Application of crotonylation modification in field of thyroid cancer

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of crotonylation modification in the field of thyroid cancer. The invention provides application of crotonylation modification in preparation of a medicine for diagnosing thyroid cancer. The invention also provides an application of crotonylation modification in preparation of a medicine for diagnosing whether the thyroid cancer is metastatic or not. On one hand, it is proved that lysine crotonylation may be a potential molecular marker for diagnosing benign and malignant thyroid nodules; on the other hand, the invention also finds that lysine crotonylation can also help clinicians to evaluate whether a thyroid cancer patient suffers from lymph node metastasis or not.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of crotonylation modification in the field of thyroid cancer. Background Art

[0002] Crotonylation (Kcr) is a new type of protein post-translational modification, which was first reported by Professor Zhao Yingming's team in Cell magazine in 2011. It refers to the transfer of crotonyl groups to lysine residues under the action of crotonyl transferase, thereby playing a regulatory role. The precise identification of crotonylation sites is mainly based on the method of constructing bioinformatics predictors based on machine learning algorithms, including the convolutional neural network framework of iKcr_CNN, DeepCap-Kcr based on convolutional neural networks and long-short-term memory capsule networks, and Sohoko-Kcr, a deep learning model based on recurrent neural networks. The initial research on crotonylation focused on histones. Studies have shown that histone crotonylation plays an important role in kidney disease, depression, embryonic development, HIV latency, diabetes, tumors, bacterial infections and other processes. With the development of mass spectrometry technology, scholars have reported more and more non-histone crotonylation modifications, which play a key role in diseases such as tumors, kidney disease, and myocardial ischemia-reperfusion.

[0003] The invention of CN116482366A "Application of a crotonyl-modified PTBP1 protein" states: A combined immunoprecipitation method was used to screen colorectal cancer tissues and adjacent tissues, and it was found that the crotonylation level of PTBP1 protein was significantly highly expressed in CRC tissues. It was then identified through conventional biochemical and molecular biological methods and cell function experiments, and it was confirmed that the crotonyl-modified PTBP1 protein can be used as a new and specific colorectal cancer marker, which can further provide new ideas for the study of the pathogenesis of colorectal cancer, the diagnosis of colorectal cancer and the prediction of patient prognosis, and also provide new targets for the treatment of colorectal cancer.

[0004] In summary, although there have been many reports on the role of crotonylation modification in the occurrence and development of tumors, there has been no in-depth research in the field of thyroid cancer. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of crotonylation modification in the field of thyroid cancer.

[0006] In order to solve the above technical problems, the present invention provides the application of crotonylation modification in the preparation of drugs for diagnosing thyroid cancer.

[0007] The present invention also provides the application of crotonylation modification in the preparation of a drug for diagnosing whether thyroid cancer has metastasized.

[0008] This paper presents the crotonylation modification profile of papillary thyroid carcinoma and preliminarily explores the potential mechanism of crotonylation modification in thyroid cancer tumorigenesis and lymph node metastasis.

[0009] As an improvement of the application of the present invention: patients with high Kcr expression have a relatively higher risk of thyroid cancer lymph node metastasis.

[0010] As a further improvement of the application of the present invention: lysine crotonylation can be used as a potential molecular marker for diagnosing benign or malignant thyroid nodules.

[0011] As a further improvement of the application of the present invention: lysine crotonylation can be used as a marker for evaluating whether lymph node metastasis occurs in thyroid cancer patients.

[0012] The technical solution adopted by the present invention is: 6 pairs of cancer and adjacent specimens are divided into a lymph node metastasis group (N1 group) and a non-lymph node metastasis group (N0 group) according to whether lymph node metastasis occurs. After protein extraction and digestion, peptide enrichment and other processes, mass spectrometer sequencing is performed. The obtained sequencing information data is then sorted and analyzed. First, they were profiled for crotonylation modification of thyroid cancer proteins, and then divided into T / N group and LNM group for bioinformatics analysis. Finally, 47 clinical samples were collected and clinically verified.

[0013] The present invention studies the potential functions and mechanisms of lysine crotonylation modification in PTC tumorigenesis and lymph node metastasis. First, during the occurrence of thyroid tumors, DEKSs and crotonyl DEPs may regulate the occurrence of thyroid tumors through metabolism, PI3K-Akt signaling pathway, cell cycle and Hippo signaling pathway. Secondly, the interaction between the proteome and the Kcr proteome indicated that the differential change trend between DEPs levels and DEKSs levels may be an interesting story and should be paid special attention to. In addition, DEKSs and crotonyl DEPs may regulate lymph node metastasis through hydrogen peroxide decomposition metabolism process and tight junction pathway. Clinical validation shows that high expression of Kcr is associated with the occurrence of thyroid tumors and lymph node metastasis. The above suggests that Kcr plays an important role in the occurrence and development of PTC. In addition, most importantly, it can be used as a potential molecular marker for diagnosing benign and malignant thyroid nodules and evaluating whether lymph node metastasis occurs in patients with thyroid cancer.

[0014] The beneficial effects of the present invention are mainly reflected in:

[0015] 1. The present invention takes lysine crotonylation as a starting point to elucidate the possible molecular mechanism of the occurrence and development of thyroid cancer from a new scientific perspective.

[0016] 2. More importantly, based on further clinical validation with an expanded sample size, the present invention has confirmed that lysine crotonylation may be a potential molecular marker for diagnosing benign or malignant thyroid nodules. On the other hand, it has also been found that lysine crotonylation may also help clinicians assess whether thyroid cancer patients have lymph node metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0018] Figure 1 This is the comprehensive Kcr map of thyroid cancer tissue and adjacent tissue:

[0019] (a) Schematic diagram of the research workflow;

[0020] (b) Pearson correlation coefficient heat map of all quantified proteins between each pair of samples (samples ending in N represent normal tissue samples, and samples ending in T represent tumor tissue samples);

[0021] (c) Bubble plot showing the number of Kcr-modified peptides, Kcr sites, and proteins identified by MS (the numbers in the circles represent the number of proteins, peptides, and sites identified or quantified);

[0022] (d) Overview of Kcr proteins and sites; blue indicates downregulated proteins or sites, and red indicates upregulated proteins or sites;

[0023] (e) Statistical analysis of the number of Kcr sites on differentially modified proteins in the tumor / normal comparison group and lymph node metastasis group.

[0024] Figure 2 Clustering of features and expression patterns of DEKSs:

[0025] (a) Heat map of the enrichment of amino acid occurrence frequency near the modification site; the frequency significance of each amino acid at a specific position was calculated by the enrichment test method, and the p-value was log10 transformed to draw a heat map; the different colors in the figure show the frequency change of each amino acid at a specified position to reflect the characteristics of the motif; red indicates high frequency and green indicates low frequency; Motif feature logo map; when a feature sequence contains more than 20 identified modification sites and the statistical test p-value is less than 1e-6, the feature sequence is considered to be a motif of a modified peptide; the motif characteristics are reflected in the height ratio of the amino acid abbreviations at a specific position.

[0026] (b) Peptides with differential crotonylation between the tumor group and the normal group; blue represents peptides downregulated in cancer tissues, and red represents peptides upregulated in cancer tissues; the names of the genes described in the peptides are marked in each column;

[0027] (c) Clustering results of expression patterns of consecutive samples; six clusters were identified using Mfuzz software; Kcr proteins in Clusters 1, 3, and 4 showed a consistent upward trend from normal tissue to N1-tumor, representing the progression of thyroid cancer; from N0-tumor to N1-tumor, Kcr proteins in Clusters 2 and 5 showed a downward trend, and Kcr proteins in Cluster 6 showed an upward trend, suggesting the occurrence of thyroid cancer.

[0028] Figure 3 Functional classification and annotation of crotonylated DEPs in thyroid cancer:

[0029] (a) Volcano plot showing the differences in crotonylation sites between tumor and normal control groups;

[0030] (b) Subcellular localization distribution of DEPs;

[0031] (c) Lollipop diagram of GO secondary functional classification of DEPs;

[0032] (d) COG / KOG functional classification histogram of DEPs.

[0033] Figure 4 KEGG and PPI for Kcr DEPs in thyroid cancer:

[0034] (a) KEGG pathway chord diagram with significant enrichment; the pie chart on the left shows the top 10 pathways enriched by up-regulated DEPs, and the pie chart on the right shows the top 10 pathways enriched by down-regulated DEPs;

[0035] (b). DEPs interaction network diagram, red nodes represent upregulated DEPs, blue nodes represent downregulated DEPs, green nodes represent both upregulated and downregulated DEPs, and the size of the dots represents the number of interacting proteins.

[0036] Figure 5 For the interaction between proteome and Kcr proteome:

[0037] (a) Quadrant plot showing the positional changes of DEPs and DEKSs in tumor / normal control groups, with each data point representing a specific site; the x-axis and y-axis represent the tumor / normal ratio of the Kcr proteome and proteome, respectively, and the numbers in the corners indicate the up-regulated or down-regulated deps and dekss in each quadrant; in addition, each quadrant highlights the top three Kcr proteins and their corresponding sites, with “Pro” indicating “protein” and “Cro” referring to “Kcr site”;

[0038] (b) Comparison of proteome changes associated with thyroid cancer and Kcr proteome changes. The figure shows the relative fold changes of 10 upregulated sites and 5 downregulated sites of Kcr in the tumor / normal control group, as well as the relative fold changes of the corresponding proteins;

[0039] (c) KEGG pathway visualization based on proteomics and crotonylation proteomics; DEPs on the left and DEKSs on the right; numbers indicate the tumor / normal ratios of crotonylation and proteomics.

[0040] Figure 6 Heat map of KEGG enrichment pathways of Kcr protein in each control group:

[0041] Red indicates up-regulated proteins, and green indicates down-regulated proteins.

[0042] Figure 7 The characteristics and pathways of Kcr DEKSs in the lymph node metastasis comparison group;

[0043] (a). Heat map of the enrichment of amino acid occurrence frequency near the modification site; the frequency significance of each amino acid at a specific position is calculated by the enrichment test method, and the p-value is log10 transformed to draw a heat map; the different colors in the figure show the frequency change of each amino acid at a specified position to reflect the characteristics of the motif; red indicates high frequency and green indicates low frequency; Motif feature logo map; when a feature sequence contains more than 20 identified modification sites and the statistical test p-value is less than 1e-6, the feature sequence is considered to be the motif of the modified peptide; the motif characteristics are reflected in the height ratio of the amino acid abbreviations at a specific position;

[0044] (b). Clustering results of expression patterns of consecutive samples. Six clusters were identified using Mfuzz software. Kcr proteins in each cluster showed a consistent upward trend from normal tissue to N1-tumor, representing the progression of thyroid cancer;

[0045] (c) Network diagram of differential interactions of crotonylated proteins. Orange nodes represent upregulated crotonylated proteins, purple nodes represent downregulated crotonylated proteins; the size of the dots represents the number of interacting proteins.

[0046] Figure 8 Clinical verification of the relationship between Kcr and the development of thyroid cancer:

[0047] (a), (b), (c) Immunohistochemistry (IHC) showed that the crotonylation level was high in thyroid cancer tissues; however, there was no significant change in the crotonylation level in thyroid cancer tissues with lymph node metastasis;

[0048] (d) and (e) calculated the ratio of Kcr and N-cadherin IHC scores in thyroid cancer tissues of the same patient, excluding 12 samples that could not be calculated; the ratio ≥ 2 was defined as the relatively high expression group, and vice versa as the relatively low expression group. The results showed that crotonylation was positively correlated with N-cadherin;

[0049] (f), (g) The difference in N-cadherin IHC scores between thyroid cancer tissue and adjacent adjacent tissue of the same patient was calculated; a difference ≥ 2 was defined as a relatively high N-cadherin expression group, and vice versa as a relatively low N-cadherin expression group.

[0050] The results showed that most N-cadherins were lowly expressed in the Kcr low expression group and highly expressed in the Kcr high expression group. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0052] 1. A comprehensive map of Kcr differentially modified proteins (DEPs) and differentially modified sites (DEKSs) in thyroid cancer was discovered

[0053] In order to detect the characteristics of protein crotonylation in thyroid cancer, the present invention collected 6 pairs of thyroid cancer and adjacent tissues, and further performed TMT-labeled LC-MS / MS ( Figure 1 a). LC-MS / MS (liquid chromatography tandem mass spectrometry data) with TMT labeling (tandem mass spectrometry labeling) is a routine technology in this industry. The 12 specimens were divided into N0 group (no lymph node metastasis) and N1 group (with lymph node metastasis) according to the presence or absence of lymph node metastasis. The Pearson correlation coefficient heat map showed that there was consistency between the 6 pairs of specimens ( Figure 1 b). 262 proteins and 702 crotonylation sites were quantified in the Kcr proteome, while 5203 proteins were quantified in the proteome ( Figure 1 c) If Figure 1 As shown in Figure d, compared with the normal group, 110 differentially expressed Kcr sites of 57 differentially modified proteins were upregulated, and 59 differentially modified sites of 43 differentially modified proteins were downregulated in the tumor group; while in the lymph node metastasis comparison group, only about 20 modification sites and modified proteins were differentially regulated.

[0054] Note: The tumor group is T, and the normal group is N; no lymph node metastasis is N0, and lymph node metastasis is N1.

[0055] In summary, it can be concluded that differential modification of crotonylation occurs in thyroid cancer.

[0056] 2. Elucidating the changing characteristics of Kcr differentially modified sites during thyroid tumorigenesis

[0057] like Figure 2 As shown, in order to establish a Kcr proteomic profile associated with the development of thyroid cancer, the present invention uses the MoMo tool to analyze the motif characteristics of crotonylation sites in tumor / normal control groups ( Figure 2 a) Among the Kcr differentially modified sites, the representative top 10 up-regulated and down-regulated DEKSs are listed in Figure 2 b. HOOK3-655, HBA1-17, and HBD-18 were upregulated. PLEC-1626, ACTN1-312, and HSP90AA1-632 were downregulated. To investigate the dynamic expression pattern of upregulated DEKSs during thyroid tumorigenesis, the 110 sites in the tumor / normal control group were divided into 6 modules using Mfuzz software ( Figure 2 c). The tumors were divided into three groups according to the tumor site: normal group (normal group N0 without lymph node metastasis and normal group N1 with lymph node metastasis), tumor group without lymph node metastasis (N0-tumor), and tumor group with lymph node metastasis (N1-tumor). The expression patterns of Cluster1, 3, and 4 gradually increased during the progression of thyroid cancer. These sites were significantly enriched in pathways such as Africantrypanosomiasis, Autoimmune thyroid disease, hemoglobin beta binding, and haptoglobin–hemoglobin complex. Notably, Cluster6 increased significantly during the transition from N0 tumor group to N1 tumor group, but did not change significantly from normal group to tumor group. These Kcr sites seemed to be particularly related to lymph node metastasis and were enriched in pathways such as Autoimmune thyroid disease, chaperone binding, and extracellular space. In addition, Cluster2 and 5 were initially upregulated during the transition from normal to tumor, but were later downregulated during the transition from N0 to N1 tumor. These related DEKSs were mainly enriched in pathways such as Ribosome, dynactin binding, Autoimmune thyroid disease and chondroitin sulfate binding.

[0058] In summary, according to Figure 2 It can be seen that in the tumor / normal control group, the levels of most DEKSs showed an increasing trend with the progression of thyroid cancer, which indicates that DEKSs and their related signaling pathways may play an important role in the occurrence and development of thyroid cancer.

[0059] 3. Preliminary exploration of the potential functions and molecular mechanisms of crotonylated DEPs in thyroid tumorigenesis

[0060] To further explore the potential function and mechanism of Kcr modification in thyroid tumorigenesis, the present invention analyzed crotonylated DEPs in tumor / normal control groups. Figure 3 As a result, 110 sites in 57 proteins were upregulated and 59 sites in 43 proteins were downregulated in the volcano plot. In addition, subcellular localization analysis, GO annotation, and COG / KOG classification analysis were performed on the upregulated and downregulated DEPs. In addition, in order to gain a deeper understanding of the possible molecular mechanism of crotonylation DEPs in tumorigenesis, the present invention performed KEGG analysis ( Figure 4 a). According to the PPI network ( Figure 4 b), all upregulated crotonylated DEPs were mainly enriched in PI3K-Akt signaling pathway, cell cycle and Hippo signaling pathway.

[0061] In summary, according to Figure 3 and Figure 4 , it can be seen that: in the tumor / normal control group, crotonylated DEPs are mainly enriched in the PI3K-Akt signaling pathway, cell cycle and Hippo signaling pathways, etc. These results suggest that Kcr modification may regulate the occurrence of thyroid tumors through these pathways.

[0062] 4. Revealing the crosstalk between proteome and Kcr proteome in thyroid tumorigenesis

[0063] In order to further study the interaction between the proteome and the Kcr proteome and its role in thyroid tumorigenesis, the present invention analyzed DEKSs and DEPs. Figure 5 a), 5 proteins and 5 Kcr sites were upregulated simultaneously, including LMNA-K417, PKM-K135, and Diablo-K146. In the first quadrant, 28 proteins were downregulated and 94 Kcr sites were upregulated, such as HBA1-K17, HBD-K18, and HBA1-K145. In the ninth quadrant, 44 proteins were upregulated and 70 Kcr sites were downregulated, including LMNA-K450, COX5A-K115, and SLC25A6-K147. Figure 5 b Lists some of the top ten upregulated and top five downregulated DEKSs in crotonylation levels and their related DEPs, such as HOOK3-K655, HBA1-K17, and PLEC-K2396.

[0064] According to the KEGG pathway clustering analysis of crotonylation DEPs between different comparison groups ( Figure 6 ), the present invention displays representative DEKSs and DEPs into enriched signaling pathways ( Figure 5 c). These crotonylated proteins were mainly enriched in thyroid hormone synthesis, pyruvate metabolism, tricarboxylic acid cycle, cell cycle and apoptosis pathways. In these pathways, most of the differentially expressed proteins were different from the differentially expressed crotonylation proteins, suggesting that the crotonylation level may affect the expression of related proteins.

[0065] In summary, according to Figure 5 and Figure 6 It can be seen that the differential modification levels of most of the sites and proteins that undergo crotonylation are opposite. When determining the possible mechanisms of thyroid cancer occurrence and development, differentially changed proteins and sites should be considered.

[0066] 5. Demonstration of Kcr proteomic characteristics and possible molecular mechanisms in thyroid cancer lymph node metastasis

[0067] In order to determine the possible functions and molecular mechanisms of the Kcr proteome in the process of thyroid cancer lymph node metastasis, the present invention pays special attention to DEKSs and DEPs in the LNM comparison group. Figure 7 As shown in a, the motif characteristics of Kcr-DEPs associated with lymph node metastasis are shown. Subsequently, the expression pattern cluster analysis of the 36 upregulated DEKSs was performed ( Figure 7 b). During the progression of thyroid cancer, all clustered areas showed a significant upward trend. These DEKSs were mainly clustered in pathways such as Complement and coagulation cascades, Autoimmune thyroid diseasea, and iodide transport. In addition, according to the PPI network of the LNM comparison group ( Figure 7 c), upregulated DEPs were enriched in hydrogen peroxide catabolism and tight junction pathways.

[0068] In summary, according to Figure 8 , it can be seen that: Based on enrichment analysis, in the LNM group, the upregulated DEPs were mainly enriched in the hydrogen peroxide decomposition metabolism process and tight junction pathway. This suggests that lymph node metastasis of thyroid cancer may be related to crotonylation modification in these pathways.

[0069] VI. Further clinical validation confirmed that Kcr levels can predict thyroid cancer lymph node metastasis

[0070] To further verify the differential expression of Kcr in thyroid cancer and the relationship between Kcr expression and the development of thyroid cancer, the sample size was expanded and 47 pairs of thyroid cancer tissues and adjacent normal tissues were collected. The expression of Kcr was detected by immunohistochemistry (IHC) and evaluated by pathologists. Representative images from patients are shown in Figure 8 a. Compared with adjacent normal tissues, Kcr was significantly more expressed in tumor tissues (t=9.792, P<0.001) ( Figure 8 b and 8c). This is consistent with the original sequencing data. N-cadherin is a marker of epithelial-mesenchymal transition (EMT), which is a marker of metastasis. Figure 8 As shown in Figures 8d and 8e, Kcr expression was positively correlated with N-cadherin expression (r=0.571, P=0.0015), suggesting that Kcr is associated with the occurrence and lymph node metastasis of thyroid cancer.

[0071] The present invention also determines whether Kcr expression is associated with clinical features. Figure 8 As shown in Figures 8f and 8g, the expression of N-cadherin in the Kcr relatively high expression group was higher than that in the Kcr relatively low expression group (χ2=18.966, P<0.001), suggesting that patients with high Kcr expression may have a higher risk of thyroid cancer lymph node metastasis. However, the relative expression of Kcr was not significantly correlated with gender, age, tumor diameter, calcification, local invasion, and combined inflammation. Similarly, the small validation set may be a limitation, and further experiments with larger sample sizes are needed.

[0072] In summary, according to Figure 8 , we can know that: Based on the experimental verification of the expanded sample size, crotonylation modification is related to the occurrence and development of thyroid cancer. It may be a potential molecular marker for diagnosing thyroid cancer and its lymph node metastasis, providing a basis for the precise diagnosis and treatment of clinicians.

[0073] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of crotonylation modification in the preparation of drugs for diagnosing thyroid cancer.

2. Application of crotonylation modification in the preparation of drugs for diagnosing whether thyroid cancer has metastasized.

3. The use according to claim 2, characterized in that: Patients with high Kcr expression have a relatively higher risk of thyroid cancer lymph node metastasis.

4. The use according to any one of claims 1 to 3, characterized in that: Lysine crotonylation can be used as a potential molecular marker for diagnosing benign and malignant thyroid nodules.

5. The use according to any one of claims 1 to 3, characterized in that: Lysine crotonylation can be used as a marker to assess whether thyroid cancer patients have lymph node metastasis.

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