Application of NOD1 truncated splicing isomer as marker and target in preparation of thyroid papillary minimal cancer detection reagent, immune targeting drug and screening method

Abstract

The invention discloses application of a NOD1 truncated splicing isomer as a marker and a target spot in preparation of a thyroid papillary minimal cancer detection reagent, an immune targeting drug and a screening method, and belongs to the technical field of biological medicine. Aiming at the problem of clinical diagnosis and treatment difficulty caused by lack of an effective lymph node metastasis prediction marker for papillary thyroid carcinoma, the invention discovers that a truncated splicing isomer NOD1-S can be used as a key transcription marker. According to the method, the transcriptional instinct of NOD1-S is specifically amplified by a specific primer pair, and a detection reagent or a kit can be prepared on the basis of the transcriptional instinct of NOD1-S; meanwhile, the NOD1-S can be used as a drug target for screening or preparing immune targeting drugs. The kit is mainly used for auxiliary detection of thyroid papillary minimal carcinoma lymph node metastasis and research and development and screening of immune targeting drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. More specifically, this invention relates to the application of the truncated splice isomer of NOD1 as a marker and target in the preparation of diagnostic reagents for papillary thyroid microcarcinoma, immunotargeting drugs, and screening methods. Background Technology

[0002] Papillary thyroid microcarcinoma (PTMC) is a subtype of papillary thyroid carcinoma (PTC) with a diameter of less than 1 cm, accounting for approximately 50% of new PTC cases. Although most PTMC cases progress slowly, more than 15% of patients exhibit aggressive behaviors such as lymph node metastasis, resulting in a significantly worse prognosis.

[0003] Currently, the core challenge in clinical practice is the lack of effective molecular markers that can accurately predict lymph node metastasis in percutaneous transurethral renal cystic thyroid carcinoma (PTMC). This directly leads to a dilemma in treatment strategies: prophylactic lymph node dissection may cause serious complications such as hypoparathyroidism; while being overly conservative may miss micrometastases, increasing the risk of recurrence. Therefore, finding reliable predictive markers is crucial for achieving individualized and precise treatment of PTMC and avoiding overtreatment or undertreatment.

[0004] From a tumor biology perspective, PTMCs are often classified as cold tumors with weak immunogenicity, characterized by the presence of effector immune cells (such as CD8+) in the tumor microenvironment. + Insufficient T cell infiltration leads to weakened immune surveillance, making tumor cells more susceptible to immune escape. This immunosuppressive microenvironment is considered an important basis for tumor invasion and metastasis. However, the key molecular mechanisms driving the formation of a cold tumor phenotype in PTMCs remain unclear.

[0005] Nucleotide-binding oligomerization domain 1 (NOD1) is an important intracellular pattern recognition receptor that participates in anti-tumor immune responses by activating signaling pathways such as NF-κB. Studies have shown that NOD1 agonists can enhance the efficacy of immunotherapy, suggesting its potential as an immunotherapeutic sensitizer. However, the role of NOD1 in the immune microenvironment remodeling of PTMC, a specific cold tumor subtype, remains completely unknown.

[0006] It is noteworthy that alternative splicing is an important mechanism for tumors to produce functionally diverse proteins, and its products can serve as specific biomarkers or therapeutic targets. The NOD1 gene also has multiple splice isoforms. Previous studies have identified a truncated splice isoform, ENSG00000106100.11_novel01 (hereinafter referred to as NOD1-S), generated from the intra-NOD1 gene with intron retention, which is associated with the lymph node metastasis phenotype of PTMC, suggesting its potential importance. However, does NOD1-S affect the progression of PTMC by regulating the tumor immune microenvironment? What are the functional similarities and differences between it and the full-length isoform NOD1-L (NM_006092.4)? What are the underlying molecular mechanisms? These questions remain unanswered, constituting a clear knowledge gap in this field.

[0007] Therefore, effective predictive biomarkers for lymph node metastasis are urgently needed in the clinical diagnosis and treatment of PTMC. Furthermore, the immune regulatory mechanisms underlying its cold tumor characteristics, particularly the role of the NOD1 gene and its key splice isoforms, require in-depth exploration. Solving these problems is of great value for developing new diagnostic tools and treatment strategies. Summary of the Invention

[0008] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0009] To achieve these objectives and other advantages according to the present invention, a truncated splice isoform of the NOD1 gene is provided as a transcriptional marker in the preparation of a detection reagent for lymph node metastasis of papillary thyroid microcarcinoma, said truncated splice isoform being NOD1-S, the transcript of which contains a sequence region that can be specifically amplified by the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0010] A kit for detecting lymph node metastasis of papillary thyroid microcarcinoma is provided, comprising primer pairs for specifically detecting NOD1-S transcriptional levels, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0011] Preferably, it also includes a primer pair for specifically detecting the transcriptional level of the CCL5 gene, the sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0012] This invention provides the application of a truncated splice isoform of the NOD1 gene, NOD1-S, as a target in the screening or preparation of immunotargeted drugs for the treatment of papillary thyroid microcarcinoma.

[0013] A method for screening immunotargeted drugs for the treatment of papillary thyroid microcarcinoma is provided, comprising the following steps: S1. Provides a stable PTC cell model transfected with the NOD1-S expression vector; S2. Treat the PTC cell model with candidate substances; S3. Detect the expression level of CCL5 in the PTC cell model, and / or detect the chemotactic ability of the conditioned medium prepared from the PTC cell model on T cells; If, compared to an untreated cell model, the candidate substance causes a statistically significant increase in CCL5 expression levels and / or a statistically significant enhancement in T cell chemotaxis, then the candidate substance is a potential immune-targeting drug.

[0014] Preferably, the PTC cell model is a cell overexpressing NOD1-S selected from the TPC-1 or BCPAP cell lines.

[0015] Preferably, the T cell chemotaxis capacity is assessed by indirect co-culturing Jurkat cells with conditioned medium derived from the PTC cell model and by detecting the number of migrating cells using the CCK-8 assay.

[0016] The present invention has at least the following beneficial effects: First, this invention reveals and verifies for the first time the molecular mechanism by which the truncated splice isoform of the NOD1 gene, NOD1-S, promotes immune escape and lymph node metastasis in papillary thyroid microcarcinoma by inhibiting CCL5-mediated T cell recruitment. Experiments show that overexpression of NOD1-S in PTC cells significantly enhances cell invasion and migration and promotes lymph node metastasis in a mouse model, with the opposite effect to that of the full-length isoform NOD1-L. Mechanistically, NOD1-S inhibits the transcription and secretion of the chemokine CCL5 by downregulating the MYBBP1A-p53-IRF1 complex, thereby weakening T cell chemotaxis towards the tumor microenvironment. This discovery elucidates a novel function of NOD1-S as a key molecule driving the malignant progression of papillary thyroid microcarcinoma, providing important theoretical basis for its potential role as a molecular marker for predicting lymph node metastasis in papillary thyroid microcarcinoma.

[0017] Secondly, based on the discovered biomarker NOD1-S and its key downstream regulator CCL5, this invention provides specific primer pairs (SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 8) for detecting its transcriptional level, and validates their effective application in cell models. This primer design provides a core component for developing a diagnostic kit for assessing the risk of lymph node metastasis in papillary thyroid microcarcinoma. By simultaneously detecting the expression of NOD1-S and CCL5, the immune escape tendency of tumors can be assessed at the transcriptional level, laying the foundation for constructing a multi-indicator joint assessment system. This technical solution points to a feasible direction for transforming NOD1-S-related biomarkers into clinical molecular detection tools.

[0018] Third, this invention breaks through the conventional understanding of NOD1-S as merely a diagnostic marker. By elucidating its novel mechanism of inhibiting T cell recruitment and promoting immune escape through the "NOD1-S / MYBBP1A / CCL5" signaling axis, it reveals for the first time NOD1-S as a potential new immunomodulatory target for the treatment of papillary thyroid microcarcinoma. This discovery provides a crucial theoretical basis and a clear pathway of action for developing new therapeutic strategies for this immune-"cold" tumor. Based on this, targeting and inhibiting the function of NOD1-S to reverse the immunosuppressive microenvironment has become a highly promising new therapeutic approach.

[0019] Fourth, during the research process, this invention constructed and validated a complete functional analysis system, including a PTC cell model stably overexpressing NOD1-S, and functional evaluation indicators centered on CCL5 expression level and T cell chemotaxis. This system was successfully applied to elucidate the tumorigenic mechanism of NOD1-S and demonstrated that it can sensitively reflect the intervention effect targeting this pathway (such as using the CCL5-CCR5 inhibitor Maraviroc). Therefore, this validated cell model and dual functional evaluation indicators provide key experimental tools and feasibility evidence for the subsequent development of screening methods for immunotherapeutic drugs specifically targeting the NOD1-S-CCL5 axis. Its approach of combining clear molecular phenotypes with functional immune readouts also provides a reference for designing similar tumor immune microenvironment regulation strategies.

[0020] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0021] Figure 1 This invention uses RT-qPCR to detect the transcriptional level of NOD1-S in TPC-1 and BCPAP overexpressing cell lines; Figure 2 This invention analyzes the effect of NOD1-S overexpression on the migration ability of TPC-1 and BCPAP cells using a scratch assay. Figure 3 This invention uses Transwell assays to analyze the effect of NOD1-S overexpression on the invasion or migration ability of TPC-1 and BCPAP cells; Figure 4 This invention uses a mouse popliteal lymph node metastasis model to analyze the effect of overexpression of NOD1-S or NOD1-L transcripts on TPC-1 cell lymph node metastasis; Figure 5 This is a schematic diagram of the preparation of the conditioned medium and the indirect co-culture model of PTC cells and Jurkat cells in this invention; Figure 6 This invention uses the CCK-8 assay to detect the effect of conditioned medium prepared from PTC cells overexpressing NOD1 splice isomers on the chemotactic ability of Jurkat cells. Figure 7 This is a volcano plot of differentially expressed genes in the NOD1 splice isoform group relative to the negative control group in RNA-seq of this invention (left: NOD1-S, right: NOD1-L) and a Venn diagram of downstream differentially expressed genes specifically regulated by the NOD1 splice isoform (padj < 0.05). Figure 8 These are the top 30 differentially expressed genes that are specifically upregulated or downregulated by the NOD1 splice isoforms of this invention. Figure 9 Venn diagram of differentially expressed NOD1 splice isoform genes used in KEGG pathway enrichment analysis according to this invention. Figure 10 This is a Sankey bubble diagram (showing the Top 10 pathways, padj < 0.05) for KEGG pathway enrichment analysis using NOD1 splice isoform-specific differentially expressed genes in this invention. Figure 11 This invention uses RT-qPCR to detect changes in CCL5 gene transcription levels after NOD1 splice isoform overexpression; Figure 12 This invention uses an ELISA method to detect changes in CCL5 secretion levels after overexpression of the NOD1 splice isoform in TPC-1 and BCPAP cells; Figure 13 This invention uses RT-qPCR to detect changes in the transcriptional level of the MYBBP1A gene after overexpression of the NOD1 splice isoform in TPC-1 cells; Figure 14 This invention uses Western blot experiments to detect changes in MYBBP1A protein levels in TPC-1 cells after overexpression of the NOD1 splice isoform. Figure 15 This invention is based on transcriptome data from TCGA-PTC (containing 513 samples from 505 patients) in the cBioPortal for cancer genomics data platform, and analyzes the expression correlation between MYBBP1A and TP53, TP53 and IRF1, and IRF1 and CCL5 genes in PTC patient samples. Figure 16 This invention utilizes the TIMER 2.0 database to analyze the correlation between MYBBP1A gene expression and the infiltration levels of various T cell subtypes in THCA tissues; Figure 17 This invention utilizes transcriptomic data from TCGA-PTC on the cBioPortal for cancer genomics data platform to analyze the correlation at the transcriptional level between the MYBBP1A gene and core genes of the TNF signaling pathway (TNFRSF1A, RIPK1, FAS, and TRADD) in PTC patient samples. Figure 18 This invention utilizes co-immunoprecipitation (Co-IP) experiments to analyze the protein interactions between MYBBP1A and TP53 and IRF1 in TPC-1 cells. Figure 19 To verify the changes in CCL5 transcriptional levels, the present invention conducted a MYBBP1A rescue experiment in a stable TPC-1 cell line overexpressing the NOD1 splice isoform. Figure 20 In this invention, conditioned medium derived from NOD1 splice isoform overexpression group cells and negative control group cells were co-cultured with Jurkat cells pretreated with Maraviroc (25 nM) or DMSO, respectively, and the differences in Jurkat cell recruitment levels were analyzed and compared using CCK-8 cell viability assay. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to examples, so that those skilled in the art can implement it based on the description.

[0023] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0024] I. Materials, Main Reagents and Carriers Cell lines: human papillary thyroid carcinoma cell lines TPC-1 and BCPAP, and human acute T-lymphoblastic leukemia cell line Jurkat. TPC-1 and Jurkat cell lines were purchased from Shanghai Fuheng Biotechnology Co., Ltd., and BCPAP cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. All cell lines were identified using short tandem repeat (STR) typing and were all negative for mycoplasma.

[0025] Main reagents and carriers: M5 Total RNA Extraction Reagent (TRIgent); chloroform, isopropanol, 75% ethanol (RNase-free); PrimeScript TM RT Master Mix (Takara, RR036A); Quantitative Real-Time PCR Kit (2x Super SYBR Green qPCR Master Mix, Mei5bio, MF013-01); Lipofectamine 2000 (Mei5 Biotechnology); DMEM medium, 1640 RPMI medium; 0.25% trypsin; fetal bovine serum; G418 screening antibiotic (Proteintech, 108321-42-2); Human Rantes / CCL5 ELISA Kit (BOSTER, EK0494); Rabbit anti-human MYBBP1A antibody (Proteintech, 14524-1-AP), mouse anti-human β-actin antibody (Proteintech, 66009-1-Ig), mouse anti-human IRF1 antibody (Novus Biologicals, MAB4830-SP, 686703), mouse anti-human p53 antibody (Proteintech, 60283-2-Ig), HRP-labeled goat anti-mouse secondary antibody (Proteintech, SA00001-1). IRDye ® 800CW goat anti-rabbit fluorescent secondary antibody (LICORbio, D50311-04). Maraviroc (TargetMolChemicals Inc. T6016); Pierce® Co-Immunoprecipitation Kit (Thermo Scientific Pierce, PI26149). Small interfering RNA (siRNA) targeting MYBBP1A (si-MYBBP1A) and negative control siRNA (si-NC) were purchased from Guangzhou Ribo Biotechnology Co., Ltd.

[0026] Plasmid vectors: pcDNA3.1-3×Flag, pcDNA3.1-NOD1-L-3×Flag (named NOD1-L), pcDNA3.4-3×Flag and pcDNA3.4-MYBBP1A-3×Flag were all purchased from Changsha HonorGene Biotechnology Co., Ltd., and an overexpression plasmid pcDNA3.1-NOD1-S-3×Flag (named NOD1-S) was constructed based on pcDNA3.1-3×Flag.

[0027] Plasmid construction: Based on the known sequence of the NOD1-S splice isoform, its open reading frame sequence was predicted using the Open Reading Frame Finder database, and a plasmid containing the NOD1-S coding region (CDS) was constructed (named NOD1-S). The empty vector pcDNA3.1-3×Flag was used as a negative control.

[0028] Primer sequences: Primers for real-time PCR of NOD1-S splice isomer: SEQ ID NO: 1:NOD1-SF: 5'-TCCCATCAGAGTCTCACCCC-3' SEQ ID NO: 2:NOD1-SR: 5'-ATGCCATGCCCGTCCCTGTC-3' Primers for quantitative real-time PCR of the CCL5 gene: SEQ ID NO: 7:CCL5-F: 5'-AGCCCTCGCTGTCATCCTCA-3' SEQ ID NO: 8: CCL5-R: 5'-ACACTTGGCGGTTCTTTCGG-3' Primers for real-time PCR of the MYBBP1A gene: SEQ ID NO: 5:MYBBP1A-F: 5'-GACCTGGTGGAGGTGCTAG-3' SEQ ID NO: 6:MYBBP1A-R: 5'-ACCCAAGTCGTGCAGTAG-3' Primers for real-time PCR of the internal reference gene ACTB (actin β): SEQ ID NO: 3:ACTB-F: 5'-CACGGCATCGTCACCAACT-3' SEQ ID NO: 4:ACTB-R: 5'-CTGGATAGCAACGTACATG-3' II. Experimental Methods and Results 1. Construction of stable and transient cell lines overexpressing NOD1-S PTC Establish a cell model for subsequent functional and mechanistic studies, including the following experiments and steps: Cell culture: TPC-1 cells were cultured in DMEM medium containing 10% fetal bovine serum, and BCPAP and Jurkat cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2.

[0029] Cell transfection: a. Take TPC-1 or BCPAP cells in the logarithmic growth phase, at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in a 6-well plate.

[0030] b. Transfection was performed when the cell confluence reached 70%. The transfection system per well was as follows: 2 µg of plasmid (pcDNA3.1-3×Flag, pcDNA3.1-NOD1-S-3×Flag, or pcDNA3.1-NOD1-L-3×Flag) and 3 µL of Lipofectamine 2000 were diluted in 100 µL of Opti-MEM, incubated at room temperature for 5 minutes, mixed, and then incubated at room temperature for 20 minutes to form the transfection complex.

[0031] c. Add the complex dropwise to the cells that have been replaced with fresh antibiotic-free complete culture medium and mix gently.

[0032] d. Six hours after transfection, replace with completely fresh culture medium and continue culturing for 48 hours.

[0033] BCPAP transient transfection of cells: a. After completing the above transfection steps, BCPAP cells were cultured for another 48 hours, and then the cells were collected for subsequent transient expression level experiments.

[0034] TPC-1 cell stable transfection and stable cell line selection: a. 48 hours after transfection, cells were passaged at a ratio of 1:10 and continuously screened by adding complete culture medium containing a final concentration of 200 µg / mL G418.

[0035] b. Change the culture medium containing G418 every 2-3 days and continue screening for about 2-3 weeks until all untransfected cells die.

[0036] c. From the surviving cell population, stable cell lines derived from single-cell expansion are obtained by limiting dilution or by picking single clones.

[0037] d. Verify the NOD1-S mRNA overexpression level in each cell line by RT-qPCR (using primers shown in SEQ ID NO: 1 and SEQ ID NO: 2). Figure 1 (As shown). Successfully constructed cell models include: stable TPC-1 pcDNA3.1, NOD1-S, and NOD1-L expression lines, and transient BCPAP pcDNA3.1, NOD1-S, and NOD1-L transfected cells.

[0038] 2. In vitro validation of NOD1-S promoting PTC cell migration and invasion To verify the effect of NOD1-S on the malignant phenotype of papillary thyroid carcinoma cells, the following experiments and procedures were performed: 2.1 Cell scratch assay (migration ability): a. The constructed cell lines (TPC-1: pcDNA3.1, NOD1-S, NOD1-L; BCPAP: pcDNA3.1, NOD1-S, NOD1-L) were distributed at 1×10⁻⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in 6-well plates and cultured until the cells were completely confluent and formed a monolayer.

[0039] b. Using a sterile 10 µL pipette tip, make two parallel "well" shaped scratches perpendicular to the long axis at the bottom of the well plate.

[0040] c. Gently rinse the cells 2-3 times with PBS to remove the scraped cell debris, and then replace with serum-free culture medium.

[0041] d. Take photographs of the same location under an inverted microscope (20× objective lens) at 0, 12, and 24 hours after scratching.

[0042] e. Use ImageJ software to measure the area of ​​the scratch region at each time point and calculate the relative cell migration area. Relative migration area = [(A0-A...]] t ) / A0], where A0 is the scratch area at hour 0, A t The area of ​​the scratch is t hours later.

[0043] Result: As Figure 2 As shown, compared with the pcDNA3.1 group, the NOD1-S overexpression group showed a significantly faster scratch healing rate, indicating that NOD1-S enhances the migration ability of PTC cells.

[0044] 2.2 Transwell Experiments (Migration and Invasion Capabilities): a. Migration experiment: using 8 µm pore size Transwell chambers without matrix gel coating.

[0045] b. Invasion test: Transwell chambers pre-coated with Matrigel (diluted 1:8 with serum-free medium) were incubated at 37°C for 1 hour to allow them to gel.

[0046] c. After starving the cells in each group with serum-free medium for 24 hours, they were digested with trypsin, resuspended in serum-free medium, counted, and the cell density was adjusted to 1.25 × 10⁻⁶. 5 per mL.

[0047] d. Add 200 µL of cell suspension to the upper chamber and 700 µL of complete culture medium containing 10% fetal bovine serum to the lower chamber as a chemotactic agent.

[0048] e. The migration experiment was incubated for 24 hours, and the invasion experiment was incubated for 48 hours (37℃, 5% CO2).

[0049] f. Remove the chamber and gently wipe away the cells on the surface of the upper chamber membrane with a cotton swab. Fix the chamber in 4% paraformaldehyde for 15 minutes, then stain with 0.5% crystal violet for 15 minutes.

[0050] g. After rinsing with PBS, three fields of view were randomly selected under a microscope (200×) to count the number of cells that crossed the membrane and reached the lower surface.

[0051] Result: As Figure 3 As shown, the number of cells that crossed the Transwell membrane in the NOD1-S overexpression group was significantly greater than that in the pcDNA3.1 group, demonstrating that NOD1-S promotes the migration and invasion of PTC cells.

[0052] 3. In vivo validation of NOD1-S promoting PTC lymph node metastasis To validate the effect of NOD1-S on lymph node metastasis at the animal model level, the following experiments and procedures were performed: Animal model construction: a. Experimental animals: Four-week-old female NCG-severe immunodeficient mice were selected and acclimatized in an SPF-grade barrier environment for one week.

[0053] b. Cell preparation: Collect the cell lines constructed in Part 1 (TPC-1: pcDNA3.1, NOD1-S, NOD1-L), resuspend in sterile PBS, and adjust the density to 2.6 × 10⁻⁶. 7 per mL.

[0054] c. Cell inoculation: Under aseptic conditions, inject the cell suspension into the left hind limb paw pad of mice twice over 3 days. Each injection volume is approximately 20 µL (containing 5 × 10⁻⁶ cells). 5 (cells), with a cumulative inoculation of 1×10 cells per mouse. 6Cells. Five mice were placed in each group.

[0055] Model evaluation: a. After cell seeding, continue to feed the cells in an SPF environment for 6-7 weeks.

[0056] b. After reaching the experimental endpoint, the mice were euthanized by cervical dislocation.

[0057] c. Dissect and separate the left popliteal fossa lymph nodes of the mouse, and measure its longest diameter (L) and shortest diameter perpendicular to it (W) using calipers.

[0058] d. Calculate the lymph node volume (V), using the approximate formula: V = 0.5 × L × W 2 .

[0059] Result: As Figure 4 As shown, the volume of the popliteal lymph nodes in the NOD1-S group was significantly larger than that in the pcDNA3.1 group, while the volume of the lymph nodes in the NOD1-L group was significantly smaller, indicating that NOD1-S promotes the lymph node metastasis of PTC in vivo, and its function is the opposite of that of NOD1-L.

[0060] 4. Verification of NOD1-S's ability to inhibit T cell recruitment by PTC cells To investigate the effects of NOD1-S on T cell chemotaxis in the tumor immune microenvironment, the following experiments and procedures were performed: Preparation of conditioned medium: a. The constructed cell lines (TPC-1: pcDNA3.1, NOD1-S, NOD1-L; BCPAP: pcDNA3.1, NOD1-S, NOD1-L) were distributed at 1×10⁻⁶ cells per well. 5 The cells were seeded at a density of 100% in 6-well plates and cultured until 90% confluence was achieved.

[0061] b. Discard the original culture medium, wash once with PBS, and replace with serum-free complete culture medium.

[0062] c. Continue culturing under standard conditions for 48 hours.

[0063] d. Collect the cell supernatant and centrifuge at 300×g for 10 minutes at 4°C to remove cell debris.

[0064] e. The supernatant is filtered through a 0.22 µm filter membrane for sterilization, and the resulting conditioned medium (CM) is aliquoted and stored at -80°C or used immediately.

[0065] In vitro T cell chemotaxis assay: a. Using the Transwell indirect co-culture system (see schematic diagram) Figure 5The upper chamber is an 8 µm Transwell chamber, and the lower chamber is a 24-well plate.

[0066] b. Resuspend Jurkat cells in serum-free RPMI 1640 medium and adjust the density to 2.5 × 10⁶ cells / year. 5 per mL.

[0067] c. Add 200 µL of Jurkat cell suspension (2.5 × 10⁻⁶) to the upper chamber. 4 (cells).

[0068] d. Add 400 µL of different groups of conditioned medium to the lower chamber.

[0069] e. Co-culture at 37℃ and 5% CO2 for 24 hours.

[0070] f. Carefully remove the upper chamber of the Transwell, collect the culture medium in the lower chamber, and centrifuge at 1000 rpm for 5 minutes to collect the cells.

[0071] g. The number of Jurkat cells migrating to the lower chamber was detected using the CCK-8 assay: Collected cells were resuspended in 300 µL of medium containing 10% CCK-8 reagent, with three replicates per group. Cells were seeded in 96-well plates and incubated for 1.5 hours. The absorbance (OD) value was then measured at 450 nm using a microplate reader. The OD value was directly proportional to the number of migrating cells.

[0072] Result: As Figure 6 As shown, compared with the conditioned medium treatment of the pcDNA3.1 group, the number of Jurkat cells migrating was significantly reduced in the conditioned medium treatment of the NOD1-S group, while the number of migrating cells migrating was significantly increased in the conditioned medium treatment of the NOD1-L group, demonstrating that NOD1-S inhibits T cell recruitment by affecting tumor cell secretion factors.

[0073] 5. Preliminary Exploration of the Downstream Regulatory Mechanism of NOD1-S Based on Transcriptome Sequencing High-throughput sequencing was used to screen for NOD1-S-specific regulated genes and signaling pathways, including the following experiments and steps: Sample preparation and sequencing: a. Culture stable cell lines of pcDNA3.1, NOD1-S, and NOD1-L separately, with three independent biological replicates for each group.

[0074] b. Total RNA was extracted from cells using TRIzol reagent, and DNase I (deoxyribonuclease I) was used to remove genomic DNA contamination.

[0075] c. Use an Agilent 2100 bioanalyzer to test RNA integrity (RIN value > 7.0 is acceptable).

[0076] d. Construct sequencing libraries using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®.

[0077] e. Perform paired-end 150 bp sequencing on the Illumina NovaSeq 6000 platform, obtaining no less than 6.0 Gb of raw data for each sample.

[0078] Bioinformatics analysis: a. After quality control, the raw data were aligned to the human reference genome GRCh38 using HISAT v2.0.5 software.

[0079] b. Use FeatureCounts v1.5.0-p3 software to calculate gene expression counts.

[0080] c. Differential expression analysis was performed using the DESeq2 R package (version 1.20.0). The NOD1-S group and pcDNA3.1 group, and the NOD1-L group and pcDNA3.1 group were compared. The differentially expressed gene screening criteria were: |log2FoldChange|>1 and corrected p-value (padj)<0.05.

[0081] d. Define spliceosome-specific differentially expressed genes: NOD1-S specific upregulated gene (NOD1-S-SU): significantly upregulated in the NOD1-S group and not significantly changed or downregulated in the NOD1-L group.

[0082] NOD1-S-specific downregulated genes (NOD1-S-SD): significantly downregulated in the NOD1-S group and not significantly changed or upregulated in the NOD1-L group.

[0083] The definition of NOD1-L-specific upregulated / downregulated genes (NOD1-L-SU / SD) is similar.

[0084] e. Use Venn diagrams to display common and specific differentially expressed genes. Figure 7 , Figure 9 ).

[0085] f. KEGG pathway enrichment analysis was performed on the specific differentially expressed gene sets of NOD1-S and NOD1-L, and the top 10 pathways with the most significant enrichment (padj < 0.05) were selected and displayed using a Sankey bubble diagram. Figure 10 ).

[0086] Result: As Figures 7 to 10 As shown, RNA-seq analysis successfully identified a large number of genes differentially regulated by NOD1-S and NOD1-L. KEGG enrichment analysis revealed that both genes were significantly enriched in the TNF signaling pathway, and the chemokine CCL5 was a key shared differential gene, suggesting that it may be a key downstream effector molecule mediating the functional differences of the NOD1 double spliceosome.

[0087] 6. Validation of the mechanism by which NOD1-S regulates T cell recruitment through the MYBBP1A-CCL5 axis. To validate the signaling pathway by which NOD1-S inhibits CCL5 expression and T cell recruitment at the molecular level, the following experiments and steps were performed. CCL5 expression level detection: a. RT-qPCR: Total RNA was extracted from cells in each group, reverse transcribed into cDNA, and then qPCR was performed using primers shown in SEQ ID NO: 7 and SEQ ID NO: 8, with ACTB (SEQ ID NO: 3 and SEQ ID NO: 4) as an internal control. -ΔΔCt The relative expression level of CCL5 mRNA was calculated using this method.

[0088] Result: As Figure 11 As shown, the CCL5 mRNA level in the NOD1-S overexpression group was significantly lower than that in the pcDNA3.1 group, while it was significantly higher in the NOD1-L group.

[0089] b. ELISA: Collect conditioned medium from each group of cells, and perform the procedure according to the Human Rantes / CCL5 ELISA kit instructions to detect the secretion concentration of CCL5 protein in the supernatant.

[0090] Result: As Figure 12 As shown, consistent with the mRNA trend, the CCL5 protein level in the supernatant of the NOD1-S group was significantly decreased, while that in the NOD1-L group was significantly increased.

[0091] Validation of the key mediator molecule MYBBP1A: a. RT-qPCR and Western Blot: MYBBP1A mRNA levels were detected using primers shown in SEQ ID NO: 5 and SEQ ID NO: 6. Figure 13 Total cellular protein was extracted, and MYBBP1A protein levels were detected by Western blotting, with β-actin as an internal control. Figure 14 ).

[0092] Results: NOD1-S overexpression significantly downregulated the transcription and translation levels of MYBBP1A, while NOD1-L significantly upregulated it.

[0093] b. Correlation analysis of public databases: Data from 513 samples in the TCGA-PTC database were analyzed using the cBioPortal platform. Results showed that the mRNA expression levels of MYBBP1A were significantly positively correlated with TP53, TP53 with IRF1, and IRF1 with CCL5. Figure 15 Analysis using the TIMER 2.0 database showed that MYBBP1A expression was associated with the infiltration levels of multiple T cell subtypes in thyroid cancer. Figure 16 Furthermore, MYBBP1A expression is also positively correlated with key genes in the TNF signaling pathway (TNFRSF1A, RIPK1, etc.). Figure 17 ).

[0094] Protein-protein interaction verification (Co-IP): a. Collect TPC-1 cells and extract total protein using RIPA lysis buffer.

[0095] b. Perform immunoprecipitation using MYBBP1A antibody according to the Pierce Co-IP kit instructions.

[0096] c. Perform SDS-PAGE electrophoresis on the immunoprecipitated products and transfer them to a membrane.

[0097] d. Western blot detection was performed using p53 antibody and IRF1 antibody, respectively.

[0098] Result: As Figure 18 As shown, p53 and IRF1 protein bands can be detected in the immunoprecipitation complex of MYBBP1A, demonstrating that MYBBP1A can interact with p53 and IRF1 in PTC cells.

[0099] Rescue Experiment: a. In NOD1-L cells, transfect MYBBP1A-specific siRNA (si-MYBBP1A) or negative control siRNA (si-NC).

[0100] b. In NOD1-S cells, transfect with MYBBP1A overexpression plasmid or empty vector control.

[0101] c. 48 hours after transfection, cellular RNA was extracted, and CCL5 mRNA levels were detected by RT-qPCR.

[0102] Result: As Figure 19As shown, knocking down MYBBP1A can partially reverse the upregulation of CCL5 by NOD1-L; while overexpression of MYBBP1A can partially reverse the downregulation of CCL5 by NOD1-S, proving that MYBBP1A is a key mediator of CCL5 regulation by the NOD1 double splice.

[0103] Functional blocking experiment of CCL5-CCR5 signal axis: a. Jurkat cells were pretreated with 25 nM of the CCR5 inhibitor Maraviroc or an equal volume of DMSO for 24 hours.

[0104] b. The pretreated Jurkat cells were used in the Transwell chemotaxis assay described in Part 4, with the lower chamber containing the conditioned medium prepared for each PTC cell group.

[0105] Result: As Figure 20 As shown, Maraviroc pretreatment significantly inhibited the chemotactic activity of Jurkat cells in each conditioned medium group. More importantly, after blocking the CCL5-CCR5 axis, the difference in chemotactic activity of NOD1-S and NOD1-L towards Jurkat cells was eliminated, demonstrating that NOD1-S mainly weakens T cell recruitment by inhibiting the CCL5-CCR5 signaling axis.

[0106] III. Application Examples Example 1: Application of NOD1-S as a detection marker An example of a method for detecting NOD1-S transcription levels in PTMC samples using the primer pairs provided by this invention includes the following steps: Sample collection and processing: Obtain fresh thyroid biopsy tissue samples or surgically removed PTMC tissue samples, quickly freeze them in liquid nitrogen, and then transfer them to -80℃ for storage.

[0107] Total RNA extraction: Using TRIzol reagent, total RNA was extracted from approximately 50 mg of tissue following the standard procedures described in Part 1 of the experiment or the reagent instructions. RNA concentration and purity were determined using NanoDrop (an A260 / A280 ratio between 1.8 and 2.0 was preferred).

[0108] Reverse transcription: Take 1 µg of total RNA and use PrimeScript. TM Reverse transcription was performed using RT Master Mix in a 20 µL volume. The reaction program was: 37 °C for 15 min, 85 °C for 5 sec, and 4 °C hold. The resulting cDNA can be used immediately or stored at -20 °C.

[0109] Real-time PCR detection: a. Reaction system (20 µL): 10 µL of 2× SYBR Green qPCR Master Mix, 1 µL of cDNA template, 0.5 µL each of NOD1-S specific primers (10 µM) shown in SEQ ID NO: 1 and SEQ ID NO: 2, and sterile water to a final volume of 20 µL.

[0110] b. Reaction procedure: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 sec, 60℃ annealing / extension for 30 sec, for a total of 40 cycles; finally, perform dissolution curve analysis.

[0111] c. Simultaneously set up reaction wells for detecting the internal reference gene ACTB (using primers SEQ ID NO: 3 and SEQ ID NO: 4) as controls.

[0112] d. Data Analysis: Using 2 -ΔΔCt The relative expression level of NOD1-S in the test samples was calculated using a method. The mean NOD1-S expression level of a certain number of pathologically confirmed PTMC samples without lymph node metastasis or adjacent normal tissue samples was typically used as a reference.

[0113] Result Interpretation: If the relative expression level of NOD1-S in the tested PTMC sample is significantly higher than the reference level, it indicates a higher risk of lymph node metastasis. Further risk assessment can be performed by combining CCL5 expression levels (detected using primers SEQ ID NO: 7 and SEQ ID NO: 8).

[0114] Example 2: Example of an immunotargeting drug screening method based on the NOD1-S target A method for screening potential drugs capable of reversing NOD1-S-mediated immunosuppression includes the following steps: A screening system was established: TPC-1 cells overexpressing NOD1-S or BCPAP cells overexpressing NOD1-S, constructed in Part 1, were used as screening models. This model simulated the immunosuppressive properties of PTC cells highly expressing NOD1-S.

[0115] Candidate treatment: Seed the screening model cells into suitable well plates (e.g., 96-well plates for initial screening), and set up a blank control group (culture medium only), a model control group (solvents such as DMSO), and treatment groups with different concentrations of candidate substances. Candidate substances can be small molecule compound libraries, natural product extracts, or derivatives of known drugs, etc. The treatment time is generally 24-72 hours.

[0116] Effect index detection (either of the following two methods can be selected or used in combination): a. Detection of CCL5 expression level: After treatment, cells were collected and the expression / secretion level of CCL5 in the cells or supernatant was detected by RT-qPCR (the method is the same as item a of "II.6. Detection of CCL5 expression level" in this specific implementation) or ELISA (the method is the same as item b of "II.6. Detection of CCL5 expression level").

[0117] b. Detection of T cell chemotaxis: Conditioned culture medium (CM) was prepared by collecting cell supernatants after treatment with the candidate substance. Then, following the experimental method in Part 4, the CM was co-cultured with Jurkat cells in Transwell, and the number of migrating Jurkat cells was detected by the CCK-8 assay.

[0118] Results Analysis and Judgment: a. Compare the CCL5 levels or T cell chemotaxis counts of each candidate substance treatment group with the model control group.

[0119] b. If a candidate substance can cause a significant increase in CCL5 expression level with statistical significance (*p<0.05), and / or cause a significant enhancement in T cell chemotaxis with statistical significance (*p<0.05), then the candidate substance can antagonize the function of NOD1-S and reverse its mediated immunosuppression, and can be identified as a potential immunotherapy candidate drug targeting the NOD1-S-CCL5 axis.

[0120] c. For candidates that are initially positive, dose-dependent experiments and validation in multiple PTC cell lines are required to confirm their effectiveness and universality.

[0121] Combination therapy potential assessment: The selected effective candidate drugs are combined with clinically used immune checkpoint inhibitors (such as anti-PD-1 antibodies) in the cell model to assess whether they can synergistically enhance T cell recruitment and anti-tumor effects, providing a basis for their development into combination therapy drugs.

[0122] In all embodiments of this invention, except for the analysis of the public database, each experiment had at least three technical replicates, each independently repeated at least three times. Experimental data are expressed as mean ± standard deviation. Student's t-test was used for comparisons between two groups. One-way ANOVA was used for comparisons among multiple groups; if variances were homogeneous, Tukey's post-hoc test was used for pairwise comparisons. All statistical analyses were performed using GraphPad Prism or SPSS software. A p-value < 0.05 was considered statistically significant, and these values ​​were labeled in the figure as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0123] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. The application of a truncated splice isoform of the NOD1 gene as a transcriptional marker in the preparation of a detection reagent for lymph node metastasis of papillary thyroid microcarcinoma, characterized in that, The truncated splice isoform is NOD1-S, whose transcript contains a sequence region that can be specifically amplified by the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. A kit for detecting lymph node metastasis of papillary thyroid microcarcinoma, characterized in that, The invention includes primer pairs for specifically detecting NOD1-S transcriptional levels, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. The kit for detecting lymph node metastasis of papillary thyroid microcarcinoma according to claim 2, characterized in that, It also includes primer pairs for specifically detecting the transcriptional level of the CCL5 gene, the sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO:

8.

4. The application of a truncated splice isoform of the NOD1 gene, NOD1-S, as a target in the screening or preparation of immunotargeted drugs for the treatment of papillary thyroid microcarcinoma.

5. A method for screening immunotargeting drugs for the treatment of papillary thyroid microcarcinoma, characterized in that, Includes the following steps: S1. Provides a stable PTC cell model transfected with the NOD1-S expression vector; S2. Treat the PTC cell model with candidate substances; S3. Detect the expression level of CCL5 in the PTC cell model, and / or detect the chemotactic ability of the conditioned medium prepared from the PTC cell model on T cells; If, compared to an untreated cell model, the candidate substance causes a statistically significant increase in CCL5 expression levels and / or a statistically significant enhancement in T cell chemotaxis, then the candidate substance is a potential immune-targeting drug.

6. The method for screening immunotargeting drugs for treating papillary thyroid microcarcinoma according to claim 5, characterized in that, The PTC cell model is a cell model selected from TPC-1 or BCPAP cell lines that overexpresses NOD1-S.

7. The method for screening immunotargeting drugs for treating papillary thyroid microcarcinoma according to claim 5, characterized in that, The T cell chemotaxis capacity was assessed by indirect co-culturing Jurkat cells with conditioned medium derived from the PTC cell model, and the number of migrating cells was detected using the CCK-8 assay.

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