserved in FBS containing 10% DMSO.

[0015] 2. Cell Transfection

[0016] MeWo cells were cultured in 60 mm dishes from the day before, and the confluence of cells in each dish was expected to be 70%-80% on the second day after plating. 5 μM siGENONESMARTpool TRAF4 siRNA (M-004712-00-0005, Horizon Discovery) or siGENOME non-targeting Control siRNA (D-001206-13-5, Horizon Discovery) was added to 500 μL Opti-MEM reduced serum medium, gently mixed, and allowed to stand for 5 min. 10 μL transfection reagent LipofectamineRNAiMax was added to 500 μL Opti-MEM reduced serum medium, gently mixed, and allowed to stand for 5 min. The above solutions were gently mixed and incubated at room temperature for 15 min to obtain the transfection solution. This mixed solution was added dropwise to the 60 mm cell culture dish in which MeWo cells were cultured and gently mixed. After 6 h of transfection, fresh complete medium was replaced and the cells were cultured in a 37°C constant temperature cell incubator with 5% CO2 for 24 h.

[0017] The siGENMONE SMARTpool TRAF6 siRNA sequence is as follows:

[0018] (1)5'-GCAAGUGAUAAUCAAGUUA-3'

[0019] (2)5'-GGUGAAAUGUCCAAAUGAA-3'

[0020] (3)5'-CAGAGCAAGUGAUAAUCAA-3'

[0021] (4) 5'- CAAGUGAUUUGCACAAUA-3'.

[0022] 3. Cell Proliferation Assay

[0023] After 24 h of cell culture, siRNA-transfected MeWo cells were collected and counted, and a portion was used for cell viability assay and a portion for Western blot analysis. 2000-4000 cells / well were seeded in 90 μL medium / well in a 96-well plate. After overnight culture, cells were treated with 10 μL retinoic acid at final concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM for 5 days. 10 μL CYQUANT reagent mixture was added directly to each well, and the plate was incubated at 37°C for 1 h. Cell viability was measured under excitation light 480 nm and emission light 520 nm. Cytotoxicity assays were performed three times at each drug concentration.

[0024] 4. Western blot analysis

[0025] After 48 hours of transfection, cells were collected and resuspended in 1×NETN lysis buffer (100 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5% (V / V) Nonidet P-40 (NP-40)) with 1* protease inhibitor and phosphatase inhibitor, and incubated on ice for 30 minutes. The above lysis mixture was sonicated and centrifuged at 12000 g for 10 min at 4°C. The supernatant was the total protein. The protein concentration was determined using Quick Start™Bradford 1x Dye reagent. After adding loading buffer to the protein sample, it was boiled at 95°C for 8 min and analyzed by western bolt. 20 μg of lysate was loaded onto a 4%-20% precast protein gel and subjected to 200V constant voltage SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis ended when the loading buffer moved to the bottom layer of the protein gel. Prepare transfer solution. Soak the polyvinylidene fluoride (PVDF) membrane in methanol for 1-2 min before use. Soak the transfer filter paper in transfer solution in advance. Use semi-dry transfer (25V, 10 min) for transfer. After transfer, place the membrane in 5% skim milk powder (in TBST) for 1 h. Use primary antibody diluted in 1% BSA and incubate the primary antibody overnight at 4 degrees. Rinse the membrane with 1×TBST for 5 min and incubate with HRP-conjugated secondary antibody diluted in 1×TBST at room temperature for 1 h. Wash the membrane 3 times with 1×TBST, 5 min each time. Use SuperSignal West Pico Chemiluminescent Substrate to identify protein bands and expose them under ChemiDoc™ Touch Imaging System.

[0026] 5. Cell clone formation analysis

[0027] According to the above cell transfection method, TRAF6 was knocked down in MeWo cells using siRNA interference technology, and siNegative (siNEG) was used as a control for the same treatment. After 24 hours of interference, TRAF4 knockdown MeWo cells and siNegative (siNEG) control MeWo cells were added to 6-well plates (500-1000 cells / well), treated with 8 μM retinoic acid for 14 days, and then gently rinsed with 1× PBS and fixed with ethanol at room temperature for 30 minutes. Stained with 0.5% w / v crystal violet overnight, the cell colonies were gently washed with 1× PBS. Colony images were taken using the ChemiDoc™ Touch Imaging System.

[0028] from Figure 1 The results of the cell proliferation assay show that, first, TRAF6 was transiently knocked out in MeWo cells using siRNA interference (RNAi) technology, and Western bolt verified that its protein content was reduced, indicating that TRAF6 was successfully knocked down. After treating the cells with retinoic acid at a final concentration of 0.125 μM-32 μM for 5 days, the CyQUANT direct cell proliferation assay was performed to determine the relative cell number. Compared with control cells, siRNA-mediated knockout of TRAF4 significantly reduced the IC50 of retinoic acid for Mewo cells (from 14.012 μM to 7.998 μM), indicating that inhibition of TRAF6 significantly increased the sensitivity of human malignant melanoma cells to retinoic acid.

[0029] At the same time, the present invention further investigates the effect of TRAF6 inhibition on cell clone formation of human malignant melanoma cells MeWo under retinoic acid treatment. Figure 2 A shows that compared with the control cells not treated with retinoic acid, the number of clones generated by siTRAF6 cells is less, indicating that knocking down TRAF6 alone has a strong inhibitory effect on the clone formation of MeWo cells, which further supports the tumorigenicity of TRAF6 in human melanoma cells. In addition, compared with the siNegative (siNEG) control group, retinoic acid + siTRAF6 treatment further significantly reduced clone formation, such as Figure 2 As shown in B. The inhibitory effect of retinoic acid combined with TRAF6 inhibition on the colony formation of MeWo cells was 10.7%, while the inhibitory effect of retinoic acid alone on the colony formation of MeWo cells was 53.9%.

[0030] like Figure 3As shown in the figure, in MeWo cells transfected with siTRAF6, combined with retinoic acid treatment can further downregulate the protein level of TRAF6 compared with knockdown of TRAF6 alone. The synergistic effect not only reduced the protein levels of Bcl-2 (anti-apoptotic factor) and Survivin (anti-apoptotic factor), but also enhanced the protein level of Caspase 9 (pro-apoptotic protein). It further illustrates that the combination of TRAF6 gene knockdown and retinoic acid can increase the expression of several pro-apoptotic factors and reduce the expression of other anti-apoptotic factors, so TRAF6 gene knockdown can enhance retinoic acid-induced apoptosis of human malignant melanoma cells. This may be that retinoic acid affects cell proliferation and growth by regulating the apoptotic signaling pathway, and TRAF6 can enhance retinoic acid-induced apoptosis of human malignant melanoma cells.

[0031] In summary, TRAF6 inhibition can increase the sensitivity of human malignant melanoma to retinoic acid. Therefore, the development of TRAF6 inhibitors is likely to provide a new strategy for the treatment of human malignant melanoma.

Claims

1. Use of a tumor necrosis factor receptor-associated factor 6 inhibitor combined with retinoic acid in the preparation of a tumor treatment drug, characterized in that: The tumor is human malignant melanoma; the tumor necrosis factor receptor-associated factor 6 inhibitor is siRNA, and the siRNA sequence is as follows: (1)5'-GCAAGUGAUAAUCAAGUUA-3' (2)5'-GGUGAAAUGUCCAAAUGAA-3' (3)5'-CAGAGCAAGUGAUAAUCAA-3' and (4) 5′- CAAGUGAUUUGCACAAAUA -3′; When the drug is used, the tumor necrosis factor receptor-associated factor 6 inhibitor is first administered, and then retinoic acid is administered.

Images