NR4A1 transcriptional activity inhibitor and application thereof

Abstract

The invention discloses an NR4A1 transcriptional activity inhibitor and application thereof, and belongs to the technical field of transcriptional activity inhibitors. The NR4A1 transcriptional activity inhibitor at least comprises a flavonoid compound, and the flavonoid compound comprises 4 ', 5, 7-trimethoxy flavonoside.The NR4A1 transcriptional activity inhibitor regulates NR4A1 gene expression and NR4A1 protein expression by inhibiting combination of NR4A1 and an NIS promoter, and can be used for preparing thyroid cancer resisting drugs or kits with NR4A1 as a target.

Description

Technical Field

[0001] The present invention relates to the technical field of transcription activity inhibitors, and in particular to an NR4A1 transcription activity inhibitor and application thereof. Background Art

[0002] Thyroid cancer is one of the most common malignant tumors of the endocrine system. Differentiated thyroid cancer (DTC) is the most common subtype of thyroid cancer. Most DTC patients receive conventional treatments, including surgery and selective radioactive iodine therapy, and have a good prognosis. However, DTC patients may have a 20% risk of local recurrence and a 10% risk of distant metastasis, progressing to iodine-refractory thyroid cancer (RAIR-DTC), with decreased sensitivity to radioactive iodine, and a poor prognosis after surgery. The main causes of RAIR-DTC are epigenetic changes and signaling pathway dysregulation, which can cause abnormal expression and localization of thyroid-specific genes, especially NIS, leading to failure of radioactive iodine therapy.

[0003] The NR4A1 gene, a member of the orphan nuclear receptor family, is significantly associated with the occurrence and development of papillary thyroid carcinoma (PTC). Studies have shown that high expression of NR4A1 is closely associated with poor prognosis in patients with PTC. NR4A1 directly binds to the promoter region of lymphoid enhancer binding factor (LEF1), affecting histone acetylation and DNA demethylation, thereby upregulating LEF1 expression at the transcriptional level, thereby promoting the expression of downstream growth-related genes in PTC. At present, there are few studies on NR4A1 in the field of iodine-refractory thyroid cancer. Previous studies have found that NR4A1 binds to the promoter of sodium iodide Tongxiang transporter (NIS) and affects the transcriptional level of NIS. At present, there are not many reported NR4A1 inhibitors, and only one proteolysis-targeted chimera (PROTAC) called NR-V04 is in the clinical trial stage. It is the first drug developed for NR4A1. NR-V04 can effectively degrade NR4A1 within a few hours of in vitro treatment, which can effectively inhibit tumors and can last for at least 4 days in vivo, showing a persistent NR4A1 degradation effect. In addition, there are some other NR4A1 inhibitors, such as CDIM8, Piperlongumine, and TMPA, which 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 transcription activity inhibitor and its application, which can inhibit the expression of NR4A1 gene, thereby improving the iodine uptake capacity of cells.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A NR4A1 transcription activity inhibitor at least includes flavonoid compounds; the flavonoid compounds at least include 4',5,7-trimethoxyflavone, and the 4',5,7-trimethoxyflavone has a flavonoid group and a methoxy group.

[0006] The NR4A1 transcription activity inhibitor 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 the NR4A1 gene and NR4A1 protein, thereby improving the iodine uptake capacity of various thyroid cancer cells. The 4',5,7-trimethoxyflavone can be used to prepare an anti-thyroid cancer drug or kit with NR4A1 as the target.

[0007] Preferably, the flavonoids include 4',5,7-trimethoxyflavone and dimethoxyphenylacetamido flavone derivatives. 4',5,7-trimethoxyflavone and dimethoxyphenylacetamido flavone derivatives are used together to effectively inhibit the binding of NR4A1 to the NIS promoter, reduce the relative expression of the NR4A1 gene, and improve the iodine uptake capacity of cells.

[0008] More preferably, the mass ratio of 4',5,7-trimethoxyflavone to the dimethoxyphenylacetamidoflavone derivative is 1:0.1-0.2.

[0009] More preferably, the dimethoxyphenylacetamidoflavonoid derivative is obtained by first reacting 3,5-dimethoxyphenylacetic acid with thionyl chloride and then reacting with 6-aminoflavonoid.

[0010] More preferably, the usage ratio of 3,5-dimethoxyphenylacetic acid and thionyl chloride is 1 g: 10-20 mL.

[0011] More preferably, the mass ratio of 6-aminoflavone to 3,5-dimethoxyphenylacetic acid is 1:1-2.

[0012] More preferably, the preparation of the dimethoxyphenylacetamido flavonoid derivative is specifically as follows: Weigh 3,5-dimethoxyphenylacetic acid, add N,N-dimethylformamide and stir, add thionyl chloride dropwise at 50-70°C, reflux for 2-5h, cool to 25-50°C after the reaction, add 6-aminoflavone, react at 1-5°C for 1-3h, let stand for 20-40min after the reaction, wash the upper organic phase with water, let stand for 20-40min, retain the water layer and add hydrochloric acid dropwise to adjust the pH to 3-4, react at 5-15°C for 1-2h, filter and add methanol, reflux for 1-3h at 60-70°C, stir at 0-5°C for 1-3h, filter, wash with water 2-5 times, and dry to obtain a dimethoxyphenylacetamidoflavone derivative.

[0013] More preferably, the usage ratio of 3,5-dimethoxyphenylacetic acid to N,N-dimethylformamide is 1 g:10-20 mL.

[0014] More preferably, the volume ratio of thionyl chloride to N,N-dimethylformamide is 1:1-2.

[0015] More preferably, the mass ratio of 6-aminoflavone to 3,5-dimethoxyphenylacetic acid is 1:1-2.

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

[0017] Preferably, an NR4A1 transcriptional activity inhibitor comprises 4',5,7-trimethoxyflavone, and at least one of a dimethoxyphenylacetamidoflavone derivative and a pyrimidine derivative. Among the NR4A1 transcriptional activity inhibitors, further using a pyrimidine derivative can further reduce the relative expression of the NR4A1 gene and improve the iodine uptake capacity of IHH4 cells.

[0018] More preferably, the mass ratio of 4',5,7-trimethoxyflavone to the dimethoxyphenylacetamidoflavone derivative is 1:0.1-0.2.

[0019] More preferably, the mass ratio of 4',5,7-trimethoxyflavone to the pyrimidine derivative is 1:0.05-0.1.

[0020] More preferably, the preparation of the pyrimidinyl derivative comprises, Take 2-(methylsulfinyl)pyrimidine, add tert-butyl alcohol, slowly add N,N-diisopropylethylamine and 3-amino-5-methoxyquinolin-2(1H)-one at room temperature, react at 70-90°C for 6-10h to obtain a pyrimidine derivative.

[0021] More preferably, the usage ratio of 2-(methylsulfinyl)pyrimidine to tert-butyl alcohol is 1 mmol:10-20 mL.

[0022] More preferably, the molar ratio of 2-(methylsulfinyl)pyrimidine to N,N-diisopropylethylamine is 1:1-5.

[0023] More preferably, the molar ratio of 2-(methylsulfinyl)pyrimidine to 3-amino-5-methoxyquinolin-2(1H)-one is 1:1-2.

[0024] The invention also discloses the use of the NR4A1 transcription activity inhibitor in preparing a medicine or a kit for inhibiting the expression of the NR4A1 gene.

[0025] The invention also discloses the use of the NR4A1 transcription activity inhibitor in preparing a medicine or a kit for inhibiting the expression of the NR4A1 protein.

[0026] The invention also discloses the use of the NR4A1 transcription activity inhibitor in preparing a medicine or a kit for inhibiting the binding of NR4A1 and NIS promoter.

[0027] The present invention also discloses the use of the NR4A1 transcription activity inhibitor in the preparation of an anti-thyroid cancer drug or a kit with NR4A1 as a target.

[0028] Since the present invention uses 4',5,7-trimethoxyflavone, dimethoxyphenylacetamido flavone derivatives and pyrimidine derivatives to form the NR4A1 transcription activity inhibitor, it has the following beneficial effects: the NR4A1 transcription activity inhibitor of the present invention significantly reduces the relative expression amount of the NR4A1 gene by inhibiting the binding of NR4A1 to the NIS promoter, thereby improving the iodine uptake capacity of thyroid cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the infrared spectrum of dimethoxyphenylacetamido flavonoid derivatives.

[0030] Figure 2 The results were reported by dual luciferase.

[0031] Figure 3 Western blot results.

[0032] Figure 4 is the relative expression level of NR4A1 gene.

[0033] Figure 5 is the relative expression level of FOXM1 gene in IHH4 cells.

[0034] Figure 6 is the relative expression level of CDK4 gene in IHH4 cells.

[0035] Figure 7 is the relative expression level of CCDN1 gene in IHH4 cells.

[0036] Figure 8 is the relative expression level of FOXM1 gene in SW579 cells.

[0037] Fig. 9 is the relative expression level of CDK4 gene in SW579 cells.

[0038] Fig.10 is the relative expression level of CCDN1 gene in SW579 cells.

[0039] Fig.11is the iodine uptake capacity of IHH4 cells.

[0040] Fig.12 is the iodine uptake capacity of SW579 cells.

[0041] Fig.13 is the iodine uptake capacity of K1 cells. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0044] Embodiment 1: An inhibitor of NR4A1 transcriptional activity, comprising 4',5,7-trimethoxyflavone (TMF).

[0045] Embodiment 2: Compared with Example 1, this example is different in that it contains an NR4A1 transcription activity inhibitor.

[0046] An NR4A1 transcription activity inhibitor comprises TMF and a dimethoxyphenylacetamido flavonoid derivative, wherein the mass ratio of TMF to the dimethoxyphenylacetamido flavonoid derivative is 1:0.2.

[0047] The preparation of dimethoxyphenylacetamido flavonoid derivatives comprises: Weigh 3,5-dimethoxyphenylacetic acid, add N,N-dimethylformamide and stir, add thionyl chloride dropwise at 60°C, reflux for 3h, cool to 45°C after the reaction, add 6-aminoflavone, react at 4°C for 2h, let stand for 30min after the reaction, wash the upper organic phase with water, let stand for 30min, retain the water layer and add hydrochloric acid dropwise to adjust the pH to 3, react at 10°C for 1h, filter and add methanol, reflux for 2h at 65°C, stir for 2h at 4°C, filter, wash 3 times with water, and dry to obtain a dimethoxyphenylacetamidoflavone 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-aminoflavone to 3,5-dimethoxyphenylacetic acid is 1:1.5; and the volume ratio of N,N-dimethylformamide to methanol is 1:1.

[0048] Embodiment 3: The difference between this embodiment and embodiment 2 is that this embodiment contains an NR4A1 transcription activity inhibitor.

[0049] A NR4A1 transcription activity inhibitor, except that the mass ratio of TMF to the dimethoxyphenylacetamido flavonoid derivative is changed to 1:0.1, other conditions are the same as those in Example 2.

[0050] Embodiment 4: The difference between this embodiment and embodiment 2 is that this embodiment contains an NR4A1 transcription activity inhibitor.

[0051] A NR4A1 transcription activity inhibitor comprises TMF, a dimethoxyphenylacetamido flavonoid derivative and a pyrimidine derivative. The mass ratio of TMF to the dimethoxyphenylacetamido flavonoid derivative is 1:0.2; the mass ratio of TMF to the pyrimidine derivative is 1:0.1.

[0052] The preparation of dimethoxyphenylacetamido flavonoid derivatives is the same as in Example 2.

[0053] Preparation of pyrimidinyl derivatives, comprising: Take 2-(methylsulfinyl)pyrimidine, add tert-butyl alcohol, slowly add N,N-diisopropylethylamine and 3-amino-5-methoxyquinolin-2(1H)-one at room temperature, react at 80℃ for 8h to obtain pyrimidinyl derivatives. The dosage ratio of 2-(methylsulfinyl)pyrimidine and tert-butyl alcohol is 1mmol:10mL; the molar ratio of 2-(methylsulfinyl)pyrimidine and N,N-diisopropylethylamine is 1:2; the molar ratio of 2-(methylsulfinyl)pyrimidine and 3-amino-5-methoxyquinolin-2(1H)-one is 1:1.

[0054] Embodiment 5: The difference between this embodiment and embodiment 4 is that this embodiment contains an NR4A1 transcription activity inhibitor.

[0055] A NR4A1 transcription activity inhibitor, except that the mass ratio of TMF to pyrimidine derivative is changed to 1:0.05, other conditions are the same as those in Example 4.

[0056] Comparative Example 1: The difference between this comparative example and Example 1 is that it contains an NR4A1 transcription activity inhibitor.

[0057] A NR4A1 transcription activity inhibitor comprises TMF and a pyrimidine derivative, wherein the mass ratio of TMF to the pyrimidine derivative is 1:0.1.

[0058] Preparation of pyrimidinyl derivatives, comprising: Take 2-(methylsulfinyl)pyrimidine, add tert-butyl alcohol, slowly add N,N-diisopropylethylamine and 3-amino-5-methoxyquinolin-2(1H)-one at room temperature, react at 80℃ for 8h to obtain pyrimidinyl derivatives. The dosage ratio of 2-(methylsulfinyl)pyrimidine and tert-butyl alcohol is 1mmol:10mL; the molar ratio of 2-(methylsulfinyl)pyrimidine and N,N-diisopropylethylamine is 1:2; the molar ratio of 2-(methylsulfinyl)pyrimidine and 3-amino-5-methoxyquinolin-2(1H)-one is 1:1.

[0059] Experimental example: 1. Material Characterization The infrared spectrum of the dimethoxyphenylacetamido flavonoid derivative prepared by the present invention was measured by Fourier transform infrared spectrometer using KBr tablet method at a wave number of 400-4000cm -1 Measurement within the range, resolution 0.06cm -1 , scanned 32 times.

[0060] Figure 1 This is the infrared spectrum of dimethoxyphenylacetamido flavonoid derivatives, 3400cm -1 The absorption peak of NH appears near 2920cm -1 、2850cm -1 The absorption peaks of saturated alkanes -CH3 and -CH2 appear near 1750cm -1 The absorption peak of C=O appears near 1620cm -1 The absorption peak of C=C appears near 1170cm -1 The absorption peak of COC appears nearby.

[0061] 2. Cytotoxicity assay Human embryonic kidney cells (293T cells) were cultured at 5 × 10 4 The cells were seeded at a density of 100 μg / L in a 24-well plate and divided into 4 groups, namely, blank group, control group, experimental group 1, experimental group 2 and experimental group 3. TMF, dimethoxyphenylacetamido flavonoid derivatives and pyrimidine derivatives were prepared into solutions with a final concentration of 40 μmol / L using dimethyl sulfoxide (DMSO) as solvent. The blank group was not treated, the control group was treated with dimethyl sulfoxide (DMSO) for 24 h, the experimental group 1 was treated with TMF for 24 h, the experimental group 2 was treated with dimethoxyphenylacetamido flavonoid derivatives for 24 h, and the experimental group 3 was treated with pyrimidine derivatives for 24 h. After the treatment, the cell survival rate of experimental group 1, experimental group 2 and experimental group 3 was evaluated by thiazole blue colorimetry (MTT method), and the absorbance of the solution at a wavelength of 570 nm was measured by an enzyme marker. Cell survival rate (%) = (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%.

[0062] The cell survival rate of the experimental group 1 was 89.4%, the cell survival rate of the experimental group 2 was 91.5%, and the cell survival rate of the experimental group 3 was 90.6%, which indicates that the TMF, dimethoxyphenylacetamido flavonoid derivatives and pyrimidine derivatives used in the NR4A1 transcription activity inhibitor of the present invention have good cell compatibility.

[0063] 3. Detection of NR4A1 binding ability to NIS promoter Plasmids were constructed, including firefly luciferase reporter gene (NISp) plasmid with full-length sequence of NIS promoter, NR4A1 overexpression plasmid, NR4A1 empty plasmid and Renilla luciferase internal reference gene plasmid. Among them, NR4A1 overexpression plasmid is pCMV-NR4A1(human)-SV40-Neo, purchased from Wuhan Miaoling Biotechnology Co., Ltd.; NR4A1 empty plasmid is pCMV-SV40-Neo, purchased from Wuhan Miaoling Biotechnology Co., Ltd.; Renilla luciferase internal reference gene plasmid is phRL-TK, purchased from Promega (Beijing) Biotechnology Co., Ltd. The construction method of NISp plasmid is as follows: using a cell genomic DNA extraction kit to extract the whole genomic DNA of IHH4 cells, the cell genomic DNA extraction kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; designing primers according to the NIS promoter sequence and the pGL 4.23 plasmid sequence, the positive primer nucleotide sequence is 5'-GGGGTACCCCTTATAAAACTTCCACGCTCAAGCG-3', and its nucleotide sequence is shown in SEQ ID NO.1, and the negative primer nucleotide sequence is 5'-CCCAAGCTTGGGAAGCTTATGCACTGTGATCCCCTGAT-3', and its nucleotide sequence is shown in SEQ ID NO.2; obtaining the target gene of NIS promoter from the whole genomic DNA by PCR amplification; using restriction endonuclease to incubate overnight, cutting the target gene and pGL 4.23 plasmid, using DNA ligase to link the cut target gene with the pGL 4.23 plasmid, and pGL 4.23 plasmid was purchased from Promega (Beijing) Biotechnology Co., Ltd.; DNA gel electrophoresis was used to separate the ligation products according to molecular weight, and the gel of about 5400 bp was collected. The ligation products were purified using a gel recovery / DNA purification kit to obtain the purified ligation products. The gel recovery / DNA purification kit was purchased from Nanjing Novogene Biotechnology Co., Ltd.; the purified ligation products were mixed with DH5α competent cells, heat-shocked for 90 seconds, incubated at 4°C for 5 minutes, added with 1 mL LB culture medium and incubated at 37°C in a constant temperature shaker for 1 hour, centrifuged at 4°C and 3000 rpm for 10 minutes, 900 μL of supernatant was discarded, and the remaining cell suspension was mixed and coated on an agar plate containing ampicillin and incubated at 37°C overnight. After selecting a single clone for expansion culture, the bacterial liquid was sent to Suzhou Junji Biotechnology Co., Ltd. for sequencing; the plasmid was extracted using a plasmid mini-extraction kit. The plasmid mini-extraction kit was purchased from Ibotek Biotechnology Co., Ltd. to obtain the NISp plasmid.

[0064] Human embryonic kidney cells (293T cells) were cultured at 5 × 10 4The cells were inoculated at a density of 1 μg in a 24-well plate and divided into 4 groups, namely group A, group B, group C and group D. Group A and group B were co-transfected with 1 μg NISp plasmid, 1 μg NR4A1 empty plasmid and 0.1 μg Renilla luciferase internal reference gene plasmid; group C and group D were co-transfected with 1 μg NISp plasmid, 1 μg NR4A1 overexpression plasmid and 0.1 μg 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 antibody. The plasmids of each group were evenly distributed in 25 μL of reduced serum medium, and then 1 μL of Lipo-3000 transfection reagent was evenly distributed in 25 μL of reduced serum medium. After standing for 5 minutes, the reduced serum medium containing mass and the reduced serum medium containing Lipo-3000 transfection reagent were mixed, and continued to stand for 20 minutes before adding to the 24-well plate for transfection, corresponding to the transfected cells of groups A, B, C, and D. After 6 hours of transfection, 0.5 mL of DMEM medium containing serum and double antibodies was replaced and cultured for 18 hours, and the NR4A1 transcription activity inhibitor of Example 1 was diluted with DMSO to a final concentration of 40 μmol / L. 5 μL of NR4A1 transcription activity inhibitor with a final concentration of 40 μmol / L was added to B and D for 24 hours, and the same volume of dimethyl sulfoxide (DMSO) was added to groups A and C for 24 hours to obtain 293T cells in groups A, B, C, and D after transfection.

[0065] The 293T cells in groups A, B, C, and D after transfection were collected and subjected to dual luciferase reporter experiments. The steps of the dual luciferase reporter experiment were as follows: the cells were washed twice with phosphate buffered saline (PBS), 100 μL of cell lysis solution was added, and the cells were shaken and lysed for 15 min. The cell lysate was collected and centrifuged at 12000 rpm for 10 min at 4 °C. 20 μL of supernatant was taken, 50 μL of firefly luciferase substrate was added, and the mixture was immediately detected by an ELISA reader, which was recorded as X1. Then 50 μL of Renilla luciferase substrate stop solution was added, and the mixture was immediately placed in an ELISA reader for detection, which was recorded as X2. Luciferase reaction intensity = X1 / X2.

[0066] Figure 2The results of dual luciferase reporting are shown in Figure 1. Compared with Group A and Group C, the luciferase reaction intensity of Group A was lower than that of Group C. This is because Group A co-transfected NISp plasmid, NR4A1 empty plasmid and Renilla luciferase internal reference gene plasmid, and Group C co-transfected NISp plasmid, NR4A1 overexpression plasmid and Renilla luciferase internal reference gene plasmid. This shows that NR4A1 can bind to the NIS promoter, and NR4A1 can increase the luciferase reaction intensity after binding to the NIS promoter. Compared with Group C and Group D, the fluorescence intensity of Group C was higher than that of Group D. This is because in the treatment of NR4A1 transcriptional activity inhibitor, Group C was treated with TMF and Group D was treated with DMSO. This shows that TMF can inhibit NR4A1 from binding to the NIS promoter.

[0067] 4. Western Blotting 293T cells were cultured at 5 × 10 5 The cells were planted in a 6-well plate at a density of 100 μg / well, and each well contained 2 mL of culture medium. A control group and an experimental group were set up. In the control group, 20 μL of DMSO was added after the cells adhered to the wall and treated for 24 h. In the experimental group, DMSO was used to dilute the NR4A1 transcription activity inhibitor of Example 1 to a final concentration of 40 μmol / L, and 20 μL of NR4A1 transcription activity inhibitor with a final concentration of 40 μmol / L was added after the cells adhered to the wall and treated for 24 h. After the treatment, the cells in the control group and the experimental group were collected and subjected to protein blotting experiments respectively. The steps of the protein blotting experiment are as follows: the cells were washed twice with PBS, nuclear protein extraction kit was used to extract nuclear protein, and the protein concentration was determined using a BCA protein quantification kit. The nuclear protein extraction kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and the BCA protein quantification kit was purchased from Shanghai Ke Aibo Biotechnology Co., Ltd. According to the protein quantification results, the corresponding volume of radioimmunoprecipitation lysis (RIPA) buffer was added, and the loading buffer was added. The loading buffer was purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd. and heated at 100°C for 10 min for denaturation. After protein samples were separated by SDS-PAGE, they were transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane and blocked with TBST buffer containing 5% skim milk powder for 1 h. The primary antibody was added and incubated at 4 °C for 12 h. Then, IgG horseradish peroxidase (HRP)-conjugated secondary antibody was added and incubated at room temperature for 2 h. The membrane was washed three times with TBST buffer and developed with enhanced chemiluminescence reagent (ECL).

[0068] Figure 3 The results of Western blotting experiments are shown in Figure 2. Figure 3 It can be seen that the expression of NR4A1 protein in the control group was higher than that in the experimental group, while the expression of TOP1 protein in the control group and the experimental group was basically the same. This shows that TMF can reduce the protein expression of NR4A1.

[0069] 5. Detection of relative expression of NR4A1 293T cells were cultured at 5 × 10 5 The cells were planted in a 6-well plate at a density of 100 / well. A control group and an experimental group were set up. In the control group, 20 μL of DMSO was added after the cells adhered to the wall and treated for 24 hours. In the experimental group, DMSO was used to dilute the NR4A1 transcription activity inhibitor of Example 1 to a final concentration of 40 μmol / L, and 20 μL of NR4A1 transcription activity inhibitor with a final concentration of 40 μmol / L was added after the cells adhered to the wall and treated for 24 hours. After the treatment, cells from each group were collected and qPCR experiments were performed separately. The qPCR experimental steps are as follows: the cells were washed twice with PBS, and the FreeZol Reagent kit was used for cell lysis and RNA extraction. The FreeZol Reagent kit was purchased from Nanjing Novozymes Biotech Co., Ltd.; cDNA was synthesized using a reverse transcription kit, and the reverse transcription kit was purchased from Nanjing Novozymes Biotech Co., Ltd. The qPCR reaction system was 5 μL cDNA template, 10 μL SYBRgreen fluorescent dye, 1 μL 10 μmol / L positive primer, 1 μL 10 μmol / L negative primer and 3 μL sterile enzyme-free water. The relative expression of NR4A1 gene in the control group and experimental group was detected using qPCR instrument.

[0070] Figure 4 is the relative expression of NR4A1 gene, S1 corresponds to the result of the control group, and S2 is the result of the experimental group. The relative expression of NR4A1 gene in the experimental group was significantly lower than that in the control group, which indicates that TMF can effectively reduce the relative expression of NR4A1 gene.

[0071] The 293T cells were treated with the NR4A1 transcriptional activity inhibitors of Examples 1-5 and Comparative Example 1 for 24 hours, and the relative expression levels of the NR4A1 genes in each group were measured after the treatment. The measurement results are shown in Table 1.

[0072] Table 1 Relative expression of NR4A1 gene

[0073] The relative expression level of Example 1 is significantly lower than that of the control group, which shows that TMF can reduce the relative expression level of the NR4A1 gene. The relative expression level of Example 2-3 is lower than that of Example 1 because, in the NR4A1 transcription activity inhibitor, Example 2-3 first uses 3,5-dimethoxyphenylacetic acid and 6-aminoflavone to react to prepare dimethoxyphenylacetamido flavonoid derivatives, and then uses the dimethoxyphenylacetamido flavonoid derivatives in combination with TMF to form the NR4A1 transcription activity inhibitor, while Example 1 only uses TMF as the NR4A1 transcription activity inhibitor; the relative expression level of Example 2 is lower than that of Example 3 because the amount of dimethoxyphenylacetamido flavonoid derivatives used in the NR4A1 transcription activity inhibitor is different. This shows that the use of dimethoxyphenylacetamido flavonoid derivatives and TMF together can reduce the relative expression level of the NR4A1 gene. The relative expression of Example 4-5 is lower than that of Example 2 because, in the NR4A1 transcription activity inhibitor, Example 4-5 further uses pyrimidine derivatives; the relative expression of Example 4-5 is lower than that of Comparative Example 1 because, in the NR4A1 transcription activity inhibitor, Comparative Example 1 only uses pyrimidine derivatives, but does not use TMF and dimethoxyphenylacetamido flavonoid derivatives; the relative expression of Example 4 is lower than that of Example 5 because the amount of pyrimidine derivatives used is different. This shows that the further use of pyrimidine derivatives in the NR4A1 transcription activity inhibitor can further reduce the relative expression of the NR4A1 gene.

[0074] 6. Detection of relative expression of downstream genes FOXM1, CDK4 and CCDN1 of NR4A1 gene 293T cells were replaced with human thyroid papillary carcinoma cells (IHH4) and human thyroid squamous cell carcinoma cells (SW579), and the relative expression levels of FOXM1, CDK4 and CCDN1 genes, the downstream genes of NR4A1 gene, were detected using qPCR instrument.

[0075] Figure 5 is the relative expression level of 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 of CDK4 gene in IHH4 cells, S1 is the control group, S2 is the experimental group, and the relative expression of the experimental group is significantly lower than that of the control group; Figure 7 is the relative expression level of 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 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; Fig. 9is the relative expression of CDK4 gene in SW579 cells, S1 is the control group, S2 is the experimental group, and the relative expression of the experimental group is significantly lower than that of the control group; Fig.10 is the relative expression of CCDN1 gene in SW579 cells, S1 is the control group, S2 is the experimental group, and the relative expression of the experimental group is significantly lower than that of the control group. This shows that TMF can reduce the relative expression of NR4A1 downstream genes in a variety of thyroid cancer cells.

[0076] 7. Detection of cellular iodine uptake capacity IHH4 cells, SW579 cells and K1 cells were collected and their iodine uptake capacity was tested respectively. The test method was as follows: cells were cultured at 2×10 6 The cells were planted at a density of 100 μL / well in a 10 cm dish, each dish containing 10 mL of culture medium. A control group and an experimental group were set up. In the control group, 100 μL DMSO was added after the cells adhered to the wall and treated for 24 h. In the experimental group, DMSO was used to dilute the NR4A1 transcription activity inhibitor of Example 1 to a final concentration of 40 μmol / L, and 20 μL of NR4A1 transcription activity inhibitor with a final concentration of 40 μmol / L was added after the cells adhered to the wall and treated for 24 h. After the treatment, the cells were washed twice with Hanks balanced salt solution containing 1 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 100 μL of Hanks balanced salt solution containing 10 μmol / L sodium iodide was added, and incubated at 37 ° C in the dark for 2 h. Washed 3 times with Hanks balanced salt solution containing 1 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES). Use 500 μL of 100 μmol / L sodium hydroxide to lyse the cells, obtain cell lysate, and use the BCA method for protein quantification. Incubate the cell lysate, ammonium cerium sulfate, and sodium arsenite at a ratio of 1:1:1 at 20°C for 30 minutes. After incubation, centrifuge at 12000 rpm for 15 minutes, and use an enzyme reader to detect the absorbance at 420 nm. Normalize the iodide ion content of each group with protein concentration, and compare the iodine uptake capacity of each group of cells. Iodide ion concentration (pmol / mL) = log 10 (A420) / (-0.0007), cellular iodine uptake capacity (nmol / g) = iodine ion concentration (pmol / mL) / protein concentration (mg / mL).

[0077] Fig.11 is the iodine uptake capacity of IHH4 cells, S1 is the result of the control group, and S2 is the result of the experimental group; Fig.12 is the iodine uptake capacity of SW579 cells, S1 is the result of the control group, and S2 is the result of the experimental group; Fig.13is the iodine uptake capacity of K1 cells, 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, which indicates that TMF can improve the iodine uptake capacity of various thyroid cancer cells.

[0078] The IHH4 cells were treated with the NR4A1 transcriptional activity inhibitors of Examples 1-5 and Comparative Example 1 for 24 hours, and the iodine uptake capacity of the IHH4 cells in each group was measured after the treatment. The measurement results are shown in Table 2.

[0079] Table 2 Cellular iodine uptake capacity (nmol / g)

[0080] As can be seen from Table 2, the iodine uptake capacity of cells in Example 1 of the present invention is significantly higher than that of the control group, which shows that TMF can improve the iodine uptake capacity of IHH4 cells. The iodine uptake capacity of cells in Example 2-3 is higher than that in Example 1 because, in the NR4A1 transcription activity inhibitor, Example 2-3 first uses 3,5-dimethoxyphenylacetic acid and 6-aminoflavone to react to prepare dimethoxyphenylacetamido flavonoid derivatives, and then uses the dimethoxyphenylacetamido flavonoid derivatives in combination with TMF to form the NR4A1 transcription activity inhibitor, while Example 1 only uses TMF as the NR4A1 transcription activity inhibitor; the iodine uptake capacity of cells in Example 2 is higher than that in Example 3 because the amount of dimethoxyphenylacetamido flavonoid derivatives used in the NR4A1 transcription activity inhibitor is different. This shows that the use of dimethoxyphenylacetamido flavonoid derivatives and TMF together can improve the iodine uptake capacity of IHH4 cells. The iodine uptake capacity of cells in Example 4-5 is higher than that in Example 2 because pyrimidine derivatives are further used in Example 4-5 among the NR4A1 transcription activity inhibitors; the iodine uptake capacity of cells in Example 4-5 is higher than that in Comparative Example 1 because only pyrimidine derivatives are used in Comparative Example 1 among the NR4A1 transcription activity inhibitors, without using TMF and dimethoxyphenylacetamido flavonoid derivatives; the iodine uptake capacity of cells in Example 4 is higher than that in Example 5 because the amount of pyrimidine derivatives used is different. This shows that the further use of pyrimidine derivatives in the NR4A1 transcription activity inhibitor can further improve the iodine uptake capacity of IHH4 cells.

[0081] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0082] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An NR4A1 transcription activity inhibitor, comprising at least a flavonoid compound; the flavonoid compound at least comprises 4',5,7-trimethoxyflavone, and the 4',5,7-trimethoxyflavone has a flavonoid group and a methoxy group.

2. The NR4A1 transcriptional activity inhibitor according to claim 1, characterized in that: The flavonoid compounds include 4',5,7-trimethoxyflavone and dimethoxyphenylacetamidoflavone derivatives.

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

2.

4. The NR4A1 transcriptional activity inhibitor according to claim 2, characterized in that: The dimethoxyphenylacetamido flavonoid derivative is obtained by first reacting 3,5-dimethoxyphenylacetic acid with thionyl chloride and then reacting with 6-aminoflavonoid.

5. The NR4A1 transcriptional activity inhibitor according to claim 4, characterized in that: The usage ratio of the 3,5-dimethoxyphenylacetic acid and thionyl chloride is 1 g: 10-20 mL.

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

7. Use of the NR4A1 transcriptional activity inhibitor according to claim 1 in the preparation of a drug or a kit for inhibiting NR4A1 gene expression.

8. Use of the NR4A1 transcriptional activity inhibitor according to claim 1 in the preparation of a drug or a kit for inhibiting NR4A1 protein expression.

9. Use of the NR4A1 transcriptional activity inhibitor of claim 1 in the preparation of a drug or a kit for inhibiting the binding between NR4A1 and NIS promoter.

10. 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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