Application of TEAD1 and CTTNBP2NL as thyroid cancer molecular marker or therapeutic target

Abstract

The invention relates to the technical field of cancer treatment, in particular to application of TEAD1 and CTTNBP2NL as thyroid cancer molecular markers or therapeutic targets. The expression of the CTTNBP2NL is in positive correlation with immune cells in THCA, and the CTTNBP2NL can be used as a biomarker to detect the development and prognosis of tumors. The invention provides new molecular pathways and biomarkers for development, prognosis and treatment of thyroid gland, and provides a brand new entry point for targeted intervention.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer treatment, and particularly to the application of TEAD1 and CTTNBP2NL as molecular markers or therapeutic targets for thyroid cancer. Background Art

[0002] Thyroid cancer (THCA) is an endocrine system malignancy with an increasing incidence in recent decades, which has triggered extensive research on its molecular basis and potential therapeutic targets. Although the prognosis of thyroid cancer is generally good, there is still a subset of patients who exhibit aggressive disease and resistance to conventional therapies; in this regard, new molecular pathways and biomarkers should be identified to improve treatment strategies.

[0003] Apoptosis is crucial for maintaining cell homeostasis and clearing damaged or malignant cells. Dysregulation of the apoptotic pathway is a hallmark of cancer, which enables uncontrolled cell proliferation and tumor growth. Therefore, understanding apoptosis in thyroid cancer cells is essential for developing targeted therapies that can restore the apoptotic mechanism and inhibit tumor progression.

[0004] The Drosophila protein, nausicaa, regulates lamellipodial actin dynamics in a cortactin-dependent manner records that CTTNBP2NL (N-terminal cortactin-binding protein 2-like) has become an important factor in apoptosis of various cancer cells. CTTNBP2NL interacts with components of the cytoskeleton and signal transduction pathways, which may affect cell survival and apoptosis. However, the role of CTTNBP2NL in thyroid cancer has not been studied. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of TEAD1 and CTTNBP2NL as molecular markers or therapeutic targets for thyroid cancer.

[0006] In order to solve the above technical problem, the technical solution adopted by the present invention is: the application of a reagent for detecting CTTNBP2NL in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0007] Another technical solution adopted by the present invention is: the application of a reagent for detecting TEAD1 in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0008] Another technical solution adopted by the present invention is: the application of a reagent for detecting TEAD1 and CTTNBP2NL in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0009] Another technical solution adopted by the present invention is: the application of a reagent for detecting the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a kit for detecting thyroid cancer.

[0010] Another technical solution adopted by the present invention is: the application of a reagent for regulating the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a drug for treating thyroid cancer.

[0011] Another technical solution adopted by the present invention is: the application of a CTTNBP2NL inhibitor in the preparation of a drug for treating thyroid cancer.

[0012] Another technical solution adopted by the present invention is: the application of a TEAD1 inhibitor in the preparation of a drug for treating thyroid cancer.

[0013] Another technical solution adopted by the present invention is: the application of a TEAD1 inhibitor and a CTTNBP2NL inhibitor in the preparation of a drug for treating thyroid cancer.

[0014] The beneficial effect of the present invention is that the expression of CTTNBP2NL is positively correlated with immune cells in THCA, and CTTNBP2NL can be used as a biomarker to detect the development and prognosis of tumors. The present invention provides a new molecular pathway and biomarker for the development, prognosis and treatment of the thyroid, and provides a new entry point for targeted intervention. Description of the Drawings

[0015] Figure 1 It is a test chart of the expression level of CTTNBP2NL in thyroid cancer in the embodiment of the present invention; Figure 2 It is a test chart of the correlation between CTTNBP2NL and tumor mutation burden (TMB) in the embodiment of the present invention; Figure 3 It is a test chart of the correlation between CTTNBP2NL and the gene expression of TEAD1 in the embodiment of the present invention; Figure 4 It is a test chart of the correlation between CTTNBP2NL and immune cells in the embodiment of the present invention; Figure 5 It is a Western blot analysis chart of the protein expression level in TPC1 cells with silenced CTTNBP2NL in the embodiment of the present invention; Figure 6 It is a test result chart of detecting the apoptosis of TPC1 cells with silenced CTTNBP2NL by flow cytometry in the embodiment of the present invention; Figure 7 It is a detection chart of the proliferation of TPC1 cells with silenced CTTNBP2NL in the embodiment of the present invention; Figure 8Detection diagram of colony formation of TPC1 cells with silenced CTTNBP2NL in the embodiments of the present invention; Figure 9 Western blot analysis diagram of apoptosis-related proteins of TPC1 cells with silenced CTTNBP2NL in the embodiments of the present invention; Figure 10 Verification result diagram of overexpression of CTTNBP2NL in TPC1 cells verified by Western blot and qPCR in the embodiments of the present invention; Figure 11 Detection result diagram of apoptosis of TPC1 cells with overexpressed CTTNBP2NL detected by flow cytometry in the embodiments of the present invention; Figure 12 Detection diagram of proliferation of TPC1 cells with overexpressed CTTNBP2NL in the embodiments of the present invention; Figure 13 Detection diagram of colony formation of TPC1 cells with overexpressed CTTNBP2NL in the embodiments of the present invention; Figure 14 Western blot analysis diagram of apoptosis-related proteins of TPC1 cells with overexpressed CTTNBP2NL in the embodiments of the present invention; Figure 15 Verification result diagram of the effect of TEAD1 on the expression of CTTNBP2NL verified by dual-luciferase reporter gene assay in the embodiments of the present invention; Figure 16 Verification result diagram of the binding of TEAD1 and CTTNBP2NL verified by chromatin immunoprecipitation in the embodiments of the present invention; Figure 17 Detection diagram of proliferation of TPC1 cells with silenced TEAD1 and overexpressed CTTNBP2NL in the embodiments of the present invention; Figure 18 Detection result diagram of apoptosis of TPC1 cells with silenced TEAD1 and overexpressed CTTNBP2NL detected by flow cytometry in the embodiments of the present invention; Figure 19 Detection diagram of colony formation of TPC1 cells with silenced TEAD1 and overexpressed CTTNBP2NL in the embodiments of the present invention. Detailed implementation mode

[0016] To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following is described in conjunction with the implementation modes and accompanied by the drawings.

[0017] Application of a reagent for detecting CTTNBP2NL in the preparation of a kit for evaluating the prognosis effect of thyroid cancer.

[0018] As can be seen from the above description, CTTNBP2NL has a high expression level in thyroid cancer. The expression of CTTNBP2NL is positively correlated with immune cells in THCA. CTTNBP2NL affects the development, prognosis and treatment of tumors through the interaction of immune cells, and can be used to detect the development and prognosis of tumors.

[0019] Another technical solution adopted by the present invention is: the use of a reagent for detecting TEAD1 in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0020] As can be seen from the above description, the beneficial effect of the present invention is that: TEA domain family member 1 (TEAD1) is another important element in cancer biology, and it participates in the Hippo signaling transduction cascade. Overexpression of TEAD1 can significantly enhance the proliferation ability of cells. Therefore, TEAD1 can be used as a biomarker to detect the development and prognosis of tumors.

[0021] Another technical solution adopted by the present invention is: the use of a reagent for detecting TEAD1 and CTTNBP2NL in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0022] As can be seen from the above description, the expressions of CTTNBP2NL and TEAD1 are positively correlated with immune cells in THCA, and can be used to evaluate the prognosis of thyroid cancer.

[0023] Another technical solution adopted by the present invention is: the use of a reagent for detecting the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a kit for detecting thyroid cancer.

[0024] As can be seen from the above description, CTTNBP2NL has a high expression level in thyroid cancer and shows low expression in various cancers including breast invasive carcinoma, and can be used as a detection marker for thyroid cancer.

[0025] Another technical solution adopted by the present invention is: the use of a reagent for regulating the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a drug for treating thyroid cancer.

[0026] Another technical solution adopted by the present invention is: the use of a CTTNBP2NL inhibitor in the preparation of a drug for treating thyroid cancer.

[0027] As can be seen from the above description, overexpression of CTTNBP2NL in thyroid cancer cells leads to a decrease in apoptosis and a significant enhancement of cell proliferation and clonal potential. Inhibiting CTTNBP2NL in thyroid cancer can inhibit cell growth and increase apoptosis of thyroid cancer cells.

[0028] Furthermore, CTTNBP2NL inhibitors increase the apoptosis of thyroid cancer cells by inhibiting CTTNBP2NL expression.

[0029] Another technical solution adopted by the present invention is: the application of TEAD1 inhibitors in the preparation of drugs for treating thyroid cancer.

[0030] As can be seen from the above description, TEAD1 can directly bind to the promoter of the CTTNBP2NL gene, thereby regulating its transcription. At the same time, although CTTNBP2NL is deleted, TEAD1 can still enhance the clonogenic potential of cells and promote cell survival and proliferation, indicating that TEAD1 plays a crucial role in the survival of thyroid cancer cells, and inhibiting TEAD1 expression can increase the apoptosis of thyroid cancer cells.

[0031] Furthermore, on the one hand, TEAD1 inhibitors increase the apoptosis of thyroid cancer cells by inhibiting TEAD1 expression, reducing the transcription of the CTTNBP2NL gene; on the other hand, by inhibiting TEAD1 expression, they reduce the clonogenic potential of cells and the proliferation of thyroid cancer cells.

[0032] Another technical solution adopted by the present invention is: the application of TEAD1 inhibitors and CTTNBP2NL inhibitors in the preparation of drugs for treating thyroid cancer.

[0033] As can be seen from the above description, overexpression of TEAD1 can compensate for the deletion of CTTNBP2NL by promoting cell proliferation, reducing apoptosis, and enhancing the clonogenic potential of TPC1 cells. This indicates that there is a complex regulatory relationship between TEAD1 and CTTNBP2NL. Simultaneously inhibiting the expression of TEAD1 and CTTNBP2NL can avoid the compensatory regulation of TEAD1 on CTTNBP2NL and more efficiently increase the apoptosis of thyroid cancer cells.

[0034] Figures 1 - 19 In ; ; . That is, when the p-value is less than 0.05, one asterisk is used to indicate that the result is statistically significant; when the p-value is less than 0.01, two asterisks are used to indicate high significance; when the p-value is less than 0.001, three asterisks are used to indicate extremely high significance.

[0035] Example 1 of the present invention is: to explore the roles of TEAD1 and CTTNBP2NL in PTC (papillary thyroid carcinoma) tissues, and the steps are as follows: 1. Materials and methods 1.1 Cancer Genome Atlas database analysis Data on gene expression levels, clinical parameters, and survival outcomes were extracted from The Cancer Genome Atlas (TCGA). The correlations of TEAD1 and CTTNBP2NL expression and their relationships with patient prognosis were analyzed using bioinformatics tools, namely cBioPortal (https: / / www.cbioportal.org / ) and GEPIA (https: / / gepia.cancer-pku.cn / ). Differential expression analysis was performed on tumor tissues and normal thyroid tissues.

[0036] 1.2 Cell culture TPC1 cells were obtained from Peking University Peizhi Company (BNCC337912, Peking University Peizhi, Beijing, China) for STR detection and mycoplasma detection. The cells were cultured in RPMI-1640 medium (11875-093, Gibco, Beijing, China) supplemented with 1% penicillin-streptomycin solution (15140-122, Gibco, Beijing, China) to prevent bacterial contamination. The cultures were incubated in a 5% CO2 incubator at 37 °C. When setting up the experiments, the cells were carefully plated at the optimal density on tissue culture-treated dishes and allowed to adhere overnight before further experimental treatment.

[0037] 1.3 Colony formation assay Cells were harvested by trypsin (trypsin-EDTA 0.25%, 25200-056, Gibco, Beijing, China), counted, and seeded into six-well plates (140675, Thermo Fisher Scientific, Beijing, China) at 500 cells per well. The plates were then incubated in a 37 °C incubator for 10 - 14 days until visible colonies appeared. The colonies were fixed in 4% paraformaldehyde (158127, Sigma-Aldrich, St. Louis, Beijing, China) for 15 minutes, rinsed with phosphate-buffered saline (PBS), and stained with 0.5% crystal violet for 30 minutes. After thorough washing with water and air-drying, the colonies were counted manually or using an automatic colony counter (ZX-400 automatic colony counter, Zhejiang Zexi Biotechnology Co., Ltd., Hangzhou, China).

[0038] 1.4 Flow cytometry According to the production guidelines of the annexin V-APC / PI fluorescence double staining cell apoptosis detection kit (P-CA-207, Pricella, Wuhan, China), the cells were rinsed with PBS, resuspended in 100 µL of binding buffer, stained with PI and annexin V-APC dyes, and 5 µL of APC and PI staining solution was added to each tube. The cells were incubated in the dark at room temperature for 30 minutes. After staining, the cells were washed and then resuspended in 500 µL of binding buffer for further analysis. Flow cytometry was evaluated using a BD FACSCantoII (BD Biosciences, San Jose, California, USA). The data were processed by FlowJo software (Treestar FlowJo 10.10.0, Becton, Dickinson and Company, New York, USA), and an accurate gating strategy was adopted to accurately identify and quantify various cell populations.

[0039] 1.5 Dual-Luciferase Reporter Assay Cells were seeded in 24-well plates and transfected with 500 ng of firefly luciferase reporter plasmid and 50 ng of Renilla luciferase control plasmid using Lipofectamine 3000 (L3000001, Thermo Fisher). After transfection, the cells were treated for 24 - 48 hours and then lysed with 1X passive lysis buffer (E1941, Promega). Luciferase activity was measured using a Dual-Luciferase Reporter Assay System (E1910, Promega), and fluorescence was detected using a Glomax Multi+ detection system (GM2000, Promega).

[0040] 1.6 Chromatin Immunoprecipitation (ChIP) The cells were crosslinked with 1% formaldehyde for 10 minutes and then the reaction was quenched with 125 mM glycine for 5 minutes. Then the cells were lysed in sodium dodecyl sulfate (SDS) buffer containing protease inhibitors (11836170001, Roche). The chromatin was fragmented into 200 - 500 bp fragments by sonication. The chromatin was incubated overnight at 4 °C with 2 - 5 µg of specific antibody (ab8580, Abcam) pre-bound to protein A / G magnetic beads (88802, Thermo Fisher). The magnetic beads were washed with low salt, high salt, LiCl, and TE buffers. The chromatin was eluted and the crosslinking was reversed by heating at 65 °C for 4 hours. The DNA was purified using a PCR kit (DP214-02, Tiangen), and the enriched regions were quantified by quantitative polymerase chain reaction (qPCR) and normalized to the input DNA.

[0041] 1.7 RNA Extraction Total RNA was isolated from TPC1 cells using TRIzol reagent (15596018, Invitrogen, Beijing, China) according to the protocol provided by the manufacturer. TRIzol reagent was added directly to the cells in the culture dish for lysis, followed by thorough homogenization. Then, chloroform was added to achieve phase separation. The aqueous phase containing RNA was carefully transferred to a new tube. Subsequently, RNA was precipitated with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water (10977015, Invitrogen, Carlsbad, USA).

[0042] 1.8 qPCR 1 μg of RNA was reverse transcribed into complementary DNA (cDNA) using the iScript cDNA Synthesis Kit (1708891, Bio-Rad, China) to effectively convert RNA into cDNA and ensure high-quality templates for subsequent qPCR. SYBR Green Master Mix containing fluorescent dye (CW3360H, Cwbio, Jiangsu) was used to detect DNA. The mixture was added to the reaction device and then loaded into the StepOnePlus Real-Time PCR System (Applied Biosystems). The primers used were as follows: TEAD1 forward, 5'-TGGCTACTTCCTGGAAGACC-3'; TEAD1 reverse, 5'-CCTTCTGCTGCTGTAGTCCT-3'; CTTNBP2NL forward, 5'-AGGAAGGAGGAGGAGGAAGG-3'; CTTNBP2NL reverse, 5'-CCTTCTGGGAGGAGGTAGTG-3'; GAPDH forward, 5'-CAAGGTCATCCATGACAACTTTG-3'; GAPDH reverse, 5'-GTCCACCACCCTGTTGCTGTAG-3'. 1.9 Cell transfection To regulate the expression of TEAD1 and CTTNBP2NL, plasmid-based overexpression and small interfering RNA-mediated knockdown methods were used. When overexpressing TEAD1, cells were transfected with the pCMV-TEAD1 plasmid (plasmid 33109, Addgene, Watertown, MA, USA). The sequence information is shown in Table S1.

[0043] 1.10 Cell proliferation ability assay Cell proliferation was evaluated using a CCK-8 kit (CK04, Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. Absorbance at a wavelength of 450 nm was recorded using a Bio-Rad microplate reader (SpectraMax i3X, Molecular Devices, Shanghai, China) to determine the proliferation rate relative to the control group.

[0044] 1.11 Protein extraction and Western blot analysis Proteins were extracted using radioimmunoprecipitation assay buffer (89900, Thermo Fisher Scientific, Waltham, USA). Cells were lysed on ice to ensure the integrity of proteins during extraction. The lysate was clarified by spinning at 12,000 rpm for 15 minutes in a refrigerated centrifuge at 4°C.

[0045] Equal amounts of protein (30 μg / sample) were loaded onto an SDS polyacrylamide gel electrophoresis gel for electrophoresis to separate proteins by molecular weight. After electrophoresis, the proteins were transferred to a PVDF membrane (IPVH, Millipore, Massachusetts, USA).

[0046] The membrane was blocked with a 5% non-fat milk solution prepared in TBS-T buffer. Then, specific TEAD1 (diluted 1:1000, ab221367, Abcam), CTTNBP2NL (diluted 1:1000, 25523-1-AP, Proteintech), and loading control GAPDH (diluted 1:5000, ab8245, Abcam), BAX monoclonal antibody (diluted 1:1000, 50599-2-Ig, Proteintech), caspase 3 / p17 / p19 monoclonal antibody (diluted 1:1000, 66470-2-Ig, Proteintech), P53 monoclonal antibody (diluted 1:1000, 60283-2-Ig, Proteintech), and human BCL2 polyclonal antibody (diluted 1:1000, 12789-1-AP, Proteintech) were used. After thorough washing to remove unbound primary antibodies, the membrane was incubated with an HRP-conjugated secondary antibody (diluted 1:5000, ab205719, Abcam) for 1 hour at room temperature.

[0047] 2. Statistical analysis Each experiment was performed in triplicate. Data were expressed as the mean ± standard deviation. Student's t-test was used for significant comparison between two groups. When analyzing the differences among multiple groups, one-way ANOVA was used, followed by Tukey's post hoc test. These analyses were performed using GraphPad Prism 9.0 software (9.0, GraphPad Software, San Diego, USA). P < 0.05 was considered statistically significant.

[0048] 3. Results 3.1 CTTNBP2NL Plays a Role in Thyroid Cancer The expression of CTTNBP2NL in tumor and normal tissues was evaluated using the TCGA database, as shown in Figure 1 (where THCA-Tumor is tumor tissue and THCA-Normol is normal tissue). It can be seen from Figure 1 that CTTNBP2NL was highly expressed in tumor tissues. High expression levels of CTTNBP2NL were observed in several cancers including thyroid cancer (THCA) in the TCGA database. In contrast, CTTNBP2NL was lowly expressed in multiple cancers including breast invasive carcinoma.

[0049] To determine the importance of CTTNBP2NL in predicting immune checkpoint inhibitors, the association between the expression level of CTTNBP2NL and two key cancer-related factors, namely tumor mutation burden and microsatellite instability, was analyzed, as shown in Figure 2 . It can be seen from Figure 2 that in cutaneous melanoma (SKCM), the expression of PDIA3 (protein disulfide isomerase A3) was positively correlated with TMB (tumor mutation burden), but in cholangiocarcinoma (CHOL), acute myeloid leukemia (LAML), lung squamous cell carcinoma (LUSC) and thyroid cancer (THCA), the expression of PDIA3 was negatively correlated with TMB.

[0050] To explore the regulatory role of CTTNBP2NL in THCA, genes related to CTTNBP2NL expression were further evaluated, as shown in Figure 3 . It was found from Figure 3 that the Pearson correlation coefficient between TEAD1 and CTTNBP2NL was as high as 0.7. In thyroid cancer, the expression of CTTNBP2NL was strongly positively correlated with the gene TEAD1, indicating a potential regulatory relationship between the two. To clarify the interaction between CTTNBP2NL and the intratumoral immune environment, the correlation between the expression level of CTTNBP2NL and infiltration of multiple immune cells was studied, as shown in Figure 4 , and it can be seen from Figure 4It can be seen that the expression of CTTNBP2NL is positively correlated with immune cells in THCA, indicating that it may affect tumor development, prognosis, and treatment through the interaction of immune cells. The infiltration levels of several immune cell types were calculated using THCA.

[0051] 3.2 Silencing CTTNBP2NL inhibits the growth of TPC1 cells The role of CTTNBP2NL in TPC1 cells was investigated by silencing the expression of CTTNBP2NL and evaluating its effects on cell behavior, referring to Figures 5 - 9 . As confirmed in the above figures, compared with the control group, silencing CTTNBP2NL significantly downregulated the protein expression level by approximately twofold (see Figure 5 ). Flow cytometry analysis was used to evaluate the effect of CTTNBP2NL silencing on apoptosis (see Figure 6 ). Compared with the control group, apoptosis was significantly increased in the CTTNBP2NL-silenced group, indicating that CTTNBP2NL may play a protective role in the apoptosis of TPC1 cells. Further studies on cell proliferation were performed using the CCK8 assay (see Figure 7 ). Compared with the control group, cell proliferation was significantly reduced in the CTTNBP2NL-silenced group, indicating that CTTNBP2NL is crucial for the proliferative ability of TPC1 cells. To evaluate the long-term proliferative potential of TPC1 cells, a colony formation assay was also performed (see Figure 8 . The results of Western blot analysis of apoptosis marker proteins BAX and Bcl-2 were consistent with those of flow cytometry (see Figure 9 ). The number of colonies formed by CTTNBP2NL-silenced cells was significantly reduced compared with the control group.

[0052] In summary, the research results indicate that CTTNBP2NL plays a crucial role in regulating the apoptosis and proliferation of TPC1 cells.

[0053] 3.3 Overexpression of CTTNBP2NL induces the growth of TPC1 cells The functional role of CTTNBP2NL in TPC1 cells was elucidated by overexpressing the CTTNBP2NL gene and detecting its effects on cells. Western blot analysis confirmed that overexpression of CTTNBP2NL led to a twofold increase in its protein level compared with the control group (see Figure 10 ).

[0054] To determine the effect of CTTNBP2NL overexpression on apoptosis, flow cytometry analysis was performed (see Figure 11), compared with the control group, apoptosis of cells overexpressing CTTNBP2NL was significantly reduced. Therefore, CTTNBP2NL may have an anti-apoptotic effect in TPC1 cells and may contribute to cell survival.

[0055] The cell proliferation was further evaluated using the CCK8 assay (see Figure 12 ). Compared with the control group, cell proliferation in the CTTNBP2NL expression group increased. This finding indicates that CTTNBP2NL may be involved in the process of promoting the proliferation ability of TPC1 cells.

[0056] A colony formation assay was performed to evaluate the long-term proliferation potential of TPC1 cells (see Figure 13 )). The results of Western blot analysis of apoptosis marker proteins BAX and Bcl-2 were consistent with those of flow cytometry (see Figure 14 ). Compared with the control group, the number of colonies formed by CTTNBP2NL-expressing cells increased significantly. This finding further confirmed the role of CTTNBP2NL in enhancing the proliferation and survival of TPC1 cells.

[0057] In summary, the research results show that overexpression of CTTNBP2NL in TPC1 cells leads to reduced apoptosis, significantly enhanced cell proliferation and clonogenic potential, making it a potential therapeutic target for PTC.

[0058] 3.4 TEAD1 regulates the expression of CTTNBP2NL in TPC1 cells To explore the regulatory relationship between TEAD1 and CTTNBP2NL in TPC1 cells. A dual-luciferase reporter assay was used to study whether TEAD1 directly affects the expression of CTTNBP2NL (see Figure 15 ). The expression of CTTNBP2NL was significantly regulated by TEAD1 (TEA domain transcription factor 1). Specifically, compared with the control group, higher luciferase activity was observed in cells co-transfected with the TEAD1 expression vector and the CTTNBP2NL promoter-luciferase construct. This finding indicates that TEAD1 can up-regulate the transcription of CTTNBP2NL.

[0059] To further confirm these findings, chromatin immunoprecipitation followed by PCR (ChIP-PCR) was performed to determine whether TEAD1 physically binds to the promoter region of the CTTNBP2NL gene (see Figure 16). ChIP-PCR results confirmed the direct interaction between TEAD1 and the CTTNBP2NL promoter. Compared with the DNA fragments immunoprecipitated with the control immunoglobulin G antibody, the DNA fragments immunoprecipitated with the TEAD1 antibody showed a significant enrichment in the CTTNBP2NL promoter region. Therefore, TEAD1 can directly bind to the promoter of the CTTNBP2NL gene, thereby regulating its transcription. This regulatory interaction emphasizes the importance of TEAD1 in maintaining the transcriptional activity of CTTNBP2NL. However, when CTTNBP2NL was silenced, overexpression of TEAD1 could compensate for this loss. CCK8 assay results showed that compared with the cells with CTTNBP2NL silenced alone, the proliferation of cells with TEAD1 overexpressed and CTTNBP2NL silenced was significantly increased. Therefore, TEAD1 may partially rescue the proliferation defect by activating alternative signaling pathways or target genes involved in cell cycle progression.

[0060] Given the previously observed role of CTTNBP2NL in promoting cell proliferation and inhibiting apoptosis, understanding the regulation of it by TEAD1 may have important implications for PTC treatment strategies targeting the TEAD1-CTTNBP2NL signaling.

[0061] 3.5 HTEAD1 regulates the growth of TPC1 cells controlled by CTTNBP2NL This study aimed to clarify the interaction between TEAD1 and CTTNBP2NL in regulating the behavior of TPC1 cells. Specifically, the effects of the combination of TEAD1 overexpression and CTTNBP2NL silencing on cell proliferation, apoptosis, and clonogenic potential were investigated.

[0062] To evaluate the effect on cell proliferation, CCK8 assay was performed (see Figure 17 ). The results showed that compared with the group with CTTNBP2NL silenced alone, the cell proliferation in the group with TEAD1 overexpressed and CTTNBP2NL silenced was significantly increased. This finding indicates that overexpression of TEAD1 can partially rescue the proliferation defect caused by CTTNBP2NL knockout, highlighting a compensatory mechanism. Flow cytometry was also used to evaluate cell apoptosis (see Figure 18), compared with the group with only CTTNBP2NL silenced, the apoptosis of cells in the group overexpressing TEAD1 and silencing CTTNBP2NL was significantly reduced. Therefore, even in the case of CTTNBP2NL silencing, TEAD1 can still play an anti-apoptotic role, indicating that TEAD1 plays a crucial role in cell survival. TEAD1 can activate downstream effectors that inhibit the apoptotic pathway, including upregulating anti-apoptotic proteins or inhibiting pro-apoptotic factors. This anti-apoptotic effect is crucial in cancer biology, enabling cancer cells to evade programmed cell death, thus leading to tumor progression and resistance to therapies.

[0063] In addition, colony formation assays were also performed to study the long-term proliferative capacity of cells (see Figure 19 ). The number of colonies formed by cells overexpressing TEAD1 and silencing CTTNBP2NL was significantly increased compared with cells with only CTTNBP2NL silenced. This finding further supports the view that, despite the absence of CTTNBP2NL, TEAD1 can still enhance the clonogenic potential of cells and promote cell survival and proliferation. This indicates that TEAD1 enhances the long-term proliferative capacity of PTC cells. This enhanced clonogenic potential suggests that TEAD1 not only supports immediate cell proliferation but also maintains the long-term proliferative capacity of cells, which is a key feature of cancer stem cells and tumorigenicity.

[0064] In summary, the above research results show that overexpression of TEAD1 can compensate for the deletion of CTTNBP2NL by promoting cell proliferation, reducing cell apoptosis, and enhancing the clonogenic potential of TPC1 cells. These results indicate that there is a complex regulatory relationship between TEAD1 and CTTNBP2NL. This understanding opens up new avenues for PTC treatment strategies targeting the TEAD1-CTTNBP2NL axis, thereby inhibiting tumor growth and improving patient prognosis.

[0065] Example two of the present invention is: the application of a reagent for detecting CTTNBP2NL in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0066] Example three of the present invention is: the application of a reagent for detecting TEAD1 in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0067] Example four of the present invention is: the application of a reagent for detecting TEAD1 and CTTNBP2NL in the preparation of a kit for evaluating the prognosis of thyroid cancer.

[0068] Example five of the present invention is: the application of a reagent for detecting the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a kit for detecting thyroid cancer.

[0069] Example 6 of the present invention is: The use of a reagent for regulating the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a drug for treating thyroid cancer.

[0070] Example 7 of the present invention is: The use of a CTTNBP2NL inhibitor in the preparation of a drug for treating thyroid cancer. The CTTNBP2NL inhibitor increases the apoptosis of thyroid cancer cells by inhibiting the expression of CTTNBP2NL.

[0071] Example 8 of the present invention is: The use of a TEAD1 inhibitor in the preparation of a drug for treating thyroid cancer. On the one hand, the TEAD1 inhibitor increases the apoptosis of thyroid cancer cells by inhibiting the expression of TEAD1, reducing the transcription of the CTTNBP2NL gene. On the other hand, by inhibiting the expression of TEAD1, the TEAD1 inhibitor reduces the clonogenic potential of cells and decreases the proliferation of thyroid cancer cells.

[0072] Example 9 of the present invention is: The use of a TEAD1 inhibitor and a CTTNBP2NL inhibitor in the preparation of a drug for treating thyroid cancer.

[0073] In summary, the present invention provides the application of TEAD1 and CTTNBP2NL as thyroid cancer molecular markers or therapeutic targets, and it is found that TEAD1 may partially rescue the proliferation defect by activating alternative signaling pathways or participating in target genes involved in cell cycle progression. The interaction between TEAD1 and CTTNBP2NL opens up a new approach for the therapeutic intervention of PTC. For example, inhibiting CTTNBP2NL while inhibiting TEAD1 may significantly reduce cell viability and tumor growth because the cells will lose a major survival signal and a compensatory pathway. Taking the TEAD1-CTTNBP2NL axis as a new therapeutic target, manipulating this pathway can restore the apoptosis process and inhibit tumor growth. Targeting this axis can significantly reduce the proliferation of tumor cells and increase the sensitivity to traditional therapies.

[0074] Understanding the extensive gene network regulated by TEAD1 can help identify more therapeutic targets. TEAD1 interacts with multiple cofactors and is involved in multiple signaling pathways, including the Hippo pathway, which plays a key role in regulating cell proliferation and apoptosis. In addition to the direct regulation of CTTNBP2NL expression by TEAD1, compensatory effects observed upon TEAD1 overexpression may involve other signaling pathways or factors. For example, TEAD1 interacts with several effector proteins in the Hippo pathway, which is crucial for regulating cell proliferation, migration, and apoptosis. TEAD1 may activate genes involved in cell cycle progression, anti-apoptotic responses, and metastasis by interacting with other transcription factors or coactivators. Furthermore, TEAD1 may also cross-talk with classical signaling pathways such as MAPK and PI3K / Akt, which are essential for the growth and survival of cancer cells. Therefore, in addition to the TEAD1-CTTNBP2NL axis, other signaling networks associated with TEAD1 can provide additional therapeutic targets. Targeting these pathways while inhibiting TEAD1 may overcome the resistance mechanisms that typically limit the effectiveness of single-target therapies, thus providing a comprehensive treatment approach. For example, simultaneous inhibition of TEAD1 and disruption of the Hippo or PI3K / Akt pathway can effectively inhibit tumor growth and induce apoptosis in cancer cells. Future studies should focus on exploring the specific roles of these alternative pathways and factors and evaluating their potential as clinical therapeutic targets.

[0075] Further research is needed to explore the detailed molecular mechanisms of the TEAD1-CTTNBP2NL interaction. The downstream targets of TEAD1, which contribute to the compensatory effect of TEAD1, should be determined through comprehensive transcriptome analysis (using RNA sequencing to identify changes in gene expression) and proteome analysis (to discover affected protein networks). Functional studies such as gene knockout or overexpression experiments can verify the roles of these downstream targets in PTC cell survival and apoptosis. In addition, in vivo studies using orthotopic or transgenic animal models of PTC are crucial for confirming the therapeutic potential of modulating the TEAD1-CTTNBP2NL axis in reducing tumor growth and metastasis potential. These models also help to test small molecule inhibitors or gene editing methods, such as CRISPR / Cas9, against TEAD1 or CTTNBP2NL. Clinical studies should aim to evaluate the expression levels of TEAD1 and CTTNBP2NL in PTC patients through immunohistochemistry or transcriptional analysis and correlate the findings with clinical outcomes such as tumor stage, recurrence, or drug resistance. Furthermore, patient-derived xenograft models can provide an excellent platform for studying the efficacy of personalized treatment approaches targeting the TEAD1-CTTNBP2NL pathway, thus paving the way for customized therapy based on individual molecular profiles.

[0076] The above are only embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent transformations made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in related technical fields, are equally included in the patent protection scope of the present invention.

Claims

1. Use of a reagent for detecting CTTNBP2NL in the preparation of a kit for evaluating the prognosis of thyroid cancer.

2. Use of a reagent for detecting TEAD1 in the preparation of a kit for evaluating the prognosis of thyroid cancer.

3. Use of a reagent for detecting TEAD1 and CTTNBP2NL in the preparation of a kit for evaluating the prognosis of thyroid cancer.

4. Use of a reagent for detecting the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a kit for detecting thyroid cancer.

5. Use of a reagent for regulating the expression level of TEAD1 and / or CTTNBP2NL in the preparation of a drug for treating thyroid cancer.

6. Use of a CTTNBP2NL inhibitor in the preparation of a drug for treating thyroid cancer.

7. The application according to claim 6, characterized in that, The CTTNBP2NL inhibitor increases apoptosis of thyroid cancer cells by inhibiting CTTNBP2NL expression.

8. Use of a TEAD1 inhibitor in the preparation of a drug for treating thyroid cancer.

9. The application according to claim 8, wherein The TEAD1 inhibitor, on the one hand, increases apoptosis of thyroid cancer cells by inhibiting TEAD1 expression, reducing CTTNBP2NL gene transcription; on the other hand, it reduces the clonogenic potential of cells and decreases the proliferation of thyroid cancer cells by inhibiting TEAD1 expression.

10. Use of a TEAD1 inhibitor and a CTTNBP2NL inhibitor in the preparation of a drug for treating thyroid cancer.

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