An NR4A1 transcriptional activity inhibitor and its application

Abstract

The present invention discloses an NR4A1 transcriptional activity inhibitor and its application, belonging to the technical field of transcriptional activity inhibitors. The NR4A1 transcriptional activity inhibitor at least includes flavonoid compounds, and the flavonoid compounds include 4',5,7-trimethoxyflavone. The disclosed NR4A1 transcriptional activity inhibitor of the present invention can be used to prepare an anti-thyroid cancer drug or kit targeting NR4A1 by inhibiting the binding of NR4A1 to the NIS promoter to regulate the expression of NR4A1 gene and NR4A1 protein.

Description

Technical Field

[0001] The present invention relates to the technical field of transcriptional activity inhibitors, and particularly relates to an NR4A1 transcriptional activity inhibitor and its application. Background Art

[0002] Thyroid cancer is one of the most common malignant tumors in the endocrine system. Differentiated thyroid cancer (DTC) is the most common subtype of thyroid cancer. Most DTC patients adopt conventional treatment methods, including surgery and selective radioactive iodine therapy, and have a good prognosis. However, there may be a risk of 20% local recurrence and 10% distant metastasis in DTC patients, progressing to radioiodine-refractory thyroid cancer (RAIR-DTC), with a decreased sensitivity to radioactive iodine and a poor prognosis for postoperative radioactive iodine therapy. The main causes of RAIR-DTC are epigenetic alterations and signal pathway dysregulation, both of which can cause abnormal expression and localization of thyroid-specific genes, especially NIS, resulting in the failure of radioactive iodine therapy.

[0003] The nuclear receptor family member NR4A1 gene is significantly associated with the occurrence and development of papillary thyroid cancer (PTC). Studies have shown that the high expression of NR4A1 is closely related to the poor prognosis of PTC patients. NR4A1 binds directly to the promoter region of lymphoid enhancer-binding factor 1 (LEF1), affects histone acetylation and DNA demethylation, and thus upregulates the expression of LEF1 at the transcriptional level, further promoting the expression of downstream growth-related genes in PTC. Currently, there is little research on NR4A1 in the field of radioiodine-refractory thyroid cancer. Previous studies have found that NR4A1 binds to the promoter of sodium iodide symporter (NIS), affecting the transcriptional level of NIS. Currently, the number of reported NR4A1 inhibitors is not large, and only one proteolysis-targeting chimera (PROTAC) named NR-V04 is in the clinical trial stage. It is the first developed drug targeting NR4A1. NR-V04 can effectively degrade NR4A1 within a few hours of in vitro treatment. It can effectively inhibit tumors and can persist in vivo for at least 4 days, showing a persistent NR4A1 degradation effect. In addition, there are some other NR4A1 inhibitors, such as CDIM8, piperlongumine, and TMPA. These inhibitors can reduce cell proliferation, but their IC50 values are relatively large. Therefore, the development and research of NR4A1 transcriptional activity inhibitors still have important clinical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide an NR4A1 transcriptional activity inhibitor and its application, which can inhibit the expression of the NR4A1 gene, thereby improving the iodine uptake ability of cells.

[0005] The technical solution adopted by the present invention to achieve the above purpose is as follows:

[0006] An inhibitor of NR4A1 transcriptional activity, which at least includes flavonoids; the flavonoids at least include 4',5,7-trimethoxyflavone, and 4',5,7-trimethoxyflavone has a flavone group and a methoxy group.

[0007] The inhibitor of NR4A1 transcriptional activity of the present invention includes 4',5,7-trimethoxyflavone. 4',5,7-trimethoxyflavone inhibits the binding of NR4A1 to the NIS promoter to reduce the expression of NR4A1 gene and NR4A1 protein, and improves the iodine uptake ability of various thyroid cancer cells, and can be used in the preparation of drugs or kits for anti-thyroid cancer targeting NR4A1.

[0008] Preferably, the flavonoids include 4',5,7-trimethoxyflavone and dimethoxyphenylacetamido flavone derivatives. The common use of 4',5,7-trimethoxyflavone and dimethoxyphenylacetamido flavone derivatives can effectively inhibit the binding of NR4A1 to the NIS promoter, reduce the relative expression level of NR4A1 gene, and improve the cell iodine uptake ability.

[0009] More preferably, the mass ratio of 4',5,7-trimethoxyflavone to dimethoxyphenylacetamido flavone derivatives is 1:0.1-0.2.

[0010] More preferably, the dimethoxyphenylacetamido flavone derivative is obtained by first reacting 3,5-dimethoxyphenylacetic acid with thionyl chloride and then reacting with 6-aminoflavone.

[0011] Even more preferably, the dosage ratio of 3,5-dimethoxyphenylacetic acid to thionyl chloride is 1 g:10-20 mL.

[0012] Even more preferably, the mass ratio of 6-aminoflavone to 3,5-dimethoxyphenylacetic acid is 1:1-2.

[0013] More preferably, the preparation of the dimethoxyphenylacetamido flavone derivative is specifically as follows.

[0014] Weigh 3,5-dimethoxybenzeneacetic acid, add it to N,N-dimethylformamide and stir. Dropwise add thionyl chloride at 50 - 70 °C, and reflux for 2 - 5 h. After the reaction is completed, cool down to 25 - 50 °C, add 6-amino flavone, and react at 1 - 5 °C for 1 - 3 h. After the reaction is completed, let it stand for 20 - 40 min, wash the upper organic phase with water, let it stand for 20 - 40 min, retain the aqueous layer and adjust the pH to 3 - 4 by dropping hydrochloric acid, react at 5 - 15 °C for 1 - 2 h, filter by suction, add methanol, reflux at 60 - 70 °C for 1 - 3 h, stir at 0 - 5 °C for 1 - 3 h, filter by suction, wash with water 2 - 5 times, and dry to obtain the dimethoxybenzeneacetamido flavone derivative.

[0015] More preferably, the dosage ratio of 3,5-dimethoxybenzeneacetic acid to N,N-dimethylformamide is 1 g : 10 - 20 mL.

[0016] More preferably, the volume ratio of thionyl chloride to N,N-dimethylformamide is 1 : 1 - 2.

[0017] More preferably, the mass ratio of 6-amino flavone to 3,5-dimethoxybenzeneacetic acid is 1 : 1 - 2.

[0018] More preferably, the volume ratio of N,N-dimethylformamide to methanol is 1 : 1 - 2.

[0019] Preferably, an NR4A1 transcriptional activity inhibitor includes 4’,5,7-trimethoxy flavone, and at least one of the dimethoxybenzeneacetamido flavone derivative and the pyrimidinyl derivative. In the NR4A1 transcriptional activity inhibitor, further using the pyrimidinyl derivative can further reduce the relative expression level of the NR4A1 gene and improve the iodine uptake ability of IHH4 cells.

[0020] More preferably, the mass ratio of 4’,5,7-trimethoxy flavone to the dimethoxybenzeneacetamido flavone derivative is 1 : 0.1 - 0.2.

[0021] More preferably, the mass ratio of 4’,5,7-trimethoxy flavone to the pyrimidinyl derivative is 1 : 0.05 - 0.1.

[0022] More preferably, the preparation of the pyrimidinyl derivative includes

[0023] Take 2-(methylsulfinyl)pyrimidine, add tert-butanol, slowly add N,N-diisopropylethylamine and 3-amino-5-methoxyquinolin-2(1H)-one at room temperature, and react at 70 - 90 °C for 6 - 10 h to obtain the pyrimidinyl derivative.

[0024] Further preferably, the dosage ratio of 2-(methylsulfinyl)pyrimidine to tert-butanol is 1 mmol: 10 - 20 mL.

[0025] Further preferably, the molar ratio of 2-(methylsulfinyl)pyrimidine to N,N-diisopropylethylamine is 1: 1 - 5.

[0026] Further preferably, the molar ratio of 2-(methylsulfinyl)pyrimidine to 3-amino-5-methoxyquinolin-2(1H)-one is 1: 1 - 2.

[0027] The present invention also discloses the application of an NR4A1 transcriptional activity inhibitor in the preparation of a drug or kit for inhibiting the expression of the NR4A1 gene.

[0028] The present invention also discloses the application of an NR4A1 transcriptional activity inhibitor in the preparation of a drug or kit for inhibiting the expression of the NR4A1 protein.

[0029] The present invention also discloses the application of an NR4A1 transcriptional activity inhibitor in the preparation of a drug or kit for inhibiting the binding of NR4A1 to the NIS promoter.

[0030] The present invention also discloses the application of an NR4A1 transcriptional activity inhibitor in the preparation of a drug or kit for anti-thyroid cancer targeting NR4A1.

[0031] Since the present invention uses 4',5,7-trimethoxyflavone, dimethoxyphenylacetamido flavone derivatives and pyrimidine derivatives to form an NR4A1 transcriptional activity inhibitor, it has the following beneficial effects: The NR4A1 transcriptional activity inhibitor of the present invention significantly reduces the relative expression level of the NR4A1 gene by inhibiting the binding of NR4A1 to the NIS promoter, and improves the iodine uptake ability of thyroid cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the infrared spectrum of dimethoxyphenylacetamido flavone derivatives.

[0033] Figure 2 It is the result of dual-luciferase reporter assay.

[0034] Figure 3 It is the result of Western blot experiment.

[0035] Figure 4 It is the relative expression level of the NR4A1 gene.

[0036] Figure 5 It is the relative expression level of the FOXM1 gene in IHH4 cells.

[0037] Figure 6 It is the relative expression level of the CDK4 gene in IHH4 cells.

[0038] Figure 7 It is the relative expression level of the CCDN1 gene in IHH4 cells.

[0039] Figure 8 It is the relative expression level of the FOXM1 gene in SW579 cells.

[0040] Figure 9 It is the relative expression level of the CDK4 gene in SW579 cells.

[0041] Figure 10 It is the relative expression level of the CCDN1 gene in SW579 cells.

[0042] Figure 11 It is the iodine uptake ability of IHH4 cells.

[0043] Figure 12 It is the iodine uptake ability of SW579 cells.

[0044] Figure 13 It is the iodine uptake ability of K1 cells. Detailed implementation manners

[0045] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0046] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0047] Example 1:

[0048] An NR4A1 transcriptional activity inhibitor, comprising 4',5,7-trimethoxyflavone (TMF).

[0049] Example 2:

[0050] Compared with Example 1, the difference in this example lies in an NR4A1 transcriptional activity inhibitor.

[0051] An NR4A1 transcriptional activity inhibitor, comprising TMF and a dimethoxyphenylacetamido flavone derivative. The mass ratio of TMF to the dimethoxyphenylacetamido flavone derivative is 1:0.2.

[0052] The preparation of the dimethoxyphenylacetamido flavone derivative includes

[0053] Weigh 3,5-dimethoxyphenylacetic acid, add it to N,N-dimethylformamide and stir. Dropwise add thionyl chloride at 60 °C, and reflux for 3 h. After the reaction is completed, cool down to 45 °C, add 6-amino flavone, and react at 4 °C for 2 h. After the reaction is completed, let it stand for 30 min. Wash the upper organic phase with water, let it stand for 30 min, retain the aqueous layer and adjust the pH to 3 by dropping hydrochloric acid. React at 10 °C for 1 h, filter by suction, add methanol, reflux at 65 °C for 2 h, stir at 4 °C for 2 h, filter by suction, wash with water 3 times, and dry to obtain the dimethoxyphenylacetamido flavone derivative. The dosage ratio of 3,5-dimethoxyphenylacetic acid to N,N-dimethylformamide is 1 g:10 mL; the dosage ratio of 3,5-dimethoxyphenylacetic acid to thionyl chloride is 1 g:10 mL; the mass ratio of 6-amino flavone to 3,5-dimethoxyphenylacetic acid is 1:1.5; the volume ratio of N,N-dimethylformamide to methanol is 1:1.

[0054] Example 3:

[0055] Compared with Example 2, the difference in this example lies in an NR4A1 transcriptional activity inhibitor.

[0056] An NR4A1 transcriptional activity inhibitor, except that the mass ratio of TMF to the dimethoxyphenylacetamido flavone derivative is changed to 1:0.1, and other conditions are the same as in Example 2.

[0057] Example 4:

[0058] Compared with Example 2, the difference in this example lies in an NR4A1 transcriptional activity inhibitor.

[0059] An NR4A1 transcriptional activity inhibitor, including TMF, dimethoxyphenylacetamido flavone derivative and pyrimidinyl derivative. The mass ratio of TMF to the dimethoxyphenylacetamido flavone derivative is 1:0.2; the mass ratio of TMF to the pyrimidine derivative is 1:0.1.

[0060] The preparation of the dimethoxyphenylacetamido flavone derivative is the same as in Example 2.

[0061] The preparation of the pyrimidinyl derivative includes

[0062] Take 2-(methylsulfinyl)pyrimidine, add tert-butanol, slowly add N,N-diisopropylethylamine and 3-amino-5-methoxyquinolin-2(1H)-one at room temperature, and react at 80 °C for 8 h to obtain the pyrimidinyl derivative. The dosage ratio of 2-(methylsulfinyl)pyrimidine to tert-butanol is 1 mmol:10 mL; the molar ratio of 2-(methylsulfinyl)pyrimidine to N,N-diisopropylethylamine is 1:2; the molar ratio of 2-(methylsulfinyl)pyrimidine to 3-amino-5-methoxyquinolin-2(1H)-one is 1:1.

[0063] Example 5:

[0064] The difference between this example and Example 4 lies in an NR4A1 transcriptional activity inhibitor.

[0065] An NR4A1 transcriptional activity inhibitor, except that the mass ratio of TMF to pyrimidine derivative is changed to 1:0.05, and other conditions are the same as in Example 4.

[0066] Comparative Example 1:

[0067] The difference between this comparative example and Example 1 lies in an NR4A1 transcriptional activity inhibitor.

[0068] An NR4A1 transcriptional activity inhibitor includes TMF and pyrimidine-based derivatives. The mass ratio of TMF to pyrimidine derivative is 1:0.1.

[0069] The preparation of pyrimidine-based derivatives includes

[0070] Take 2-(methylsulfinyl)pyrimidine, add tert-butanol, slowly add N,N-diisopropylethylamine and 3-amino-5-methoxyquinolin-2(1H)-one at room temperature, and react at 80 °C for 8 h to obtain pyrimidine-based derivatives. The dosage ratio of 2-(methylsulfinyl)pyrimidine to tert-butanol is 1 mmol:10 mL; the molar ratio of 2-(methylsulfinyl)pyrimidine to N,N-diisopropylethylamine is 1:2; the molar ratio of 2-(methylsulfinyl)pyrimidine to 3-amino-5-methoxyquinolin-2(1H)-one is 1:1.

[0071] Experimental Example:

[0072] 1. Material Characterization

[0073] The infrared spectrum of the dimethoxybenzeneacetamido flavone derivative prepared by the present invention was measured using a Fourier transform infrared spectrometer, and the KBr tablet method was adopted. The measurement was carried out in the wavenumber range of 400 - 4000 cm -1 with a resolution of 0.06 cm -1 , and scanned 32 times.

[0074] Figure 1 is the infrared spectrum diagram of the dimethoxybenzeneacetamido flavone derivative. An absorption peak of N-H appears near 3400 cm -1 , and absorption peaks of saturated alkane -CH -1 and -CH -1 appear near 2920 cm 3 and 2850 cm 2 , an absorption peak of C=O appears near 1750 cm -1 , and an absorption peak appears near 1620 cm -1An absorption peak of C=C appears nearby, 1170 cm -1 An absorption peak of C-O-C appears nearby.

[0075] 2. Cytotoxicity detection

[0076] Human embryonic kidney cells (293T cells) were seeded in 24-well plates at a density of 5×10 4 and divided into 4 groups: blank group, control group, experimental group 1, experimental group 2, and experimental group 3. Using dimethyl sulfoxide (DMSO) as a solvent, TMF, dimethoxybenzeneacetamido flavone derivatives, and pyrimidinyl derivatives were respectively prepared into solutions with a final concentration of 40 μmol / L. The blank group was not treated, the control group was treated with dimethyl sulfoxide (DMSO) for 24 h, experimental group 1 was treated with TMF for 24 h, experimental group 2 was treated with dimethoxybenzeneacetamido flavone derivatives for 24 h, and experimental group 3 was treated with pyrimidinyl derivatives for 24 h. After the treatment, the cell viability of experimental group 1, experimental group 2, and experimental group 3 was evaluated by the thiazolyl blue tetrazolium bromide colorimetric method (MTT method), and the absorbance value of the solution at a wavelength of 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0077] The cell viability of experimental group 1 was 89.4%, the cell viability of experimental group 2 was 91.5%, and the cell viability of experimental group 3 was 90.6%. This indicates that TMF, dimethoxybenzeneacetamido flavone derivatives, and pyrimidinyl derivatives used in the NR4A1 transcriptional activity inhibitor of the present invention all have good cell compatibility.

[0078] 3. Detection of the binding ability of NR4A1 to the NIS promoter

[0079] Construct plasmids, which include the firefly luciferase reporter gene (NISp) plasmid with the full-length sequence of the NIS promoter, the NR4A1 overexpression plasmid, the NR4A1 empty plasmid, and the Renilla luciferase internal reference gene plasmid. Among them, the NR4A1 overexpression plasmid is pCMV-NR4A1(human)-SV40-Neo, purchased from Wuhan Miaoling Biotechnology Co., Ltd.; the NR4A1 empty plasmid is pCMV-SV40-Neo, purchased from Wuhan Miaoling Biotechnology Co., Ltd.; the Renilla luciferase internal reference gene plasmid is phRL-TK, purchased from Promega (Beijing) Biotechnology Co., Ltd. The construction method of the NISp plasmid is as follows: Extract the genomic DNA of IHH4 cells using a cell genomic DNA extraction kit, which is purchased from Nanjing Novozymes Biotech Co., Ltd.; Design primers according to the NIS promoter sequence and the pGL 4.23 plasmid sequence. The nucleotide sequence of the forward primer is 5’-GGGGTACCCCTTATAAAACTTCCACGCTCAAGCG-3’, and its nucleotide sequence is shown in SEQ ID NO.1. The nucleotide sequence of the reverse primer is 5’-CCCAAGCTTGGGAAGCTTATGCACTGTGATCCCCTGAT-3’, and its nucleotide sequence is shown in SEQ ID NO.2; Obtain the target gene of the NIS promoter from the genomic DNA by PCR amplification; Incubate overnight with restriction enzymes to cut the target gene and the pGL 4.23 plasmid, and use DNA ligase to link the cut target gene with the pGL 4.23 plasmid, and the pGL 4.23 plasmid is purchased from Promega (Beijing) Biotechnology Co., Ltd.; Separate the ligation products by molecular weight through DNA gel electrophoresis, collect the gel around 5400bp, and use a gel extraction / DNA purification kit to purify the ligation products to obtain the purified ligation products. The gel extraction / DNA purification kit is purchased from Nanjing Novozymes Biotech Co., Ltd.; Mix the purified ligation products with DH5α competent cells, heat shock for 90s, incubate at 4°C for 5 min, add 1 mL of LB medium and incubate in a 37°C constant temperature shaker for 1h, centrifuge at 4°C and 3000 rpm for 10 min, discard 900 μL of the supernatant, and spread the remaining cell suspension on an agar plate containing ampicillin and incubate overnight at 37°C. After picking monoclonal colonies for expansion culture, send the bacterial solution to Suzhou Junji Biotechnology Co., Ltd. for sequencing; Extract the plasmid using a plasmid miniprep kit, which is purchased from Abclonal Biotechnology Co., Ltd., to obtain the NISp plasmid.

[0080] Seed human embryonic kidney cells (293T cells) at 5×10 4Inoculate at a density into 24-well plates, divided into 4 groups, denoted as group A, group B, group C and group D. Group A and group B were co-transfected with 1 μg of NISp plasmid, 1 μg of empty NR4A1 plasmid and 0.1 μg of Renilla luciferase internal reference gene plasmid; Group C and group D were co-transfected with 1 μg of NISp plasmid, 1 μg of overexpressed NR4A1 plasmid and 0.1 μg of Renilla luciferase internal reference gene plasmid. After the cells adhered, the cell culture medium was replaced with 0.5 mL of DMEM medium without serum and double antibiotics. The plasmids of each group were evenly distributed in 25 μL of serum-reduced medium, and then 1 μL of Lipo-3000 transfection reagent was evenly distributed in 25 μL of serum-reduced medium. After standing for 5 min, the serum-reduced medium containing the plasmids and the serum-reduced medium containing the Lipo-3000 transfection reagent were mixed, and then continued to stand for 20 min and then added to the 24-well plates for transfection, corresponding to the transfected cells in groups A, B, C and D. After 6 h of transfection, replace with 0.5 mL of DMEM medium containing serum and double antibiotics and culture for 18 h. Dilute the NR4A1 transcriptional activity inhibitor of Example 1 with DMSO to a final concentration of 40 μmol / L. Add 5 μL of the NR4A1 transcriptional activity inhibitor with a final concentration of 40 μmol / L to B and D for treatment for 24 h, and add the same volume of dimethyl sulfoxide (DMSO) to groups A and C for treatment for 24 h to obtain 293T cells in groups A, B, C and D after transfection.

[0081] Collect the 293T cells in groups A, B, C and D after transfection, and perform dual-luciferase reporter assays separately. The steps of the dual-luciferase reporter assay are as follows: Wash the cells twice with phosphate buffered saline (PBS), add 100 μL of cell lysate, shake and lyse for 15 min, collect the cell lysate and centrifuge at 12,000 rpm for 10 min at 4 °C, take 20 μL of the supernatant, add 50 μL of firefly luciferase substrate, mix well and immediately detect with a microplate reader, denoted as X1; Then add 50 μL of the substrate stop solution of Renilla luciferase, mix well and immediately place it in the microplate reader for detection, denoted as X2. Luciferase reaction intensity = X1 / X2.

[0082] Figure 2For the dual-luciferase reporter results, when comparing group A and group C, the luciferase reaction intensity in group A was lower than that in group C. This was because during transfection, group A co-transfected the NISp plasmid, the empty NR4A1 plasmid, and the Renilla luciferase internal reference gene plasmid, while group C co-transfected the NISp plasmid, the NR4A1 overexpression plasmid, and the Renilla luciferase internal reference gene plasmid. This indicates that NR4A1 can bind to the NIS promoter, and after binding of NR4A1 to the NIS promoter, it can increase the luciferase reaction intensity. When comparing group C and group D, the fluorescence intensity in group C was higher than that in group D. This was because during the treatment with the NR4A1 transcriptional activity inhibitor, group C was treated with TMF, and group D was treated with DMSO. This indicates that TMF can inhibit the binding of NR4A1 to the NIS promoter.

[0083] 4. Western Blot Experiment

[0084] Seed 293T cells in a 6-well plate at a density of 5×10 5 cells / well, with 2 mL of medium in each well. Set up a control group and an experimental group. In the control group, after the cells adhered, add 20 μL of DMSO and treat for 24 h. In the experimental group, dilute the NR4A1 transcriptional activity inhibitor of Example 1 with DMSO to a final concentration of 40 μmol / L, and after the cells adhered, add 20 μL of the NR4A1 transcriptional activity inhibitor with a final concentration of 40 μmol / L and treat for 24 h. After the treatment, collect the cells of the control group and the experimental group and perform Western Blot experiments separately. The steps of the Western Blot experiment are as follows: Wash the cells twice with PBS, extract nuclear proteins using a nuclear protein extraction kit, and measure the protein concentration using a BCA protein quantification kit. The nuclear protein extraction kit is purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the BCA protein quantification kit is purchased from Shanghai Kaimibo Biotechnology Co., Ltd. According to the protein quantification results, add the corresponding volume of radioimmunoprecipitation assay (RIPA) buffer, add the loading buffer (the loading buffer is purchased from Shanghai Yemei Biopharmaceutical Technology Co., Ltd.), and heat and denature at 100 °C for 10 min. After separating the protein samples by SDS-PAGE, transfer them to a 0.45 μm polyvinylidene fluoride (PVDF) membrane, block the membrane with TBST buffer containing 5% skim milk powder for 1 h, add the primary antibody and incubate at 4 °C for 12 h, then add the IgG horseradish peroxidase (HRP)-conjugated secondary antibody and incubate at room temperature for 2 h, wash 3 times with TBST buffer, and develop and image using enhanced chemiluminescence reagent (ECL).

[0085] Figure 3 For the Western Blot experiment results. From Figure 3 it can be seen that the expression level of NR4A1 protein in the control group was higher than that in the experimental group, while the expression levels of TOP1 protein in the control group and the experimental group were basically the same. This indicates that TMF can reduce the protein expression of NR4A1.

[0086] 5. Detection of relative expression level of NR4A1

[0087] Seed 293T cells in a 6-well plate at a density of 5×10 5 cells / well. Set up a control group and an experimental group. In the control group, after the cells adhered, add 20 μL of DMSO and treat for 24 h. In the experimental group, dilute the NR4A1 transcriptional activity inhibitor of Example 1 with DMSO to a final concentration of 40 μmol / L, and after the cells adhered, add 20 μL of the NR4A1 transcriptional activity inhibitor with a final concentration of 40 μmol / L and treat for 24 h. After the treatment, collect the cells of each group and perform qPCR experiments respectively. The steps of the qPCR experiment are as follows: Wash the cells twice with PBS, use the FreeZol Reagent kit for cell lysis and RNA extraction, and the FreeZol Reagent kit is purchased from Nanjing Novoprotein Science and Technology Co., Ltd.; use the reverse transcription kit to synthesize cDNA, and the reverse transcription kit is purchased from Nanjing Novoprotein Science and Technology Co., Ltd. The qPCR reaction system is 5 μL of cDNA template, 10 μL of SYBRgreen fluorescent dye, 1 μL of forward primer with a concentration of 10 μmol / L, 1 μL of reverse primer with a concentration of 10 μmol / L, and 3 μL of sterile and enzyme-free water. Use a qPCR instrument to detect the relative expression level of the NR4A1 gene in the control group and the experimental group.

[0088] Figure 4 Let be the relative expression level of the NR4A1 gene, S1 corresponds to the result of the control group, and S2 is the result of the experimental group. The relative expression level of the NR4A1 gene in the experimental group is significantly lower than that in the control group, indicating that TMF can effectively reduce the relative expression level of the NR4A1 gene.

[0089] Treat 293T cells with the NR4A1 transcriptional activity inhibitors of Examples 1-5 and Comparative Example 1 for 24 h, and after the treatment, measure the relative expression level of the NR4A1 gene in each group. The measurement results are shown in Table 1.

[0090] Table 1 Relative expression level of NR4A1 gene

[0091]

[0092] The relative expression level in Example 1 was significantly lower than that in the control group, indicating that TMF can reduce the relative expression level of the NR4A1 gene. The relative expression levels in Examples 2-3 were lower than that in Example 1 because in the NR4A1 transcriptional activity inhibitor, in Examples 2-3, 3,5-dimethoxyphenylacetic acid and 6-aminoflavone were first used to prepare dimethoxyphenylacetamido flavone derivatives, and then the dimethoxyphenylacetamido flavone derivatives were used in combination with TMF to form the NR4A1 transcriptional activity inhibitor, while in Example 1, only TMF was used as the NR4A1 transcriptional activity inhibitor; the relative expression level in Example 2 was lower than that in Example 3 because in the NR4A1 transcriptional activity inhibitor, the usage amounts of the dimethoxyphenylacetamido flavone derivatives were different. This shows that the dimethoxyphenylacetamido flavone derivatives and TMF used together can reduce the relative expression level of the NR4A1 gene. The relative expression levels in Examples 4-5 were lower than that in Example 2 because in the NR4A1 transcriptional activity inhibitor, pyrimidine derivatives were further used in Examples 4-5; the relative expression levels in Examples 4-5 were lower than that in Comparative Example 1 because in the NR4A1 transcriptional activity inhibitor, in Comparative Example 1, only pyrimidine derivatives were used and TMF and dimethoxyphenylacetamido flavone derivatives were not used; the relative expression level in Example 4 was lower than that in Example 5 because the usage amounts of the pyrimidine derivatives were different. This shows that further using pyrimidine derivatives in the NR4A1 transcriptional activity inhibitor can further reduce the relative expression level of the NR4A1 gene.

[0093] 6. Detection of the relative expression levels of the downstream genes FOXM1, CDK4, and CCDN1 of the NR4A1 gene

[0094] Replace 293T cells with human papillary thyroid carcinoma cells (IHH4) and human squamous thyroid carcinoma cells (SW579), and use a qPCR instrument to detect the relative expression levels of the downstream genes FOXM1, CDK4, and CCDN1 genes of the NR4A1 gene.

[0095] Figure 5 is the relative expression level of the FOXM1 gene in IHH4 cells, S1 is the control group, S2 is the experimental group, and the relative expression level of the experimental group is significantly lower than that of the control group; Figure 6 is the relative expression level of the CDK4 gene in IHH4 cells, S1 is the control group, S2 is the experimental group, and the relative expression level of the experimental group is significantly lower than that of the control group; Figure 7 is the relative expression level of the CCDN1 gene in IHH4 cells, S1 is the control group, S2 is the experimental group, and the relative expression level of the experimental group is significantly lower than that of the control group; Figure 8 is the relative expression level of the FOXM1 gene in SW579 cells, S1 is the control group, S2 is the experimental group, and the relative expression level of the experimental group is significantly lower than that of the control group; Figure 9It is the relative expression level of the CDK4 gene in SW579 cells. S1 is the control group, and S2 is the experimental group. The relative expression level in the experimental group is significantly lower than that in the control group; Figure 10 It is the relative expression level of the CCDN1 gene in SW579 cells. S1 is the control group, and S2 is the experimental group. The relative expression level in the experimental group is significantly lower than that in the control group. This indicates that TMF can reduce the relative expression levels of genes downstream of NR4A1 in multiple thyroid cancer cells.

[0096] 7. Detection of cell iodine uptake ability

[0097] Collect IHH4 cells, SW579 cells and K1 cells, and detect the iodine uptake abilities of IHH4 cells, SW579 cells and K1 cells respectively. The detection method is as follows: Seed the cells at a density of 2×10 6 cells / well in a 10 cm dish, with each dish containing 10 mL of medium. Set up a control group and an experimental group. In the control group, after the cells adhered, add 100 μL of DMSO and treat for 24 h. In the experimental group, dilute the NR4A1 transcriptional activity inhibitor of Example 1 with DMSO to a final concentration of 40 μmol / L, and after the cells adhered, add 20 μL of the NR4A1 transcriptional activity inhibitor with a final concentration of 40 μmol / L and treat for 24 h. After the treatment, wash the cells twice with Hanks balanced salt solution containing 1 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), add 100 μL of Hanks balanced salt solution containing 10 μmol / L sodium iodide, and incubate in the dark at 37 °C for 2 h. Wash three times with Hanks balanced salt solution containing 1 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Lyse the cells with 500 μL of 100 μmol / L sodium hydroxide to obtain cell lysates, and perform protein quantification using the BCA method. Incubate the cell lysates, ammonium cerium(IV) sulfate, and sodium arsenite at a ratio of 1:1:1 at 20 °C for 30 min. After the incubation, centrifuge at 12,000 rpm for 15 min, and use a microplate reader to detect the absorbance at 420 nm. Normalize the iodine ion content of each group with the protein concentration, and compare the iodine uptake abilities of the cells in each group. Iodide ion concentration (pmol / mL) = log 10 (A420) / (-0.0007), cell iodine uptake ability (nmol / g) = iodide ion concentration (pmol / mL) / protein concentration (mg / mL).

[0098] Figure 11 It is the iodine uptake ability of IHH4 cells. S1 is the result of the control group, and S2 is the result of the experimental group; Figure 12 It is the iodine uptake ability of SW579 cells. S1 is the result of the control group, and S2 is the result of the experimental group; Figure 13K1 cell iodine uptake capacity, S1 is the result of the control group, and S2 is the result of the experimental group. In IHH4 cells, SW579 cells, and K1 cells, the iodine uptake capacity of the experimental group cells was higher than that of the control group, indicating that TMF can improve the iodine uptake capacity of multiple thyroid cancer cells.

[0099] Treat IHH4 cells with the NR4A1 transcriptional activity inhibitors of Examples 1-5 and Comparative Example 1 for 24 h, and measure the iodine uptake capacity of each group of IHH4 cells after the treatment. The measurement results are shown in Table 2.

[0100] Table 2 Cell iodine uptake capacity (nmol / g)

[0101]

[0102] As can be seen from Table 2, the iodine uptake capacity of the cells in Example 1 of the present invention was significantly higher than that of the control group, indicating that TMF can improve the iodine uptake capacity of IHH4 cells. The iodine uptake capacity of the cells in Examples 2-3 was higher than that in Example 1 because in the NR4A1 transcriptional activity inhibitor, in Examples 2-3, 3,5-dimethoxybenzeneacetic acid and 6-aminoflavone were first used to prepare dimethoxybenzeneacetamido flavone derivatives, and then the dimethoxybenzeneacetamido flavone derivatives were used in combination with TMF to form the NR4A1 transcriptional activity inhibitor, while in Example 1, only TMF was used as the NR4A1 transcriptional activity inhibitor; the iodine uptake capacity of the cells in Example 2 was higher than that in Example 3 because the usage amounts of the dimethoxybenzeneacetamido flavone derivatives were different in the NR4A1 transcriptional activity inhibitor. This indicates that the common use of dimethoxybenzeneacetamido flavone derivatives and TMF can improve the iodine uptake capacity of IHH4 cells. The iodine uptake capacity of the cells in Examples 4-5 was higher than that in Example 2 because in the NR4A1 transcriptional activity inhibitor, pyrimidine derivatives were further used in Examples 4-5; the iodine uptake capacity of the cells in Examples 4-5 was higher than that in Comparative Example 1 because in the NR4A1 transcriptional activity inhibitor, only pyrimidine derivatives were used in Comparative Example 1, and TMF and dimethoxybenzeneacetamido flavone derivatives were not used; the iodine uptake capacity of the cells in Example 4 was higher than that in Example 5 because the usage amounts of the pyrimidine derivatives were different. This indicates that the further use of pyrimidine derivatives in the NR4A1 transcriptional activity inhibitor can further improve the iodine uptake capacity of IHH4 cells.

[0103] The conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be elaborated here.

[0104] The above-described embodiments have described the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and do not limit the present invention. Any modifications, supplements, or similar substitutions made within the principle scope of the present invention shall be included within the protection scope of the present invention.

Claims

1. An NR4A1 transcriptional activity inhibitor, comprising at least a flavonoid compound; the flavonoid compound comprises a 4',5,7-trimethoxyflavone and a dimethoxyphenylacetamido flavonoid derivative, wherein the 4',5,7-trimethoxyflavone has a flavonoid group and a methoxy group; in the preparation of the dimethoxyphenylacetamido flavonoid derivative, 3,5-dimethoxyphenylacetic acid is weighed, N,N-dimethylformamide is added and stirred, thionyl chloride is added dropwise at 50-70°C, refluxed for 2-5h, and cooled after the reaction is completed. The mixture was heated to 25-50°C, 6-aminoflavone was added, and the mixture was reacted at 1-5°C for 1-3h. After the reaction was completed, the mixture was allowed to stand for 20-40min. The upper organic phase was washed with water, and the mixture was allowed to stand for 20-40min. The aqueous layer was retained and hydrochloric acid was added dropwise to adjust the pH to 3-4. The mixture was allowed to react at 5-15°C for 1-2h. After filtration, methanol was added. The mixture was refluxed at 60-70°C for 1-3h. The mixture was stirred at 0-5°C for 1-3h. The mixture was filtered, washed with water for 2-5 times, and dried to obtain a dimethoxyphenylacetamidoflavone derivative.

2. The NR4A1 transcriptional activity inhibitor according to claim 1, characterized in that: The mass ratio of the 4',5,7-trimethoxyflavone to the dimethoxyphenylacetamidoflavone derivative is 1:0.1-0.

2.

3. The NR4A1 transcriptional activity inhibitor according to claim 1, characterized in that: The usage ratio of the 3,5-dimethoxyphenylacetic acid and thionyl chloride is 1 g: 10-20 mL.

4. The NR4A1 transcriptional activity inhibitor according to claim 1, characterized in that: The mass ratio of the 6-aminoflavone to 3,5-dimethoxyphenylacetic acid is 1:1-2.

5. Use of the NR4A1 transcriptional activity inhibitor of claim 1 in the preparation of an anti-thyroid cancer drug or kit targeting NR4A1.

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