Application of iodine-containing composition in preparation of thyroid cancer treatment product

Abstract

The invention relates to the technical field of preparations for treating thyroid cancer patients, in particular to application of an iodine-containing composition in preparation of products for treating thyroid cancer. At present, a specific guidance scheme about the optimal iodine intake does not exist for patients with differentiated thyroid cancer (DTC), especially patients with structural persistent lesions after surgery and / or radioiodine treatment. In China, although definite evidences are lacked, a large number of patients still select lifelong low-iodine diets and try to avoid iodine-induced relapse. The application of the invention overcomes prejudice and proves that low-iodine diet can promote biochemical progress of thyroid cancer on the contrary, and iodine-suitable diet can better help patients with differentiated thyroid cancer to recover, inhibit RYR1 gene expression and weaken tumor angiogenesis and cell migration ability, thereby inhibiting tumor progress.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of thyroid cancer patient treatment preparations, and particularly relates to application of an iodine-containing composition in preparation of a product for treating thyroid cancer. BACKGROUND

[0002] Iodine is an indispensable element for the synthesis of thyroid hormones and plays an important physiological role in maintaining normal thyroid function. In the 19th century, iodine deficiency was first confirmed as the cause of endemic goiter and cretinism, and iodized salt has been widely used to prevent iodine deficiency diseases. The World Health Organization (WHO) recommends that the daily iodine intake of adults should be controlled at 150-300 micrograms to maintain normal thyroid function.

[0003] In addition, the association between abnormal iodine intake and thyroid cancer has also been extensively studied, but the conclusions are controversial. Some researchers have found that since the implementation of the universal salt iodization policy, the incidence of thyroid cancer (especially papillary thyroid cancer, PTC) has shown an upward trend.

[0004] Currently, there is no clear recommendation for the optimal iodine intake for thyroid cancer patients (especially those who have undergone total thyroidectomy and radioactive iodine therapy but still have structural persistent lesions). However, we have observed in the process of clinical follow-up that many thyroid cancer patients (especially those with local persistent lesions or distant metastasis) often choose to have a lifelong low-iodine diet due to concerns about the "iodine-induced thyroid cancer recurrence" risk reported in previous studies. The widespread avoidance of iodine by thyroid cancer patients raises new issues, such as the risk of insufficient iodine intake for their family members and the long-term impact on the patients' own lives. Despite numerous previous studies, the exact impact of low-iodine diet on thyroid cancer is still unclear, and in-depth mechanism research and prospective studies are urgently needed. SUMMARY

[0005] The purpose of the present application is to provide the application of the iodine-containing composition in the preparation of the product for treating thyroid cancer. The technical problems to be solved are not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0006] In the first aspect, the present application provides the application of the above-mentioned iodine-containing composition in any one of the following A1) to A7): A1) preparing a product for inhibiting the volume or weight of thyroid cancer tumor in a subject; A2) preparing a product for inhibiting the growth rate of thyroid cancer tumor in a subject; A3) preparing a product for reducing the necrotic area of thyroid cancer tumor in a subject; A4) preparing a product for reducing the volume or cross-sectional area of thyroid follicle in a subject; A5) preparing a product that reduces the number, density or activity of blood vessels or microvessels in a tumor in a subject; A6) preparing a product that inhibits the expression of RYR1 gene in a subject; A7) preparing a product that treats or adjuvant treats thyroid cancer.

[0007] In one embodiment, the iodine-containing composition is a solid, a solution or a suspension.

[0008] In another embodiment, the iodine-containing composition is a capsule, a tablet, a powder, a dispersion, a solution or a suspension.

[0009] In one embodiment, the iodine-containing composition further comprises a protein, a carbohydrate or a lipid. In particular, the iodine-containing composition comprises an iodine-containing edible solid and an iodine-containing drinkable liquid; the iodine concentration in the iodine-containing edible solid is 50 μg / kg. The iodine concentration in the iodine-containing drinkable liquid is between 0 and 3000 μg / L, and the iodine concentration in the drinkable liquid is not 0.

[0010] In another embodiment, the iodine-containing composition is for administration via a gastrointestinal route; the iodine-containing composition further comprises an acceptable carrier, diluent or adjuvant.

[0011] In one embodiment, the adjuvant treatment is inhibiting the progression of thyroid cancer; in particular, the progression of thyroid cancer is induced by a low-iodine diet.

[0012] In another embodiment, the thyroid cancer is differentiated thyroid cancer.

[0013] In one embodiment, the thyroid cancer is papillary thyroid cancer or / and follicular thyroid cancer.

[0014] In a particular embodiment, the thyroid cancer is a structurally persistent differentiated thyroid cancer; in particular, the thyroid cancer is a structurally persistent differentiated thyroid cancer after total thyroidectomy or radioiodine therapy.

[0015] In one embodiment, the subject is a mammal. In particular, the mammal is a rodent, a primate or a human.

[0016] In a second aspect, the present application provides a product for treating or adjuvant treating thyroid cancer, including but not limited to: the above-mentioned iodine-containing composition and pharmaceutically acceptable adjuvants.

[0017] In one embodiment, the adjuvant treatment is inhibiting the progression of thyroid cancer; in particular, the progression of thyroid cancer is induced by a low-iodine diet.

[0018] In a third aspect, the present application provides a method for constructing an animal model for promoting thyroid cancer progression, transplanting thyroid cancer cells or thyroid cancer tissue into an immunodeficient animal, and administering a low-iodine content composition; the iodine content of the low-iodine content composition is lower than that of the above-mentioned iodine-containing composition.

[0019] In one embodiment, the iodine content of the low-iodine content composition is ≤ 50 μg / kg.

[0020] In another embodiment, the low-iodine content composition is administered for at least 7 days.

[0021] In a fourth aspect, the present application provides the use of a product for inhibiting the expression of the RYR1 gene in any one of B1) to B3) below: B1) preparing a product for inhibiting the migration ability of thyroid cancer cells; B2) preparing a product for inhibiting the invasion ability of thyroid cancer cells; B3) preparing a product for treating or adjuvant treating thyroid cancer.

[0022] In a fifth aspect, the present application provides the use of a substance for detecting the expression of the RYR1 gene in any one of C1) to C2) below: C1) preparing a product for evaluating whether a subject with thyroid cancer is at risk of progression C2) preparing a product for evaluating whether a subject with thyroid cancer is in a low-iodine diet state.

[0023] In a sixth aspect, the present application provides a method for adjuvant treating thyroid cancer, continuously administering an effective amount of the above-mentioned iodine-containing composition, or the above-mentioned product for treating or adjuvant treating thyroid cancer, or the above-mentioned product for inhibiting the expression of the RYR1 gene, to inhibit the progression of thyroid cancer.

[0024] Compared with the prior art, the present application has the following beneficial effects: The present study explores the effects of a low-iodine diet on biomarkers in patients with structural persistent lesions differentiated thyroid cancer, and further reveals the potential mechanisms through preclinical studies, aiming to provide a reference for the development of iodine intake regimens for thyroid cancer patients. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A schematic diagram of the experimental design for Example 1.

[0026] Figure 2This study aimed to monitor the dynamic growth of tumors in mice fed a low-iodine (DI) diet and an adequate-iodine (AI) diet. Figure A shows a comparison of the average tumor volume of the two groups of nude mice at each time point; Figure B shows the tumor volume growth curves of the two groups of nude mice; Figure C shows a comparison of the average maximum tumor volume measured by ultrasound of the two groups of nude mice; Figure D shows a comparison of the tumor weight of the two groups of nude mice; Figure E shows a comparison of the tumor necrosis rate of the two groups of nude mice; and Figure F shows the in situ tumors in the DI and AI groups.

[0027] Figure 3 This study compares tumor angiogenesis in mice fed a low-iodine (DI) diet and an adequate-iodine (AI) diet based on multimodal imaging; where A represents thyroid cancer-bearing mice. 68 A) Representative images from Ga-NOTA-3PRGD2 PET imaging, with red arrows indicating subcutaneous tumors; B) Representative images from ultrasound imaging, with grayscale images in the left column and monochrome ultramicrovascular imaging (mSMI) images in the right column, with red arrows indicating intratumoral vessels; C) Semi-quantitative analysis comparison of the ratio of the highest SUVmax of the tumor to the SUVmax of the liver in the DI and AI groups; D) Semi-quantitative analysis comparison of the ratio of the SUVmax of the tumor at the 40% volume of interest (VOI) threshold to the SUVmax of the liver in the DI and AI groups; E) Comparison of Adler grading of intratumoral blood flow assessed by ultrasound mSMI in the DI and AI groups; F) Representative images from tumor histopathology; G) Quantitative analysis comparison of the mean tumor vascular density in the DI and AI groups.

[0028] Figure 4 This study presents histopathological analysis of thyroid tissue in mice fed a low-iodine (DI) diet and an adequate-iodine (AI) diet. In the image, A shows representative histopathological images of thyroid tissue from the two groups of nude mice, and B shows a comparison of the cross-sectional area of ​​thyroid follicles between the two groups of nude mice.

[0029] Figure 5 Transcriptomic analysis of xenografted thyroid tumors in mice fed a low-iodine diet (DI) and an adequate-iodine diet (AI) was performed. In this study, A is a bubble chart showing the top 10 Kyoto Gene and Genome Encyclopedia (KEGG) pathways involved in the comparison between the two groups; B is a volcano plot of differentially expressed genes (DEGs) (fold change > 2), with red dots representing significantly upregulated genes, blue dots representing significantly downregulated genes, and gray dots representing genes with no significant difference; C and D are the genes upregulated in the DI group relative to the AI ​​group, obtained from RNA sequencing analysis; E and F are box plots showing the expression distribution of upregulated genes in the DI group in thyroid cancer tissues and paired normal thyroid tissues from the Cancer Genome Atlas (TCGA) database obtained through gene expression profile interaction analysis (GEPIA). The database contained 512 tumor tissue samples and 337 normal tissue samples.

[0030] Figure 6Effects of RYR1 gene knockdown on the proliferation and migration abilities of thyroid cancer cells; wherein A is the CCK-8 experiment to evaluate the effect of RYR1 gene knockdown on the proliferation ability of TPC-1 cells; B is the comparison of the migration rate of the RYR1 gene knockdown group and the negative control group (NC group) at different time points in the scratch healing experiment; C is the comparison of the number of migrated cells between the RYR1 gene knockdown group and the NC group in the Transwell experiment; D is the scratch healing experiment to evaluate the effect of RYR1 gene knockdown on the wound healing ability of TPC-1 cells; E is the Transwell experiment to evaluate the effect of RYR1 gene knockdown on the migration ability of TPC-1 cells. DETAILED DESCRIPTION

[0031] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0032] In the following examples, the experimental methods are conventional methods, unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0033] The human thyroid cancer cell lines B-CPAP and TPC-1 used in the following examples were purchased from the American Type Culture Collection (ATCC). B-CPAP cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS), and the culture conditions were 37°C, 5% carbon dioxide, and subcultured twice a week. After the cells grew to the logarithmic growth phase, they were collected by trypsin digestion.

[0034] All animal experiments in the following examples were strictly in accordance with the experimental protocol and approved by the Institutional Animal Care and Use Committee (IACUC) of the Experimental Animal Research Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (IBMS) (Approval No.: XHDW-2023-102). The 12 BALB / c nude mice used in the experiments were purchased from HFK Animal Center, Beijing Huafukang Biotechnology Co., Ltd.

[0035] Example 1: Iodine diet treatment experiment The experimental design is shown in Figure 1 .

[0036] All nude mice were initially fed with feed containing 50 μg / kg of iodine (purchased from Ready Dietech Company). B-CPAP cells (5 x 106 One cell was dissolved in 100 μL of phosphate buffer. Tumor formation could be observed 2-5 days after inoculation, and the tumor volume gradually increased over time. Tumors were successfully formed in all 12 nude mice after inoculation with B-CPAP cells.

[0037] On the 5th day after inoculation, 12 nude mice were randomly divided into a low-iodine diet group (DI group) and an iodine-appropriate diet group (AI group), with 6 mice in each group. Different iodine intervention treatments were started in each group. The nude mice in the DI group drank purified water, while the nude mice in the AI ​​group drank drinking water with an iodine content of 350 μg / L [prepared by dissolving potassium iodide (KI, purchased from Solarbio) in purified water].

[0038] Continuous iodine intervention for 11 days; during the 16-day observation period, all experimental nude mice survived, and the tumor inoculation success rate was 100%. Tumor volume changes in thyroid cancer-bearing mice in the DI and AI groups were dynamically monitored at different time points using calipers. Tumor volume (mm²) was recorded. 3 According to the formula "Volume = (Length × Width)", 2 ÷ 2” is used for calculation.

[0039] like Figure 2 A and Figure 2 As shown in Figure B, there were significant differences in tumor volume changes between the DI and AI groups of thyroid cancer-bearing mice. In the later stages of the experiment (from day 12 after inoculation / day 7 after iodine intervention), the tumor volume of nude mice in the DI group was significantly larger than that in the AI ​​group (all P < 0.050). Figure 2 As shown in Figure A). The tumor growth curves also show that from day 12, the tumor growth rate in the DI group was significantly faster than that in the AI ​​group, and the difference in tumor growth rate between the two groups gradually increased over time (e.g., ...). Figure 2 As shown in B).

[0040] Then, the nude mice were subjected to... 68 Ga-NOTA-3PRGD2 positron emission tomography (PET) and ultrasound (US) examinations were performed. Nude mice were then euthanized, and subcutaneous tumor and thyroid tissues were collected. After measuring tumor weight, paraffin sections of the tumor and thyroid tissues were prepared for histopathological analysis; simultaneously, a portion of the tumor tissue was collected for RNA sequencing.

[0041] At the end of the experiment, ultrasound examination showed that the largest tumor volume in the DI group was significantly larger than that in the AI ​​group (P=0.031); at the same time, the tumor weight in the DI group was also significantly higher than that in the AI ​​group (P=0.012). Figure 2 C and Figure 2 (As shown in D). Furthermore, histopathological analysis showed that the proportion of necrotic areas in the tumors of the DI group was significantly higher than that of the AI ​​group (P=0.049) (as shown in D). Figure 2 E is shown.

[0042] Pathological sections of thyroid tissue from nude mice in each group showed that, compared with the AI ​​group, the thyroid follicle volume of nude mice in the DI group was increased (e.g., Figure 4 As shown in Figure A), this is consistent with previous findings. Statistical analysis of thyroid tissue showed that the cross-sectional area of ​​thyroid follicles in the DI group was significantly larger than that in the AI ​​group (0.702 vs. 0.288, P=0.0005) (as shown in Figure A). Figure 4 (As shown in B), suggesting that a low-iodine diet can induce goiter formation.

[0043] Example 2: 68 Ga-NOTA-3PRGD2 Small Animal PET Imaging The aforementioned positron emission tomography (PET) technique targeting tumors uses a small molecule antagonist peptide containing the arginine-glycine-aspartic acid (RGD) sequence as an imaging agent to reflect tumor angiogenesis. Nude mice were scanned using a small animal PET scanner (Siemens, Germany) on day 16 post-inoculation. The NOTA-3PRGD2 precursor was provided by the Institute of Medical Isotopes, Peking University. 68 Ga-NOTA-3PRGD2 was synthesized on-site at the experimental site.

[0044] Each nude mouse was injected with 3.7 MBq (100 μCi) via the tail vein. 68 After 30 minutes of Ga-NOTA-3PRGD2 scanning, nude mice were anesthetized with 2.5% isoflurane and fixed prone on a scanning table for scanning. Static scanning parameters were set as follows: acquisition energy window 350-650 keV, slice thickness 0.78 mm, matrix 128×128, and scanning time 10 minutes. The 3D ordered subset maximum expectation algorithm (OSEM 3D) was used for iterative reconstruction of the scanned images. After two iterations, the region of interest (ROI) was delineated in the image processing system.

[0045] A semi-quantitative analysis method was used to assess tumor metabolic activity. The maximum standardized uptake value (SUVmax) was used as the indicator. The tumor (T) was set as the target area and the liver (L) was set as the background area. SUVmax was recorded and the tumor / liver (T / L) ratio was calculated.

[0046] SUVmax is calculated using two methods: 1) Manually delineate a small region of 1 cm³ within the region of interest and take the average value of the highest RGD uptake within that region; 2) Use commercial software to semi-automatically determine the volume of interest (VOI) of the tumor with 40% of SUVmax as the threshold.

[0047] Example 3: Ultrasound Microvascular Imaging (SMI) Tumor microvascular ultrasound assessment was performed on all nude mice by an ultrasound surgeon with over 5 years of experience in thyroid ultrasound operation, using an Aplio 500 ultrasound system (Canon Medical Systems, Japan) equipped with a 5-14MHz linear array probe. The nude mice were kept in a lateral decubitus position under mild isoflurane anesthesia to fully expose the tumor site.

[0048] First, grayscale ultrasound was performed to observe the size and composition of the tumor. Then, microvascular imaging (mSMI) was used to assess the tumor's microvascular characteristics. During the procedure, the pressure of the probe on the tumor was minimized to avoid vascular collapse. Due to the high sensitivity of monochrome microvascular imaging (mSMI), this application uses it as the primary reference standard to preserve the image with the richest microvascular blood flow signal in the tumor.

[0049] The Adler blood flow grading method was used to classify tumor blood flow in mSMI images. The specific criteria are as follows: Grade 0 (no blood flow); Grade I (1-2 short strip or rod-shaped blood flows); Grade II (3-4 short strip or rod-shaped blood flows, or 1 clear long strip blood flow); Grade III (more than 5 short strip blood flows, or more than 2 long strip blood flows or reticular blood flows).

[0050] The applicant passed 68 Ga-NOTA-3PRGD2 small animal PET imaging and ultrasound microvascular imaging (SMI) were used to assess intratumoral vascular density and activity (e.g., Figure 3 A and Figure 3 (as shown in B) to evaluate the effect of a low-iodine diet on tumor angiogenesis.

[0051] right 68 Semi-quantitative analysis of Ga-NOTA-3PRGD2 PET images showed that, whether calculated using the highest tumor uptake (1.584 vs. 1.283, P=0.013) or using the 40% isoprofile threshold analysis (1.576 vs. 1.228, P=0.009), tumor uptake in the DI group was significantly higher than that in the AI ​​group (e.g., ...). Figure 3 C and Figure 3 (as shown in D).

[0052] Monochromatic ultramicrovascular imaging (mSMI) results combined with Adler grading assessment showed that the DI group had a greater number of tumor microvessels and a significantly higher Adler grade than the AI ​​group (P=0.015). Figure 3 B and Figure 3 As shown in E), this indicates that the blood flow within the tumor is richer in the DI group.

[0053] Meanwhile, quantitative analysis of histopathological images showed that the tumor vascular density in the DI group was significantly higher than that in the AI ​​group (P=0.005). Figure 3 F and Figure 3As shown in G).

[0054] The results of the above multimodal studies, including PET imaging, ultrasound SMI imaging, and histopathological analysis, consistently show that tumor angiogenesis was significantly greater in the DI group than in the AI ​​group.

[0055] Example 4: RNA extraction and RNA sequencing library construction Total RNA was extracted using the standard Trizol method (catalog number 15596026, Ingenie Biosciences, Carlsbad, USA). RNA libraries were constructed from 12 independent biological replicates using the NEBNext Small RNA Library Preparation Kit (for Illumina platform, catalog number E7420, New England Biological Laboratory, Ipswich, USA) according to the kit instructions, and sequenced using an Illumina HiSeq 2500 sequencer (Illumina, San Diego, USA).

[0056] Example 5: Bioinformatics Analysis of RNA Sequencing Data Bubble charts and volcano plots were generated for the RNA sequencing data. The criteria for screening differentially expressed genes (DEGs) were set as follows: Log2 fold change (Log2FC) > 1 and corrected P-value < 0.05. Subsequently, the `enrichKEGG` function in the `clusterProfiler` R package was used to analyze Kyoto Gene and Genome Encyclopedia (KEGG) pathways related to molecular characteristics among different groups.

[0057] Analysis of differentially expressed genes (DEGs) showed significant clustering differences in the transcriptomes of the DI and AI groups. Figure 5 A). Compared to the AI ​​group, the DI group had 16 upregulated genes and 49 downregulated genes (e.g., Figure 5 (As shown in B). The genes upregulated in the DI group were mainly enriched in biological processes related to cancer transcriptional dysregulation (such as genes like HIST1H3G and MAF) and calcium signaling pathways (such as genes like RYR1 and NOS2), which is consistent with the results of the volcano plot analysis (e.g., Figure 5 (As shown in B). Box plot analysis confirmed that, compared with the AI ​​group, the mRNA expression levels of HIST1H3G and RYR1 were significantly upregulated in the DI group (P=0.004; P=0.016) (as shown in B). Figure 5 C and Figure 5 (as shown in D).

[0058] The applicant further validated the expression of these two genes in thyroid cancer patients using a clinical database—Gene Expression Interaction Analysis (GEPIA). The results showed that the RYR1 gene, upregulated under low-iodine dietary conditions, exhibited significantly higher expression levels in thyroid cancer tissues than in paired normal thyroid tissues; while the expression of the HIST1H3G gene showed no statistically significant difference between thyroid cancer and normal thyroid tissues (e.g., Figure 5 E and Figure 5 As shown in F).

[0059] Example 6: Small interfering RNA (siRNA) transfection TPC-1 cells (Shanghai Stem Cell Bank, China) were transfected with siRNA in 12-well plates. The transfection system was prepared as follows: 100 μL of Opti-MEM medium was mixed with 2 μL of RNA transfection reagent (Invigentech), and another 100 μL of Opti-MEM medium was mixed with 2.5 μL of siRNA. The two mixtures were gently mixed and incubated at room temperature for 5 minutes. Finally, the resulting transfection complex was added to TPC-1 cells along with 500 μL of DMEM medium. The siRNA sequence is shown in Table 1.

[0060]

[0061] (1) CCK-8 proliferation assay: TPC-1 cells were cultured at a density of 1.5 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 96-well plates, with each well containing DMEM medium with 15% fetal bovine serum (8 replicates per group). After transfection with siRNA, 10 μL of CCK8 reagent (CCK-8; Beijing Aoqing Biotechnology Co., Ltd., China) was added to each well at 24, 48, and 72 hours. After incubation in an incubator for 1.5 hours, the absorbance (OD450) at 450 nm was measured using a microplate reader (BioTek, Winusch & Co., Ltd., USA) to assess cell proliferation.

[0062] (2) Scratch healing assay: TPC-1 cells were seeded in 12-well plates (3 replicates per group) and transfected with siRNA 24 hours later. 24 hours after transfection, the cells were scratched with a sterile 200 μL pipette tip, rinsed with phosphate-buffered saline (PBS), and then cultured in serum-free medium. The scratched areas were photographed at 0, 24, 48, and 72 hours, and the cell migration area was calculated using ImageJ software.

[0063] The migration rate calculation formula is: Migration rate = [(0-hour scratch area - scratch area at different time points (24 hours / 48 hours / 72 hours)) ÷ 0-hour scratch area] × 100%.

[0064] (3) Transwell migration assay: TPC-1 cells were seeded in 6 cm culture dishes. After 24 hours of transfection, the cells were collected and 2 × 10⁶ cells were transferred to a culture dish. 4 One cell was resuspended in 200 μL of serum-free DMEM medium and added to the upper chamber of each Transwell chamber (catalog number 3422, Costa Biotech, Washington, D.C.); 600 μL of DMEM medium containing 15% fetal bovine serum was added to the lower chamber, and the cells were incubated at 37°C and 5% carbon dioxide for 24 hours.

[0065] After incubation, unmigrated cells in the upper chamber were gently wiped away with cotton swabs. Cells on the lower surface of the lower chamber were fixed with 4% paraformaldehyde (catalog number 1004965000, Sigma-Aldrich, St. Louis, USA) and then stained with crystal violet (catalog number 46364, Sigma-Aldrich, St. Louis, USA). Cell images of each group were taken using an inverted microscope, and migrating cell counting analysis was performed.

[0066] To gain a deeper understanding of the biological significance of the RYR1 gene, the applicant achieved RYR1 gene knockdown by transfecting thyroid cancer cells (TPC-1 cells) with si-RNA and conducted functional verification experiments. CCK-8 cell proliferation assays showed no significant difference in absorbance at 450 nm between the si-RYR1 group and the negative control group (NC group) (e.g., Figure 6 As shown in Figure A, this indicates that RYR1 gene knockdown has no significant effect on the proliferation of thyroid cancer cells.

[0067] The scratch healing assay results showed that, compared with the NC group, TPC-1 cells transfected with si-RYR1 exhibited significantly reduced scratch healing ability at 24 hours (P=0.017), 48 hours (P=0.025), and 72 hours (P=0.002). Figure 6 B and Figure 6 (As shown in D). Similarly, Transwell migration assays showed that after RYR1 gene knockdown, the number of TPC-1 cells migrating to the lower chamber of the Transwell within 24 hours was significantly less than that in the NC group (P=0.001). Figure 6 C and Figure 6 E is shown.

[0068] These results collectively suggest that RYR1 may be a key driver of thyroid cancer cell migration, and its expression pattern is closely related to the effect of a low-iodine diet on thyroid cancer progression.

[0069] This application employs an integrated research design, combining animal models and molecular mechanism validation to form a rigorous chain of evidence. Furthermore, the application of multimodal imaging technologies (PET and ultrasound) enhances the reliability and persuasiveness of the research results. This application challenges the prevalent lifelong low-iodine diet practice in patients with structurally persistent differentiated thyroid cancer and provides preliminary evidence for developing rational iodine intake protocols in clinical practice.

[0070] This application addresses the long-standing issue of low iodine intake in patients with structurally persistent differentiated thyroid cancer. Unexpectedly, it was found that, contrary to the expectation that "low iodine intake may inhibit the progression of differentiated thyroid cancer," low iodine intake may actually significantly promote the progression of thyroid cancer. This provides a basis for rational iodine intake in thyroid cancer patients.

[0071] In an animal model of iodine-dependent dietary intervention, tumor-bearing mice in the low-iodine diet group exhibited more aggressive tumor behavior compared to the adequate-iodine diet group: not only did their tumors grow faster and heavier, but they also showed a higher proportion of intratumoral necrosis, suggesting that a low-iodine diet may promote tumor proliferation and hypoxia-induced necrosis. Furthermore, multimodal angiography showed increased tumor angiogenesis in the low-iodine diet group, indicating that a low-iodine diet may activate pro-angiogenic processes. These results consistently confirm that a low-iodine diet has a significant pro-cancer effect on thyroid cancer.

[0072] The potential mechanisms underlying the carcinogenic effects of low-iodine diets are complex and involve multiple factors: First, chronic iodine deficiency leads to elevated thyroid-stimulating hormone (TSH) levels, which in turn stimulates the proliferation of thyroid follicular cells. Enhanced cell proliferation makes cells more susceptible to carcinogenic factors; therefore, for patients with structurally persistent differentiated thyroid cancer (especially well-differentiated types), a low-iodine diet may be a factor stimulating thyroid cancer progression. Second, iodine has antioxidant properties, and iodine deficiency leads to the accumulation of reactive oxygen species (ROS). Elevated ROS levels stabilize hypoxia-inducible factor-1α (HIF-1α), which can bind to the VEGF-A promoter, promoting VEGF gene transcription and angiogenesis.

[0073] This application further investigated RNA sequencing of tumor samples. The results showed that, unlike the HIST1H3G gene, the RYR1 gene was significantly expressed higher in the low-iodine diet group (DI group) than in the adequate-iodine diet group (AI group), and its expression in thyroid cancer tissue was also significantly higher than in normal tissue. Functional experiments further confirmed that knocking down the RYR1 gene significantly inhibited the migration ability of thyroid cancer cells, indicating that this gene may be related to tumor invasiveness. This finding suggests that the RYR1 gene may play a key role in the development and progression of differentiated thyroid cancer, and changes in its mRNA expression level may be associated with the progression of low-iodine diet-induced differentiated thyroid cancer.

[0074] In conclusion, for thyroid cancer patients (especially those with persistent structural lesions), a low-iodine diet or deliberate restriction of iodine intake may promote tumor progression by upregulating RYR1 gene expression, enhancing tumor angiogenesis and cell migration.

[0075] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of an iodine-containing composition in any one of A1) to A7): A1) to make a product that inhibits the volume or weight of a thyroid cancer tumor in a subject; A2) to make a product that inhibits the growth rate of a thyroid cancer tumor in a subject; A3) to make a product that reduces the necrotic area of a thyroid cancer tumor in a subject; A4) to make a product that reduces the volume or cross-sectional area of a thyroid follicle in a subject; A5) to make a product that reduces the number, density, or activity of blood microvessels in a tumor in a subject; A6) to make a product that inhibits the expression of the RYR1 gene in a subject; A7) to make a product that treats or adjuvantly treats a thyroid cancer.

2. Use according to claim 1, characterized in that, The iodine-containing composition is a solid, a solution, or a suspension.

3. Use according to claim 1, characterized in that, The iodine-containing composition comprises an iodine-containing edible solid and an iodine-containing drinkable liquid; The iodine-containing edible solid has an iodine concentration of 50 μg / kg; The iodine-containing drinkable liquid has an iodine concentration of 0 to 3000 μg / L, and the iodine concentration in the drinkable liquid is not 0.

4. Use according to claim 1, characterized in that, The iodine-containing composition is for administration via a gastrointestinal route.

5. The use according to claim 1, characterized in that, The adjuvant treatment is to inhibit the progression of a thyroid cancer.

6. Use according to claim 1, characterized in that, The progression of the thyroid cancer is induced by a low-iodine diet.

7. Use according to claim 1, characterized in that, The thyroid cancer is a differentiated thyroid cancer.

8. A product for the treatment or adjunctive treatment of thyroid cancer, characterised in that, The use of the iodine-containing composition and pharmaceutically acceptable excipients, including but not limited to, any one of claims 1 to 7.

9. A method for constructing an animal model for promoting progression of thyroid cancer, characterized by, A thyroid cancer cell or thyroid cancer tissue is transplanted into an immunodeficient animal, and a low-iodine content composition is administered; the low-iodine content composition has an iodine content of < 50 μg / kg.

10. Use of a product that inhibits the expression of the RYR1 gene in any one of B1) to B3): B1) to make a product that inhibits the migration ability of a thyroid cancer cell; B2) to make a product that inhibits the invasion ability of a thyroid cancer cell; B3) to make a product that treats or adjuvantly treats a thyroid cancer.

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