Use of integrin alpha 2 beta 1 inhibitors and agents for detecting integrin alpha 2 beta 1

Abstract

The present application relates to the field of biological medicine technology, and in particular to the use of integrin alpha2 beta1 inhibitor and reagent for detecting integrin alpha2 beta1, and further to the use of integrin alpha2 beta1 inhibitor in preparing a drug for improving the iodine treatment sensitivity of anaplastic thyroid cancer and reducing the migration / invasion ability of anaplastic thyroid cancer, and the use of reagent for detecting integrin alpha2 beta1 in preparing a drug for detecting the iodine treatment sensitivity and migration / invasion ability of anaplastic thyroid cancer. The present application first proves that the expression of integrin alpha2 beta1 gene is closely related to the iodine treatment sensitivity and migration / invasion ability of anaplastic thyroid cancer, and provides a new way for the treatment of anaplastic thyroid cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the use of integrin α2β1 inhibitor and reagent for detecting integrin α2β1, and further relates to the use of integrin α2β1 inhibitor in preparing a drug for improving the iodine treatment sensitivity of anaplastic thyroid cancer and reducing the migration ability / invasion ability of anaplastic thyroid cancer, and the use of reagent for detecting integrin α2β1 in preparing a drug for detecting the iodine treatment sensitivity of anaplastic thyroid cancer and the migration ability / invasion ability of anaplastic thyroid cancer. BACKGROUND

[0002] Anaplastic thyroid cancer is the most malignant type of thyroid cancer, accounting for about 10-15% of all thyroid cancers. Anaplastic thyroid cancer develops rapidly and grows quickly, and can infiltrate and metastasize in the early stage, with high malignancy and poor prognosis. Due to the insidious onset and rapid progression, most anaplastic thyroid cancer is diagnosed when it has invaded surrounding tissues or organs, such as trachea, esophagus, nerves, and muscles, and about 50% of patients have distant metastasis to lung, bone, liver, brain, etc., which is often difficult to treat by surgery. Moreover, the expression of thyroid-specific genes such as sodium iodide symporter (NIS) is significantly reduced in anaplastic thyroid cancer, resulting in low iodine uptake ability of anaplastic thyroid cancer. Its growth is also not affected by thyroid stimulating hormone (TSH), which makes radioiodine therapy and TSH suppression therapy difficult to produce obvious effects. In addition, anaplastic thyroid cancer is not sensitive to traditional chemotherapy, which leads to the failure of existing chemotherapy strategies to effectively control disease progression. Although various new treatment methods have been adopted for anaplastic thyroid cancer in recent years, the treatment effect is poor, and the prognosis of anaplastic thyroid cancer has not been significantly improved. Therefore, it is crucial to find new treatment options for anaplastic thyroid cancer to improve the prognosis of anaplastic thyroid cancer. SUMMARY

[0003] Therefore, the technical problem to be solved by the present application is to overcome the defects of the prior art, i.e., the poor treatment effect of the existing treatment options for anaplastic thyroid cancer and the poor improvement effect on the prognosis of anaplastic thyroid cancer, and to provide the use of integrin α2β1 inhibitor and reagent for detecting integrin α2β1, and specifically provide the use of integrin α2β1 inhibitor in preparing a drug for improving the iodine treatment sensitivity of anaplastic thyroid cancer and reducing the migration ability / invasion ability of anaplastic thyroid cancer, and the use of reagent for detecting integrin α2β1 in preparing a drug for detecting the iodine treatment sensitivity of anaplastic thyroid cancer and the migration ability / invasion ability of anaplastic thyroid cancer.

[0004] To this end, the present application provides the use of integrin α2β1 inhibitor in preparing a drug for improving the iodine treatment sensitivity of anaplastic thyroid cancer.

[0005] Optionally, the drug is a drug for enhancing the iodine uptake ability of the anaplastic thyroid cancer cell.

[0006] The present application also provides the use of an integrin α2β1 inhibitor in the preparation of a drug for reducing the migration / invasion ability of an anaplastic thyroid cancer.

[0007] Optionally, the integrin α2β1 inhibitor comprises an agent for inhibiting the expression of integrin α2β1 gene and / or an agent for inhibiting the activity of integrin α2β1 protein.

[0008] Optionally, the integrin α2β1 inhibitor comprises diallyl trisulfide.

[0009] The present application also provides the use of an agent for detecting integrin α2β1 in the preparation of a product for detecting the iodine treatment sensitivity of an anaplastic thyroid cancer.

[0010] The present application also provides the use of an agent for detecting integrin α2β1 in the preparation of a product for detecting the migration / invasion ability of an anaplastic thyroid cancer.

[0011] Optionally, the product comprises at least one of a drug, a kit, a chip, a test paper, or a high-throughput sequencing platform.

[0012] Optionally, the agent for detecting integrin α2β1 comprises an agent for detecting the expression amount of integrin α2β1 gene.

[0013] Optionally, the agent for detecting integrin α2β1 comprises an agent for quantitatively detecting integrin α2β1 gene mRNA and / or an agent for quantitatively detecting integrin α2β1 protein.

[0014] Optionally, the agent for quantitatively detecting integrin α2β1 gene mRNA can function based on a known detection method using nucleic acid molecules. For example, the agent for quantitatively detecting integrin α2β1 gene mRNA can quantitatively detect integrin α2β1 gene mRNA by at least one of a PCR method, a Southern hybridization method, a Northern hybridization method, a dot hybridization method, a fluorescence in situ hybridization method, a DNA microarray method, an ASO method, and a high-throughput sequencing platform method.

[0015] Further, the PCR method can be a known method, for example, can be an rt-PCR (reverse transcription PCR) method, a nested PCR method, and an ARMS (Amplification Refractory Mutation System) method, etc. The amplified nucleic acid can be detected by using a conventional method such as a dot blot hybridization method, a surface plasmon resonance method, a PCR-RFLP method, an in situ rt-PCR method, a PCR-SSO method, a PCR-SSP method, an AMPFLP method, a MVR-PCR method, and a PCR-SSCP method, etc.

[0016] Optionally, the reagent for quantitatively detecting the mRNA of the integrin α2β1 gene comprises a primer capable of specifically amplifying the integrin α2β1 gene or its transcript, and / or a probe capable of specifically recognizing the integrin α2β1 gene or its transcript.

[0017] Optionally, the reagent for quantitatively detecting the mRNA of the integrin α2β1 gene is a primer capable of specifically amplifying the integrin α2β1 gene or its transcript, for example, can be a q-PCR primer shown in SEQ ID NO. 1-4 and / or a PCR primer shown in SEQ ID NO. 5-8.

[0018] Optionally, the reagent for quantitatively detecting the integrin α2β1 protein can function based on a known detection method using an antibody, for example, the reagent for quantitatively detecting the integrin α2β1 protein can quantitatively detect the integrin α2β1 protein by an ELISA method, an immunohistochemical method, and a Western blot method.

[0019] Optionally, the reagent for quantitatively detecting the integrin α2β1 protein can comprise an antibody or an antibody fragment specifically binding to the integrin α2β1 protein, and / or a nucleic acid encoding the antibody or the antibody fragment, and / or a vector comprising the nucleic acid, and / or a cell carrying the vector.

[0020] Optionally, the antibody can be a monoclonal antibody or a polyclonal antibody, and can be obtained by a method known to those skilled in the art. For example, a hybridoma cell capable of secreting an antibody or an antibody fragment can be obtained by a hybridoma technique, and then the antibody is collected from the hybridoma cell culture.

[0021] Optionally, the biological sample for detecting the integrin α2β1 in the present application can be selected from (but not limited to) a tissue, peripheral blood, a lymph node, a cell, etc.

[0022] The technical scheme of the present application has the following advantages:

[0023] The present application first proves that integrin α2β1 gene expression is closely related to the iodine treatment sensitivity and migration / invasion ability of anaplastic thyroid carcinoma, specifically, the higher the integrin α2β1 gene expression, the worse the iodine treatment sensitivity and the stronger the migration / invasion ability of anaplastic thyroid carcinoma, therefore, the iodine treatment sensitivity and migration / invasion ability of anaplastic thyroid carcinoma can be detected by detecting the expression of integrin α2β1 gene, and the iodine treatment sensitivity of anaplastic thyroid carcinoma can be improved and the migration / invasion ability can be reduced by inhibiting the expression of integrin α2β1 gene. Therefore, the present application provides a new way for the treatment of anaplastic thyroid carcinoma.

[0024] In addition, the present application first proves that the garlic extract diallyl trisulfide can inhibit the expression of integrin α2β1 gene, which provides a medicinal basis for the application of diallyl trisulfide in the treatment of anaplastic thyroid carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0026] Figure 1 is the analysis result of integrin α2β1 expression level of different thyroid tissues in Example 1 of the present application;

[0027] Figure 2 is the protein level detection result of integrin α2β1 of each cell line in Example 2 of the present application;

[0028] Figure 3 is the real-time fluorescent quantitative PCR detection result in Example 3 of the present application;

[0029] Figure 4 is the polymerase chain reaction-agarose gel electrophoresis analysis result in Example 3 of the present application;

[0030] Figure 5 is the protein immunoblot analysis result of each cell in Example 4 of the present application;

[0031] Figure 6 is the detection result of membrane protein and cytoplasmic protein of each cell in Example 5 of the present application;

[0032] Figure 7 is the Transwell migration / invasion experiment detection result in Example 6 of the present application;

[0033] Figure 8is the expression detection result of integrin α2β1 in each thyroid tissue in Example 7 of the present application;

[0034] Figure 9 is the expression detection result of integrin β1 and NIS in thyroid tissue in Example 8 of the present application;

[0035] Figure 10 is the Transwell migration / invasion experiment detection result of each cell line in Example 9 of the present application;

[0036] Figure 11 is the iodine uptake capacity detection result of each cell in Example 10 of the present application. DETAILED DESCRIPTION

[0037] The following examples are provided to better further understand the present application, and are not limited to the best mode, and do not constitute a limitation on the content and protection scope of the present application, and any person under the inspiration of the present application or the combination of the present application with other prior art features, any product same or similar to the present application falls within the protection scope of the present application.

[0038] The specific experimental steps or conditions are not indicated in the examples, and can be operated according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, and are conventional reagent products that can be obtained by market purchase.

[0039] The gene sequences of the two subunits of "integrin α2β1" involved in the examples of the present application ITGA2 (Chromosome 5, NC_000005.10 (52989352..53094779)) and ITGB1 (Chromosome 10, NC_000010.11 (32900318..32958230, complement)) can be queried in the NCBI database.

[0040] The sources of each cell line involved in the examples of the present application are as follows:

[0041] Normal thyroid epithelial cell line Nthy-ori-3.1: European Collection of Authenticated Cell Cultures (ECACC);

[0042] Undifferentiated thyroid carcinoma cell line 8505C: European Collection of Authenticated Cell Cultures (ECACC);

[0043] Undifferentiated thyroid carcinoma cell line C643: Chinese Academy of Sciences Typical Culture Collection Committee Shanghai Cell Bank (NCACC);

[0044] Anaplastic thyroid carcinoma cell line FRO: Chinese Academy of Sciences Typical Culture Collection Shanghai Cell Bank (NCACC);

[0045] Anaplastic thyroid carcinoma cell line CAL-62: Chinese Academy of Sciences Typical Culture Collection Shanghai Cell Bank (NCACC).

[0046] Example 1

[0047] This example is used to illustrate the correlation between integrin α2β1 and anaplastic thyroid carcinoma:

[0048] 1. Experimental method

[0049] The raw microarray unit intensity files (CEL format) of microarray datasets GSE33630, GSE29265, GSE76039, GSE65144, GSE82208 and GSE53157 were obtained from the Gene Expression Omnibus (GEO) database and were executed on their corresponding chip platform GPL570 (http: / / www.ncbi.nlm.nih.gov / geo / ). These files were background-adjusted and normalized using the multi-chip average method (http: / / rmaexpress.bmbolstad.com), batch effects were removed by ComBat, and all probes were mapped to the latest version of the Affymetrix NetAffx annotation file. For gene sets represented by multiple probes, their average values were taken as the expression levels. The joint GEO cohort analyzed in this example included 81 normal thyroid tissues (Normal), 76 papillary thyroid carcinoma tissues (PTC), 31 follicular thyroid carcinoma tissues (FTC), 22 poorly differentiated thyroid carcinoma tissues (PDTC) and 52 anaplastic thyroid carcinoma tissues (ATC).

[0050] Unbiased correlation analysis was performed by GEPIA (http: / / gepia.cancer-pku.cn / detail.php) and Spearman correlation analysis method was used to analyze the correlation between the expression of integrins in the TCGA-THCA database and the genotype-tissue expression (GTEx) database.

[0051] 2. Experimental results

[0052] As Figure 1 A and Figure 1As shown in Figure B, analysis of the RMA expression value of integrin α2β1 in different thyroid tissues from the GEO database revealed that integrin α2 (ITGA2) expression was significantly upregulated in papillary thyroid carcinoma (PTC), poorly differentiated thyroid carcinoma (PDTC), and undifferentiated thyroid carcinoma (ATC). Figure 1 A), while integrin β1 (ITGB1) expression was significantly upregulated in papillary thyroid carcinoma (PTC) and undifferentiated thyroid carcinoma (ATC). Figure 1 B), and the upregulation of integrin α2β1 was more significant in undifferentiated thyroid carcinoma (ATC) tissue.

[0053] Example 2

[0054] Total cellular proteins were collected from normal thyroid epithelial cell line Nthy-ori-3.1 and undifferentiated thyroid cancer cell lines 8505C, C643, FRO, and CAL-62, and the content of integrin α2 protein in each cell line was detected by Western blotting analysis.

[0055] 1. Western Blot Analysis of Proteins

[0056] Each cell line was cultured to a cell concentration of approximately 10. 6 After collecting cells per well, discard the cell culture medium, add 1 mL of PBS pre-cooled to 4°C, collect cells with a cell scraper, centrifuge at 8,000 rpm for 5 min at 4°C, collect the cell pellet, add 50 μL of cell lysis buffer (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 1% polyethylene glycol octylphenyl ether Triton X-100) containing 1% protease inhibitor (phenylmethylsulfonyl fluoride PMSF) and 1% phosphatase inhibitor (C4H4Na2O6·2H2O), vortex to mix, repeat freeze-thaw cycle 3 times, centrifuge at 12,000 rpm for 15 min at 4°C, collect the supernatant and transfer it to a PCR tube for storage to obtain the protein solution of each cell line.

[0057] For each protein solution of each cell line, the protein concentration of each protein solution was determined by Coomassie Brilliant Blue method, and then each protein solution containing an equal amount of protein was mixed with 1 / 5 sample volume of 6x loading buffer (30 mM ethylenediaminetetraacetic acid EDTA; 36% glycerol; 0.05% xylene cyanol FF; 0.05% bromophenol blue) and loaded into a PCR tube. After heating at 95°C metal bath for 5 min to fully denature and stretch the protein, it was loaded, SDS-PAGE gel electrophoresis, wet transfer, blocking in TBST buffer containing 5% skim milk at room temperature for 1 h, incubating the corresponding primary antibody (anti-integrin α2, source: Biyun; anti-GAPDH, source: Shenguo Biotechnology) and shaking at 4°C overnight; wash the strip with appropriate TBST buffer for 3 times, 10 min each time, incubate the corresponding secondary antibody (goat anti-mouse IgG, source: Biyun; rabbit IgG-HRP, source: Biyun) at room temperature for 1 h. ECL method of chemical luminescence, with Bole ChemiDocXRS+ system for photosensitive development, the results are shown in Figure 2 A. The corresponding pictures were collected and analyzed by Image J. The relative protein expression of integrin in the sample was obtained by comparing the gray scale of the integrin protein band with that of the corresponding GAPDH (glyceraldehyde-3-phosphate dehydrogenase) band. The results are shown in Figure 2 B.

[0058] 2. Experimental results

[0059] The experimental results are shown in Figure 2 A and Figure 2 B. As can be seen from Figure 2 A and Figure 2 B, based on Western Blot analysis, the protein level of integrin α2 in thyroid cell lines can be detected by antibody, and compared with normal thyroid epithelial cell line Nthy-ori-3.1, the expression of integrin α2 in thyroid anaplastic carcinoma cell lines 8505C, C643, FRO and CAL-62 is significantly up-regulated.

[0060] Example 3

[0061] This example uses qPCR method and PCR method to verify the inhibitory effect of diallyl trisulfide (DATS) on the expression of integrin α2β1 gene in thyroid anaplastic carcinoma:

[0062] (1) Real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qRT-PCR)

[0063] The 8505C cells were treated with DATS at 5 μM, 10 μM and 20 μM and control solvent (dimethyl sulfoxide DMSO) respectively for 24 h, and then 1 mL of total RNA extraction reagent (TRIzol) was added to each culture dish to lyse the cells and separate the RNA, which was then transferred to an RNase-free 1.5 mL EP tube; after standing for 5 min, 1 / 5 volume of chloroform was added, and after mixing thoroughly, the mixture was stood for 5 min, centrifuged at 10,000 rpm for 15 min at 4°C, and the water phase layer containing the RNA was aspirated; an equal volume of isopropanol was added, the mixture was mixed thoroughly and stood for 5 min, and then centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was discarded; 1 ml of 75% anhydrous ethanol was added, the mixture was gently shaken, and then centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was discarded; the RNA was allowed to dry and precipitate at room temperature (the EP tube was inverted on filter paper), and 30 μL of DEPC water was added to the EP tube to obtain the total RNA.

[0064] 1 μL of Oligo (dT) and 2 μg of RNA were added to a PCR tube, and DEPC water was added to make a total volume of 12 μL, which was then placed in a 65°C water bath for 5 min and then placed on ice, 4 μL of 5×Reaction Buffer, 1 μL of Recombinant RNase Inhibitor, 2 μL of 10 mM dNTP mix and 1 μL of Reverse transcriptase M-MLV were added, the mixture was mixed, and then subjected to 42°C metal bath for 60 min, followed by 80°C metal bath for 10 min, to obtain the cDNA.

[0065] The obtained cDNA was diluted 5 times with RNase-free water and used as sample DNA. The Gapdh gene was used as an internal reference gene, and real-time fluorescent quantitative PCR amplification reaction was performed on an ABI PRISM 7500 real-time PCR instrument using the q-PCR primers shown in Table 1, wherein the amplification reaction system (20 μL) included 5 μL of sample DNA, 4 μL of RNase-free water, 10 μL of UltraSYBR Mixture, and 0.5 μL of upstream primer solution (the final concentration of the primer was 0.2 μM) and 0.5 μL of downstream primer solution (the final concentration of the primer was 0.2 μM); the amplification reaction program was as follows: 95°C pre-denaturation for 10 min, 95°C for 15 s, 60°C for 1 min, and cycling for 40 times.

[0066] Table 1 q-PCR primer table

[0067]

[0068]

[0069] After the amplification, the appropriate threshold line (0.2075-0.3497) was adjusted to obtain the CT value corresponding to each sample. Three repeated groups were set for each test, and the geometric mean value was taken for data analysis. The relative mRNA expression was calculated by the 2 -ΔΔCt The results are shown in Table 2 and Figure 3

[0070] Table 2: Average CT value and mRNA expression of each sample

[0071]

[0072] (2) Polymerase Chain Reaction (PCR)

[0073] The 8505C cells were treated with 5 μM, 10 μM and 20 μM DATS and the control solvent (DMSO) for 24 h. After the cells were lysed and the RNA was separated by adding 1 mL TRIzol to each culture dish and transferred to an RNase-free 1.5 mL EP tube, 1 / 5 Trizol volume of chloroform was added after standing for 5 min. After mixing thoroughly, the mixture was stood for 5 min, centrifuged at 10,000 rpm for 15 min at 4°C, and the RNA-containing aqueous phase was aspirated and an equal volume of isopropanol was added. After mixing thoroughly and standing for 5 min at 4°C, the mixture was centrifuged at 12,000 rpm for 15 min, the supernatant was discarded, 1 ml of 75% anhydrous ethanol was added, and the mixture was gently shaken and centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was discarded, and the RNA was dried and precipitated at room temperature (the EP tube was inverted on filter paper). Then, 30 μL of DEPC water was added to the EP tube to obtain total RNA.

[0074] 1 μL of Oligo(dT), 2 μg of RNA, and DEPC water were added to a PCR tube to a total volume of 12 μL. After 5 min of water bath at 65°C and standing on ice, 4 μL of 5×Reaction Buffer, 1 μL of Recombinant RNase Inhibitor, and 2 μL of 10 mM dNTPmix were added. After mixing, the mixture was incubated at 42°C in a metal bath for 60 min and at 80°C in a metal bath for 10 min to obtain cDNA.

[0075] ​PCR (polymerase chain reaction) detection was performed using the PCR primers shown in Table 3, with the Human Actin gene as the internal reference gene, wherein the PCR system (50 μL) included 36.5 μL sterile water, 5 μL 10 x PCR amplification buffer (10 x PCR Buffer), 4 μL dNTP (2.5 mM each), 0.5 μL Taq enzyme, 2 μL cDNA, 1 μL upstream primer solution (20 mM), and 1 μL downstream primer solution (20 mM); the PCR amplification conditions were as follows: denaturation at 95 °C for 5 min; in the cycling process, denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, for 35 cycles, with the annealing temperature of the internal reference gene being 60 °C and the number of cycles being 30 cycles; and finally extension at 72 °C for 5 min.

[0076] Table 3 PCR primer table

[0077]

[0078] The PCR amplification product was stored at 4 °C and subjected to agarose gel electrophoresis analysis in a timely manner. Agarose gel DNA electrophoresis: 18 μL of the PCR product was mixed with 2 μL of DNA loading buffer (purchased from Yeasen) and subjected to electrophoresis in a 1.5% (w / v) agarose gel pre-added with EB nucleic acid dye. The agarose gel after electrophoresis was observed and photographed under ultraviolet projection in a Tian Neng gel imaging system. The results of the agarose gel electrophoresis analysis are shown in Figure 4

[0079] As can be seen from Table 2, Figure 3 and Figure 4 It can be seen that the mRNA expression level of integrin a2b1 in the anaplastic thyroid cancer 8505C cells can be detected using the qPCR primers shown in SEQ ID NO. 1-4 or the PCR primers shown in SEQ ID NO. 5-8, and it can be seen from the detection results that, compared with the control solvent group (SC group), the mRNA expression level of integrin a2b1 in the 8505C cells significantly decreased after treatment with diallyl trisulfide (DATS), indicating that diallyl trisulfide (DATS) can effectively target integrin a2b1 at the transcriptional level.

[0080] Example 4

[0081] In this example, the Western blot analysis was used to verify the inhibitory effect of diallyl trisulfide (DATS) on the expression of integrin a2b1 protein in anaplastic thyroid cancer:

[0082] ​(1) After 8505C cells were treated with 5 μM, 10 μM and 20 μM of DATS and control solvent (DMSO) for 24 h, the culture medium was discarded, 1 mL of pre-cooled PBS at 4 ℃ was added to the culture dish, and the cells were scraped off the dish with a cell scraper to detach the cells from the dish. The PBS containing the cells was transferred to a 1.5 mL EP tube, centrifuged at 8,000 rpm for 5 min at 4 ℃ to obtain cell precipitate, the supernatant was discarded and 50 μL of cell lysis buffer (50 mM Tris-HCl pH8.0; 150 mM NaCl; 1% Triton X-100) containing 1% protease inhibitor (phenylmethylsulfonyl fluoride PMSF) and 1% phosphatase inhibitor (C4H4Na2O6·2H2O) was added, mixed well, and then repeated freezing and thawing for 3 times. The mixture was centrifuged at 12,000 rpm for 15 min at 4 ℃, and the supernatant was transferred to a PCR tube for storage. The whole protein solution of each cell line was obtained.

[0083] (2) The protein concentration of each protein solution was determined by Coomassie Brilliant Blue method, and then each protein solution containing an equal amount of protein was mixed with 1 / 5 sample volume of 6x loading buffer (30 mM ethylenediaminetetraacetic acid EDTA; 36% glycerol; 0.05% xylene cyanol FF; 0.05% bromophenol blue) and loaded into a PCR tube. After heating at 95 ℃ metal bath for 5 min to fully denature and stretch the protein, it was loaded, SDS-PAGE gel electrophoresis, wet transfer, blocking in TBST buffer containing 5% skim milk at room temperature for 1 h, incubating the corresponding primary antibody (anti-integrin α2, source: Biyun; anti-integrin β1, source: Biyun; anti-GAPDH, source: Shenguo) and shaking overnight at 4 ℃; washing the strip with appropriate amount of TBST buffer for 3 times, 10 min each time, incubating the corresponding secondary antibody (goat anti-mouse IgG, source: Biyun; rabbit IgG-HRP, source: Biyun) at room temperature for 1 h. ECL method chemiluminescence, ChemiDocXRS+ system of Bole was used for photosensitive development, and the results are shown in Figure 5 A. The corresponding pictures were collected and analyzed by Image J. The relative expression of integrin in the sample was obtained by comparing the gray value of integrin protein band with that of corresponding GAPDH band, and the results are shown in Figure 5 B.

[0084] As shown in Figure 5 A and Figure 5 B, the protein level of integrin α2β1 in thyroid cell lines can be detected by antibody. After treatment with diallyl trisulfide (DATS), the protein level of integrin α2β1 in 8505C cells significantly decreased compared with the control solvent group (SC group).

[0085] Example 5

[0086] This example is used to verify the inhibitory effect of diallyl trisulfide (DATS) on the membrane expression (Mem) and cytoplasmic expression (Cyto) of integrin α2β1 in anaplastic thyroid cancer cells:

[0087] (1) After 8505C cells were treated with 20 μM DATS for 24 h, the cell membrane protein and cytoplasmic protein of 8505C cells were extracted and separated by using a cell membrane protein and cytoplasmic protein extraction kit (manufacturer: Biyun Tian, model: P0033), and the operation was performed according to the instructions thereof. The membrane protein and cytoplasmic protein of each cell were obtained, respectively. At the same time, an equal amount of control solvent (DMSO) was used to replace DATS, and the above operation was repeated as a solvent control group. (2) The integrin α2β1 protein expression in each cell membrane protein and cytoplasmic protein was detected according to the method of operation (2) in Example 4, and the results are shown in Figure 6 A and Figure 6 B.

[0088] As can be seen from Figure 6 A and Figure 6 B, compared with the control solvent group (SC group), diallyl trisulfide (DATS) can inhibit the membrane expression of integrin α2β1 protein in 8505C cells.

[0089] Example 6

[0090] This example is used to verify the inhibitory effect of diallyl trisulfide (DATS) on the migration / invasion ability of anaplastic thyroid cancer cells:

[0091] (1) Transwell invasion experiment:

[0092] Matrigel matrix glue was diluted with serum-free medium (MEM medium) according to a volume ratio of 1:40 to obtain a Matrigel diluent; 100 μL of the above Matrigel diluent was added to the upper chamber of the transwell, and coated in a 37°C incubator for 1 h. After coating, the culture medium separated in the upper chamber of the transwell was discarded; 500 μL of MEM medium containing 10% (v / v) newborn calf serum was added to the lower chamber, and was ready for use;

[0093] 8505C cells were treated with 5 μM, 10 μM and 20 μM DATS and control solvent (DMSO) for 24 h, and each treated cell was added to the upper chamber of the transwell obtained in operation (1), and the amount added to each well was about 1 x 10 5Cells were added to MEM medium until the final medium volume in each well was 200 μL, with a solvent control group included. After addition, the cells were incubated at 37°C for 24 h. The medium in the upper chamber was then discarded, and the chambers were carefully removed. The matrix gel and uninvaded cells at the bottom of the chambers were carefully removed with a cotton swab, and the cells were rinsed three times with PBS. The cells at the bottom of the chambers were fixed with methanol for 15 min, the methanol was discarded, and the cells were allowed to air dry. They were then stained with 0.1% crystal violet for 30 min; deep purple cells were identified as invading cells. The cells were observed and photographed under an inverted microscope. The results are shown below. Figure 7 As shown in Figure A; the number of invasive cells was counted and the relative migration rate was calculated using ImageJ software, and the results are as follows. Figure 7 As shown in C.

[0094] (2) Transwell migration experiment: The Transwell invasion experiment in procedure (1) was performed, except that the transwell chambers were not coated with Matrigel, and the migration time of the cells in the 37°C incubator was 2 hours. The remaining procedures were the same as the invasion experiment. The results are as follows: Figure 7 As shown in B and 7D.

[0095] (3) Experimental Results

[0096] Experimental results are as follows Figure 7 As shown, by Figure 7 It can be seen that, compared with the control solvent group (SC group), the number of 8505C cells that passed through the chamber was significantly reduced after treatment with the drug DATS, that is, the migration and invasion ability of 8505C cells was weakened, indicating that DATS can effectively inhibit the migration / invasion ability of thyroid undifferentiated cancer cells 8505C.

[0097] Example 7

[0098] This example is used to verify the differential expression of integrin β1 in various thyroid tissues:

[0099] Normal thyroid tissue, papillary thyroid carcinoma tissue, and iodine-resistant thyroid cancer tissue were collected and processed as follows:

[0100] After the tissue is fixed with paraffin, paraffin sections are prepared, the sections are pasted on glass slides, dried and baked in a 65℃ oven, and then quickly put into the deparaffinization solution I (manufacturer: Wuxi Jiangyuan Industrial and Trade Co., Ltd.; model 211129) for more than 5 minutes, and then put into the deparaffinization solution II (manufacturer: Wuxi Jiangyuan Industrial and Trade Co., Ltd.; model 211129) for 5 minutes. After deparaffinization, the sections are sequentially put into anhydrous ethanol, 95% ethanol and 80% ethanol for 3 minutes each for rehydration. After rehydration, the tissue sections are washed under running water for 1 minute, and then soaked in citric acid antigen retrieval solution and boiled for 3 times. After natural cooling, the sections are washed under running water and rinsed with distilled water, and then the outline of the tissue on the glass slide is drawn with a histological pen. Then, 1% BSA is added and incubated for 1 hour. After blocking, the primary antibody (anti-integrin β1, source: Biyun Tian) is added and incubated overnight at 4℃. The next day, the corresponding secondary antibody (rabbit IgG-HRP, source: Biyun Tian) is added and incubated at room temperature for 1 hour. After incubation, the sections are washed with PBS for 3 times, and then colored with DAB staining solution until brown or brownish. The color development is stopped by washing with running water, and then the sections are rinsed with distilled water. Then, the sections are re-stained with hematoxylin and washed with running water to stop the staining. Then, the sections are dehydrated by soaking in 95% ethanol, anhydrous ethanol 1 and anhydrous ethanol 2 for 3 minutes each, and then soaked in transparency solution (manufacturer: Wuxi Jiangyuan Industrial and Trade Co., Ltd.; model 211129) I to IV (I and IV for 3 minutes each, and II and III for 5 minutes each) for transparency. The sections are mounted and photographed for analysis.

[0101] The experimental results are shown in Figure 8 As can be seen from Figure 8 It can be seen that the immunohistochemical staining method can directly show the expression level of the protein in normal thyroid, thyroid papillary carcinoma and iodine-resistant thyroid cancer tissues and the staining results. After comparison, the expression level of integrin β1 in different tissues has obvious differences. The expression level of integrin β1 in normal thyroid tissue is very low, while there is obvious high expression of integrin β1 in thyroid cancer and iodine-resistant thyroid cancer, and the expression level of integrin β1 in the most malignant iodine-resistant tissue is higher. This indicates that integrin β1 can not only indicate the occurrence of thyroid cancer, but also its expression level is positively correlated with the malignant degree of the tumor and the iodine resistance level of undifferentiated cancer. Therefore, the expression level of integrin β1 can be detected to screen the poor prognosis of thyroid cancer.

[0102] Example 8

[0103] This example is used to verify the correlation between the expression levels of integrin β1 and sodium-iodine transporter (NIS) in thyroid cancer tissues:

[0104] Take thyroid cancer tissue (19 cases), according to the method of Example 7, the expression level of integrin β1 and the expression level of sodium iodine transporter (NIS) in it are detected, wherein the first antibody used for detecting the expression level of integrin β1 is (anti-integrin β1, source: Biyun Tian), and the first antibody used for detecting the expression level of sodium iodine transporter (NIS) is (anti-NIS, source: Proteintech).

[0105] The experimental results are shown in Figs. Figure 9 A and 9B, and Figure 9 It can be seen that the expression level of integrin β1 of the detected 19 cases of thyroid cancer tissue is high, which is strong expression according to immunohistochemical score, and the expression level of sodium iodine transporter (NIS) is low, which is moderate expression according to immunohistochemical score, and the expression level of NIS protein is high, indicating that the iodine transport capacity of the tissue cells is strong, that is, the iodine uptake capacity is strong. Therefore, Figure 9 The results show that the NIS expression of thyroid cancer tissue with high expression of integrin β1 is low, which indicates that the high expression of integrin β1 is positively correlated with the iodine resistance treatment of thyroid cancer, and the expression of integrin β1 can provide a reference for whether the thyroid cancer patient receives radioiodine treatment.

[0106] Example 9

[0107] According to the method of Example 6, the migration / invasion ability of normal thyroid epithelial cell line Nthy-ori-3.1 and thyroid undifferentiated cancer cell lines 8505C, C643, FRO and CAL-62 involved in Example 2 are detected, and the detection results are shown in Figs. Figure 10 .

[0108] It can be seen from Figure 2 and Figure 10 that the expression level of integrin α2 in thyroid cells is basically consistent, the migration and invasion ability of normal thyroid epithelial cells Nthy-ori-3.1 with low expression of integrin α2 is poor, while the migration and invasion ability of four thyroid undifferentiated cancer cells with high expression of integrin α2 is strong, and the strength of the migration and invasion ability of the cells is basically consistent with the expression level of integrin α2 in Figure 2 .

[0109] Example 10

[0110] Take 8505C cells as a model to carry out cell iodine uptake experiment.

[0111] 2×10 6Cells were seeded in 12-well plates and stimulated with 20 mU / mL rTSH before being divided into a control group and a drug-treated group. The drug-treated group was treated with 20 μmol / L DATS and washed three times with PBS. Both the control and drug-treated groups were then further divided into subgroups. For the same group of cells, one subgroup was pretreated with NaClO4 (300 μmol / L), a competitive inhibitor of the NIS channel, for 30 min before radioactive iodine incubation. After pretreatment, both subgroups were treated with 1 mL of NaClO4 containing 1 μCi NaClO4. 131 Incubate cells with PBS solution of 10 μmol / L non-radioactive NaI at 37°C for 1 h, then wash three times with pre-cooled PBS. The above culture process is repeated once in parallel for cell counting: trypsin digestion and hemocytometer counting. Cells in all four subgroups are lysed with 1 mL of 0.33 mol / L NaOH solution, and cell lysates are collected. Radioactivity counts per minute (CPM) are measured using a gamma counter, and the results are expressed as CPM / 10-1. 6 Cell is a unit of measurement for iodine uptake by cells.

[0112] Experimental results are as follows Figure 11 As shown, by Figure 11 It can be seen that 8505C cells with high expression of integrin α2β1 have poor basal iodine uptake capacity, and the NIS channel competitive inhibitor NaClO4 has no significant effect on their iodine uptake capacity, indicating that the low NIS expression level of 8505C cells leads to their poor iodine uptake capacity (control group). After DATS treatment, the iodine uptake capacity of 8505C cells is enhanced, and NaClO4 can inhibit their iodine uptake capacity, indicating that the NIS expression level in 8505C cells is upregulated after DATS treatment, thereby enhancing iodine uptake capacity (drug-treated group). Combining the results in Examples 3 and 4, it shows that DATS can downregulate the expression of integrin α2β1 in 8505C cells. Therefore, combining Examples 3, 4, and 10, it is shown that DATS can inhibit the expression of integrin α2β1, thereby upregulating the expression level of NIS protein and improving the iodine uptake capacity of undifferentiated thyroid cancer cells.

[0113] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of an integrin α2β1 inhibitor in the preparation of a medicament for increasing the sensitivity of anaplastic thyroid cancer to iodine treatment. The integrin α2β1 inhibitor includes a diallyl trisulfide.

2. Use according to claim 1, characterized in that, The medicament is a medicament for enhancing the iodine uptake ability of anaplastic thyroid cancer cells.

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