Application of TRIM33 and miR-BART8-3p as target points in preparation of late nasopharyngeal carcinoma drugs

Abstract

The application discloses application of TRIM33 and miR-BART8-3p as target points in preparation of a drug for advanced nasopharyngeal carcinoma. The application first discloses that in the advanced nasopharyngeal carcinoma, EBV encoded miR-BART8-3p directly targets and inhibits the expression of TRIM33, up-regulates the expression of PD-L1, and finally promotes the molecular mechanism of immune escape. This discovery is particularly aimed at patients with advanced metastatic nasopharyngeal carcinoma, and fills the blank of the mechanism research of immunotherapy drug resistance of the population. Therefore, by up-regulating TRIM33 or inhibiting miR-BART8-3p, the antitumor immunity of the patient with advanced nasopharyngeal carcinoma can be effectively regulated, and a new strategy for treating advanced nasopharyngeal carcinoma, reversing immunotherapy drug resistance and enhancing the curative effect of a PD-1 inhibitor is formed. The application provides a complete solution from mechanism research to treatment application and precise prediction for the advanced nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of TRIM33 and miR-BART8-3p as targets in the preparation of drugs for advanced nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor closely associated with Epstein-Barr virus (EBV) infection; recent data show that approximately 85% of newly diagnosed NPC cases are related to EBV infection. In China, non-keratinizing NPC accounts for over 95% of all NPC cases, and EBV is present in almost 100% of non-keratinizing NPC tissues. Currently, radiotherapy combined with chemotherapy is the main treatment, but the prognosis remains poor for patients with advanced recurrence or metastasis. In recent years, immune checkpoint inhibitors (such as PD-1 inhibitors) have shown potential in the treatment of advanced metastatic NPC, but clinical studies show that their objective response rate as monotherapy is only about 20-30%, indicating that most patients have primary or secondary drug resistance. Therefore, elucidating the deep molecular mechanisms of immune escape in NPC and, based on this, identifying new therapeutic targets and predictive biomarkers that can reverse drug resistance and improve efficacy are urgent clinical challenges.

[0003] EBV-encoded BART family microRNAs (miRNAs) play an important role in the development and progression of nasopharyngeal carcinoma. Among them, miR-BART8-3p has been shown to be highly expressed in nasopharyngeal carcinoma tissues, promoting nasopharyngeal carcinoma invasion and metastasis (Lin et al., EBV-miR-BART8-3p induces epithelial-mesenchymal transition and promotes metastasis of nasopharyngeal carcinoma cells through activating NF-κB and Erk1 / 2 pathways, J Exp Clin Cancer Res, 2018 Nov 26, 37(1): 283.). Patent CN111334579A discloses a method for detecting miR-BART8-3p in plasma for the diagnosis and prognostic assessment of nasopharyngeal carcinoma. miR-BART8-3p is a tumor marker for efficacy and prognosis prediction in patients with non-metastatic, early, and intermediate-to-late-stage nasopharyngeal carcinoma (Lin et al., Plasma Epstein-Barr Virus MicroRNA BART8-3p as a Diagnostic and Prognostic Biomarker in Nasopharyngeal Carcinoma, TheOncologist, 2022 Apr 5, 27(4): e340-e349 and Lin et al., Plasma Epstein-Barrvirus microRNA BART8-3p as a potential biomarker for detection and prognostic prediction in early nasopharyngeal carcinoma. Sci Rep, 2024 May 28, 14(1):7433). Patients with high expression of BART8-3p before treatment have shorter survival and poorer prognosis.In addition, miR-BART8-3p can also activate the NF-κB and Erk1 / 2 signaling pathways by targeting the RNF38 gene, thereby promoting epithelial-mesenchymal transition (EMT) and tumor metastasis (Lin et al., EBV-miR-BART8-3p induces epithelial-mesenchymal transition and promotes metastasis of nasopharyngeal carcinoma cells through activating NF-κB and Erk1 / 2 pathways, J Exp Clin Cancer Res, 2018 Nov 26, 37(1): 283.).

[0004] However, current technologies have not revealed any function of miR-BART8-3p in the immunomodulation of nasopharyngeal carcinoma. Whether and how it participates in tumor immune escape, whether it affects PD-L1 expression, and whether it can serve as a potential target for reversing immunotherapy resistance are all currently unknown. Furthermore, the expression of miR-BART8-3p in plasma in patients with advanced, metastatic nasopharyngeal carcinoma (stage IVB) is currently unclear, and the correlation between miR-BART8-3p and the efficacy of immunotherapy in advanced, metastatic nasopharyngeal carcinoma, as well as its prognostic significance, remains ambiguous.

[0005] Tripartite motif-containing protein 33 (TRIM33) is a protein with E3 ubiquitin ligase activity, involved in transcriptional regulation, signal transduction, and cell differentiation. While it has been reported as a tumor suppressor gene or oncogene in other tumors, the expression and function of TRIM33 in nasopharyngeal carcinoma, particularly its association with EBV infection and the immune microenvironment, have not been reported in any studies.

[0006] In summary, while existing technologies have revealed a pathway by which miR-BART8-3p promotes nasopharyngeal carcinoma metastasis, its role in regulating the tumor immune microenvironment and influencing the efficacy of immunotherapy remains a blank. In particular, addressing the challenge of immunotherapy resistance in advanced metastatic nasopharyngeal carcinoma, exploring novel mechanisms of EBV miR-BART8-3p in immune escape, and developing new treatment strategies and predictive tools based on these mechanisms, is the core technical problem this invention aims to solve. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art. In view of the current situation that the resistance rate of immunotherapy in advanced nasopharyngeal carcinoma is high, the function of miR-BART8-3p in immune regulation is unknown, and the mechanism of action of TRIM33 in nasopharyngeal carcinoma is blank, this invention provides the application of TRIM33 and miR-BART8-3p as targets in the preparation of drugs for advanced nasopharyngeal carcinoma, and for the first time reveals and verifies a brand-new immune escape regulatory axis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of a regulator of the TRIM33 protein in the preparation of a medicament for treating EB virus-positive nasopharyngeal carcinoma, wherein the regulator downregulates the expression of PD-L1 by upregulating the expression of the TRIM33 protein in tumor cells.

[0009] Furthermore, the regulator is an agonist that overexpresses the TRIM33 gene, and the agonist upregulates the expression of TRIM33 protein by infecting tumor cells.

[0010] Furthermore, the drug is used in combination with immune checkpoint inhibitors to enhance the efficacy of immunotherapy for EBV-positive nasopharyngeal carcinoma.

[0011] Furthermore, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0012] In a second aspect, the present invention provides the use of an inhibitor of miR-BART8-3p encoded by EB virus in the preparation of a medicament for treating EB virus-positive nasopharyngeal carcinoma, wherein the inhibitor upregulates TRIM33 protein and downregulates PD-L1 expression by inhibiting miR-BART8-3p.

[0013] Furthermore, the drug is used in combination with immune checkpoint inhibitors to enhance the efficacy of immunotherapy for EBV-positive nasopharyngeal carcinoma.

[0014] Furthermore, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0015] Thirdly, the present invention provides a pharmaceutical composition for treating EBV-positive nasopharyngeal carcinoma, comprising: (1) TRIM33 agonists or miR-BART8-3p inhibitors; (2) PD-1 / PD-L1 inhibitors.

[0016] Fourthly, the present invention provides the use of miR-BART8-3p and / or TRIM33 as biomarkers in the preparation of products for detecting or predicting the therapeutic effect of PD-1 inhibitors in EB virus-positive nasopharyngeal carcinoma.

[0017] Fifthly, the present invention provides the use of reagents for detecting the expression levels of miR-BART8-3p and / or TRIM33 in the preparation of products for predicting the efficacy of PD-1 inhibitors in EB virus-positive nasopharyngeal carcinoma.

[0018] In a sixth aspect, the present invention provides a method for screening candidate compounds for treating advanced nasopharyngeal carcinoma, comprising the following steps: (a) Provide the compound to be tested; (b) Contact the test compound with a test system selected from the group consisting of (i) cells transfected with a reporter gene vector containing the miR-BART8-3p binding site in the 3'UTR of the TRIM33 gene; or (ii) EB virus-positive nasopharyngeal carcinoma cell lines. (c) Detect the response indicators of the test system: for system (i), detect the activity of the reporter gene; for system (ii), detect the expression level of PD-L1 protein; (d) Select compounds that can significantly increase the reporter gene activity of system (i) or significantly downregulate the PD-L1 protein expression level of system (ii) as candidate compounds.

[0019] In a seventh aspect, the present invention provides a kit for predicting the response of patients with advanced nasopharyngeal carcinoma to PD-1 inhibitor therapy, comprising: (1) a first detection component for detecting TRIM33 protein expression in tumor tissue samples; and / or (2) a second detection component for detecting EB virus miR-BART8-3p expression in plasma samples; and (3) an instruction manual containing judgment criteria; wherein the judgment criteria include: a reference value for TRIM33 protein expression in tumor tissue of H-score 120; and / or a reference value for miR-BART8-3p expression in plasma of 3531 copies / mL; and wherein, when the detection value is lower than the reference value for TRIM33 protein and / or higher than the reference value for miR-BART8-3p, it suggests that the patient is likely to have a poor response to PD-1 inhibitor therapy.

[0020] This invention reveals and verifies for the first time the molecular mechanism by which EBV-encoded miR-BART8-3p promotes nasopharyngeal carcinoma escape and tumor progression by targeting and inhibiting TRIM33 expression, thereby leading to upregulation of tumor cell programmed death ligand 1 (PD-L1) expression. Figure 1 (As shown). Importantly, this invention experimentally demonstrates that this regulatory effect of miR-BART8-3p on PD-L1 is independent of its known pathway of promoting metastasis via RNF38, constituting another novel dimension of the pathogenic mechanism of this viral miRNA in nasopharyngeal carcinoma.

[0021] Therefore, upregulating the functional level of TRIM33 protein in tumor cells or inhibiting miR-BART8-3p can regulate PD-L1 expression, thereby enhancing the body's anti-tumor immunity.

[0022] In summary, this invention not only reveals that miR-BART8-3p directly targets TRIM33 and upregulates PD-L1 expression, but more importantly, it is the first to systematically construct and validate a complete immune regulatory axis of miR-BART8-3p→TRIM33→PD-L1. Through a series of rigorous and complementary experiments, including target binding validation, upstream and downstream gene expression regulation, in vitro and in vivo functional validation, and clinical cohort analysis, the biological function of this pathway and its key role in immunotherapy resistance in advanced nasopharyngeal carcinoma have been fully demonstrated.

[0023] The beneficial effects of this invention are as follows: (1) Innovative Mechanism. This invention elucidates and verifies for the first time a novel immune escape axis by which EB virus-encoded miR-BART8-3p regulates PD-L1 expression by targeting and inhibiting TRIM33. This discovery fills a gap in existing technology and provides a new perspective for understanding the immunosuppressive microenvironment of advanced nasopharyngeal carcinoma.

[0024] (2) Providing a novel treatment strategy. Based on the above mechanism, this invention proposes a novel treatment strategy that targets TRIM33 or miR-BART8-3p to reverse PD-L1-mediated immunosuppression. This provides a direct target and clear direction for developing novel drugs to treat advanced, especially drug-resistant, nasopharyngeal carcinoma.

[0025] (3) Pioneering precision combination therapy. The proposed combination therapy of the above-mentioned targeted drugs with existing PD-1 / PD-L1 inhibitors provides an innovative synergistic treatment pathway to overcome the common clinical problem of immunotherapy resistance, and is expected to significantly improve the treatment response rate of advanced nasopharyngeal carcinoma.

[0026] (4) Establishing companion diagnostic tools. Based on the correlation between TRIM33 and miR-BART8-3p expression levels and the efficacy of immunotherapy, this invention provides corresponding predictive kits. This helps to identify potential beneficiaries before treatment, enabling precise and individualized immunotherapy for advanced nasopharyngeal carcinoma, improving treatment efficiency and avoiding ineffective treatment. Attached Figure Description

[0027] Figure 1 : A schematic diagram of the molecular mechanism by which miR-BART8-3p targets TRIM33 and upregulates PD-L1 as disclosed in this invention.

[0028] Figure 2miR-BART8-3p regulates PD-L1 and its clinical relevance. (A) Proteomic results; (B) Western blotting results of PD-L1 expression after inhibiting miR-BART8-3p expression in EBV(+) nasopharyngeal carcinoma C666-1; (C) Western blotting results of miR-BART8-3p overexpression in EBV(-) nasopharyngeal carcinoma (CNE-1, CNE2); (D) Spearman correlation analysis of the correlation between miR-BART8-3p expression and PD-L1 expression in nasopharyngeal carcinoma tissues.

[0029] Figure 3 miR-BART8-3p targets and regulates TRIM33 and its clinical relevance. (A) TargetScan software predicts conserved binding sites complementary to the miR-BART8-3p seed sequence of TRIM33 3'UTR; (B) Dual-luciferase reporter gene validation; (C) Western blotting results of TRIM33 expression in NP69 and C666-1 cells (left); Western blotting results of TRIM33 expression after inhibiting miR-BART8-3 expression in EBV(+) nasopharyngeal carcinoma C666-1 cells (right); (D) Spearman correlation analysis shows the correlation between BART8-3p expression and TRIM33 expression in 20 clinical nasopharyngeal carcinoma tissues.

[0030] Figure 4 : Validation of TRIM33 regulation of PD-L1 expression. (A) Western blotting results of PD-L1 expression after constructing TRIM33 overexpression or knockdown stable transgenes in C666-1 cells using lentivirus; (B) Spearman correlation analysis of the correlation between TRIM33 expression and PD-L1 expression in 20 clinical nasopharyngeal carcinoma tissues.

[0031] Figure 5 Schematic diagram of Western blotting results verifying the core role of miR-BART8-3p in regulating PD-L1 expression via TRIM33.

[0032] Figure 6 In vivo experiments demonstrated that knocking down TRIM33 promotes tumor growth and weakens the efficacy of anti-PD-1 therapy. (A) Tumor growth curves in each group of humanized mouse models; (B) Statistical analysis of tumor weights removed at the end of the experiment.

[0033] Figure 7 : Receiver operating characteristic (ROC) curve of TRIM33 expression as a predictive biomarker for the efficacy of immunotherapy in advanced metastatic nasopharyngeal carcinoma.

[0034] Figure 8 Schematic diagram of the synergistic anti-tumor effect in vivo by combination therapy of TRIM33 overexpression and PD-1 inhibitor.

[0035] Figure 9 ROC curves predicting the efficacy of plasma miR-BART8-3p immunotherapy in advanced metastatic nasopharyngeal carcinoma. Detailed Implementation

[0036] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0037] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0039] In the following examples, the Gene ID of TRIM33 is 51592, and the sequence of miR-BART8-3p is 5'-GUCACAAUCUAUGGGGUCGUAGA-3'.

[0040] The miR-BART8-3p downregulated lentivirus, miR-BART8-3p overexpression lentivirus, TRIM33 overexpression lentivirus, and TRIM33 knockdown lentivirus involved in the following embodiments are all commercially available reagents and can be obtained directly through commercial channels. The lentivirus construction process will not be described in detail in this embodiment. Those skilled in the art can use conventional techniques to construct lentiviruses that achieve the above-mentioned regulatory objectives, as long as they can achieve the technical effects described in this invention.

[0041] The general experimental methods involved in the various embodiments of this invention, such as cell line construction, Western blotting identification, Real-Time PCR detection, dual-luciferase reporter system verification, and immunohistochemical detection, are as follows, and will not be repeated in the embodiments: Construction of stable lentiviral cell lines: 5 × 10 5Target cells (CNE-1, SUNE-1, or C666-1) at a specific well density were seeded into six-well plates, and 10% FBS RPMI 1640 medium was added. The plates were incubated overnight at 37°C and 5% CO2. When the cells reached approximately 50% confluence, viral infection was performed. The culture medium was discarded, and the plates were refilled with 500 μl of lentivirus, 500 μl of complete culture medium, and an appropriate amount of polybrene (final concentration 5 μg / ml). After gentle shaking, the plates were placed in an incubator for further incubation. Depending on the cell condition, 12-16 hours after infection, the virus-containing cell culture medium was discarded, and 2 ml of fresh complete culture medium was added. Cell fluorescence was observed under a fluorescence microscope after 3 days; the appearance of green fluorescence indicated successful viral infection. Subsequently, a low concentration of puromycin was added for selection, gradually increasing the puromycin concentration until more than 95% of cells showed green fluorescence intensity, and this puromycin concentration was maintained.

[0042] (2) Western Blot identification: Collect cells in the logarithmic growth phase, add cell lysis buffer to lyse, centrifuge and collect supernatant, use Bradford method to quantify protein, take the quantified protein, add sample loading buffer and boil the sample for 5 min to denature, perform SDS-PAGE electrophoresis, the protein loading amount is 50-100 μg, perform electrotransfer using semi-dry method, block with 10% skim milk powder for 2 h, add primary antibody and incubate overnight at 4°C, wash the membrane 3 times with TTBS, 5 min each time, add HRP-labeled secondary antibody, incubate at room temperature for 2 h, wash the membrane 3 times with TTBS as above, develop in dark room ECL, scan the results, use GAPDH as internal reference to perform quantitative analysis of protein expression level.

[0043] (3) Real-Time PCR detection of PD-L1 and TRIM33 in nasopharyngeal carcinoma cell lines and tissues: Total RNA was extracted from tissues using TRIZOL reagent, and the RNA concentration was detected by an ultra-micro spectrophotometer; cDNA reverse transcription synthesis was performed according to the recommended method of the miScript II RT Kit (Qiagen); quantification was performed according to the recommended method of the miScript SYBR Green PCR Kit (Qiagen). GAPDH was used as an internal control for the mRNA reaction; 2 −ΔΔCt The relative concentration of mRNA was calculated using this method. Each experiment was performed in triplicate, and the experiment was repeated three times.

[0044] (4) Real-Time PCR detection of miR-BART8-3p in nasopharyngeal tissue: Total RNA was extracted from the tissue using TRIZOL reagent, and the RNA concentration was detected using a micro spectrophotometer. RNA was thawed on ice, and 5× miScript RT Buffer and RNase-free water were thawed at room temperature (15-25ºC). A reverse-transcription mastermix was prepared on ice. The miScript Reverse Transcriptase Mix was aliquoted into each tube, and 1 μg of RNA was added. The mixture was gently mixed, briefly centrifuged, and then stored on ice. The mixture was incubated at 37°C for 60 minutes and then at 95°C for 5 minutes to inactivate the miScript Reverse Transcriptase Mix. The mixture was then stored on ice to complete the cDNA synthesis of the miRNA. 2× QuantiTect SYBR Green PCR Master Mix, 10× miScript Universal Primer, 10× miScript Primer Assay, template cDNA, and RNase-free water were thawed and prepared according to the manufacturer's instructions to prepare the reaction mix. Add template cDNA to each tube, mix thoroughly, and then aliquot the mixture into tubes containing cDNA. Briefly centrifuge at 3000× g for 2 minutes. Set up the PCR program for quantitative PCR detection; the experiment is set up with three replicates, each performed independently three times.

[0045] (5) The steps for validating the dual-gene luciferase reporter system are as follows: 1) Construction of luciferase reporter vector: Purchase luciferase reporter vector containing TRIM33 3'UTR wild-type (WT) and mutant (MUT) sequences from Shanghai Jikai Gene Technology Co., Ltd. 2) Plating: 293T cells in logarithmic growth phase were prepared into a cell suspension and seeded into 24-well culture plates. The cells were cultured at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 60%. 3) Plasmid transfection: Using ROCHE (X-tremegene HP transfection reagent), dissolve the HP transfection reagent and the wild-type or mutant luciferase reporter vector plasmid in 100 μL opti-MEM. Add miR-BART8-3p mimics or negative control (NC) to both groups, mix well, and incubate at room temperature for 20 min. Adjust the volume according to the ratio of 1 μg plasmid to 2 μL X-tremegene HP, and remove 300 μL of culture medium from the culture wells. Add the plasmid and X-tremegene HP mixture, and incubate at 37°C and 5% CO2 for 5-6 h. Then, add 200 μL of complete culture medium containing 10% serum to ensure that the entire culture well contains 500 μL of culture medium. 4) Observe the expression of fluorescently labeled genes on the plasmid 24-48 h after transfection to determine the transfection efficiency; 5) 48 hours after cell transfection, the luciferase activity of each group of cells was detected according to the instructions of the luciferase detection kit.

[0046] (6) Immunohistochemical detection of TRIM33 in paraffin-embedded nasopharyngeal carcinoma tissue: 4 μm thick paraffin sections of nasopharyngeal carcinoma tissue were prepared, and 3.0% H2O2 solution was added. The sections were incubated at room temperature for 10 min to block endogenous peroxidase activity, and then washed three times with PBS. Anti-TRIM33 (1:1000) was added and incubated at 37℃ for 1 h, followed by three washes with PBS. Horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature for 30 min, followed by three washes with PBS. 50.0 μl of streptomycin-avidin-peroxidase solution was added and incubated at room temperature for 30 min, followed by three washes with PBS. 100.0 μl of freshly prepared DAB solution was added. (3,3'-Diaminobenzidine hydrochloride) chromogenic solution; thorough rinsing under tap water, hematoxylin counterstaining, differentiation with 0.1% hydrochloric acid alcohol, rinsing with PBS to return to blue; graded alcohol dehydration and drying, followed by xylene clearing, and mounting with neutral resin; prepare corresponding positive controls, and use homology-matched IgG antibodies to stain nasopharyngeal carcinoma tissue as negative controls instead of primary antibodies; photograph under a microscope and calculate the TRIM33 H-score: multiply the protein expression intensity and the score of the proportion of positive cells to obtain a semi-quantitative score. Use the average H score (H-score) of all patient samples as the cutoff value to determine TRIM33 positivity.

[0047] Example 1: miR-BART8-3p regulation of PD-L1 and its clinical relevance to PD-L1 This embodiment systematically verifies the regulation of PD-L1 by miR-BART8-3p and its clinical relevance to PD-L1 at the molecular, cellular, and histological levels, respectively. The specific methods are as follows: At the molecular level, Epstein-Barr virus-positive nasopharyngeal carcinoma cell line C666-1 was transfected with miR-BART8-3p downregulated lentivirus (experimental group) and empty vector lentivirus (control group), with three replicates in each group. TMT (tumor-to-tumor) proteomic proteomic analysis was used to investigate the expression relationship of miR-BART8-3p, TRIM33, and PD-L1 at the molecular level. Results are as follows: Figure 2 As shown in Figure A, inhibiting miR-BART8-3p expression in C666-1 cells significantly downregulated PD-L1 protein expression and upregulated TRIM33 protein expression.

[0048] Cellular level experiments further confirmed that, compared with the control group (In-NC), the experimental group (In-BART8-3p group) inhibited miR-BART8-3p expression and decreased PD-L1 expression in C666-1 cells. Figure 2 B). Conversely, in EBV(-) nasopharyngeal carcinoma cell lines (CNE-1, CNE2), overexpression of miR-BART8-3p and increased PD-L1 expression were observed. Figure 2 C).

[0049] In addition, PCR detection was performed on 20 clinical nasopharyngeal carcinoma tissue samples, and Spearman correlation analysis showed that miR-BART8-3p expression was positively correlated with PD-L1 expression (r = 0.540, p = 0.002), meaning that patients with high miR-BART8-3p expression also had high PD-L1 expression, and vice versa. Figure 2 D).

[0050] Example 2: miR-BART8-3p targeted regulation of TRIM33 and its clinical relevance to TRIM33. Target prediction using TargetScan bioinformatics software revealed a conserved binding site complementary to the miR-BART8-3p seed sequence in the TRIM33 3'UTR region. Figure 3 A). Luciferase reporter vectors containing the wild-type (WT) or mutant (MUT) site were constructed and co-transfected with miR-BART8-3p mimics into HEK293T cells. The results showed that, compared with the negative control group, miR-BART8-3p significantly inhibited the luciferase activity of the WT reporter vector. Figure 3B), while having no significant effect on the MUT vector, confirming the direct targeting relationship between miR-BART8-3p and TRIM33. This result is consistent with the proteomic results in Example 1, namely, inhibiting miR-BART8-3p expression in the C666-1 cell line significantly upregulates TRIM33 (B). Figure 2 A).

[0051] PCR and Western blot analysis further showed that TRIM33 levels were significantly decreased in EBV-positive C666-1 cells compared to EBV-negative NP69 nasopharyngeal cells; conversely, inhibition of miR-BART8-3p expression in C666-1 cells significantly upregulated TRIM33. Figure 3 C). Furthermore, PCR detection was performed on 20 clinical nasopharyngeal carcinoma tissues, and Spearman correlation analysis showed that miR-BART8-3p expression was negatively correlated with TRIM33 expression (r = -0.454, p = 0.012), meaning that patients with high miR-BART8-3p expression had low TRIM33 expression, and vice versa. Figure 3 D).

[0052] Example 3: Validation of TRIM33 regulation of PD-L1 expression In EBV(+) nasopharyngeal carcinoma C666-1 cells, a stable TRIM33 overexpression cell line (TRIM33 group) and an empty vector control cell line (NC group) were constructed using lentivirus; in CNE-2 cells, a stable TRIM33 knockdown cell line (sh-TRIM33) and a corresponding empty vector control (sh-NC group) were constructed. Western blot analysis showed that the total PD-L1 protein level was significantly reduced in the TRIM33 group cells, while PD-L1 expression was significantly increased in the sh-TRIM33 cells. Figure 4 A). Overexpression of TRIM33 significantly downregulated PD-L1 protein expression, while knockout of TRIM33 significantly upregulated PD-L1 protein expression. PCR detection of 20 clinical nasopharyngeal carcinoma tissues and Spearman correlation analysis showed a significant negative correlation between TRIM33 and PD-L1 expression (r = -0.499, p = 0.005). Figure 4 (B) This result suggests that low TRIM33 expression is associated with high PD-L1 expression.

[0053] Example 4: Verifying the core role of miR-BART8-3p in regulating PD-L1 expression through TRIM33 To confirm that TRIM33 is a functional mediator essential for miR-BART8-3p regulation of PD-L1 expression, EBV-positive C666-1 cells were selected and divided into experimental and control groups. The experimental group was transfected with the TRIM33 overexpression vector, while the control group was transfected with the empty control plasmid. Forty-eight hours after transfection, cells were collected, and the protein expression levels of TRIM33 and PD-L1 were detected by Western blotting. Results are as follows: Figure 5 As shown, overexpression of TRIM33 can reverse the PD-L1 upregulation induced by miR-BART8-3p overexpression, restoring PD-L1 levels to near the control group level. This result demonstrates that restoring TRIM33 expression can counteract the upregulation of PD-L1 by miR-BART8-3p, establishing TRIM33's central role in the miR-BART8-3p-mediated PD-L1 regulatory pathway and ruling out the possibility that miR-BART8-3p affects PD-L1 through other major pathways independent of TRIM33.

[0054] Example 5: In vivo experiment on the effect of TRIM33-targeted immunotherapy First, a humanized mouse model was constructed: 5×10 6 C666-1 cells stably expressing luciferase were subcutaneously inoculated into NSG mice. One week after inoculation, 1×10⁶ cells / mL were injected via the tail vein. 7 cells / mL of healthy human peripheral blood mononuclear cells (PBMCs) to rebuild the human immune system. The tumor volume was increased to approximately 100 mm. 3 Mice were randomly divided into 4 groups (n=8), and the treatment for each group was as follows: (1) sh-NC (2 mg / kg) + isotype control IgG (10 mg / kg): control group; (2) sh-NC (2 mg / kg) + anti-PD-1 antibody (10 mg / kg): immunotherapy alone group; (3) sh-TRIM33 (2 mg / kg) + isotype control IgG (10 mg / kg): sh-TRIM33 alone group; (4) sh-TRIM33 (2 mg / kg) + anti-PD-1 antibody (10 mg / kg): sh-TRIM33 combined with immunotherapy group. All groups were administered via intraperitoneal injection twice a week, using PBS buffer (pH=7.2) as the solvent.

[0055] The results are as follows Figure 6As shown, compared with the control (Group 1), TRIM33 knockdown alone (Group 3) significantly accelerated tumor growth (p < 0.05); anti-PD-1 therapy (Group 2) showed a significant tumor-suppressive effect (p < 0.05). However, in TRIM33 knockdown tumors (Group 4), the tumor-suppressive effect of anti-PD-1 therapy was significantly weakened. This indicates that low TRIM33 expression leads to an immunosuppressive microenvironment and makes the tumor insensitive to anti-PD-1 therapy. The above in vivo experimental results show that low TRIM33 expression weakens the efficacy of anti-PD-1 therapy. Therefore, the combined use of the TRIM33 modulator or miR-BART8-3p inhibitor of the present invention with a PD-1 inhibitor can synergistically enhance anti-tumor immunity, especially for nasopharyngeal carcinoma patients with low TRIM33 expression, and can serve as a strategy to overcome immunotherapy resistance.

[0056] Example 6: Validation of TRIM33 expression level as a predictive biomarker for the efficacy of immunotherapy in advanced metastatic nasopharyngeal carcinoma. Pretreatment tumor tissue samples were collected from 50 patients with metastatic nasopharyngeal carcinoma (stage IVB) who received first-line PD-1 inhibitor therapy. Patients were divided into an objective response group (ORR, n=38) and a non-response group (Non-ORR, n=12) according to RECIST 1.1 criteria. TRIM33 protein expression in all samples was detected by immunohistochemistry (IHC) using a specific antibody against TRIM33, and the H-score (used to quantify protein expression intensity) was calculated.

[0057] The results showed that the median TRIM33 H-score in the ORR group (180) was significantly higher than that in the Non-ORR group (85), and the difference was statistically significant. To further verify the predictive value of TRIM33 for the efficacy of PD-1 inhibitors, receiver operating characteristic (ROC) curves were plotted. The area under the curve (AUC) of TRIM33 H-score predicting objective response to immunotherapy was 0.76 (95% CI: 0.62-0.90). Figure 7Using an H-score of 120 as the optimal cutoff value, the predictive sensitivity and specificity were 84.2% and 83.3%, respectively. This result demonstrates that the reagent for detecting TRIM33 protein expression can be used to prepare products for predicting the efficacy of PD-1 inhibitors in nasopharyngeal carcinoma patients (such as a test kit containing this antibody and corresponding judgment criteria). In this embodiment, the "reference value" is H-score = 120. Based on the above data, those skilled in the art can prepare kits containing anti-TRIM33 antibodies, instructions for use, and the aforementioned judgment criteria (H-score = 120) to assist in clinical decision-making. This predictive method includes: detecting the expression level of TRIM33 in a patient's tumor sample and comparing it with a reference value (e.g., 120) to assess the likelihood of the patient benefiting from PD-1 inhibitor treatment.

[0058] Example 7: Synergistic antitumor effect in vivo of TRIM33 overexpression combined with PD-1 inhibitor therapy To verify the combination therapy strategy based on the regulatory mechanism of the present invention, this embodiment further evaluated the synergistic anti-tumor effect of TRIM33 functional activation and PD-1 inhibitor.

[0059] C666-1 cells stably overexpressing TRIM33 (TRIM33 group) and its empty vector control (NC group) were selected and subcutaneously seeded with 1×10⁻⁶ cells. 7 The cells / mL mentioned above were used in NSG mice to reconstruct the human immune system and to establish an in vivo nasopharyngeal carcinoma model. The tumor volume was allowed to grow to approximately 100 mm. 3 At that time, they were randomly divided into 4 groups (n=8): G1: NC + isotype control IgG; G2: NC + anti-PD-1 antibody (10 mg / kg); G3: TRIM33 + isotype control IgG; G4: TRIM33 + anti-PD-1 antibody. All groups were administered the drug via intraperitoneal injection twice weekly.

[0060] The results are as follows Figure 8 As shown, compared with G1, TRIM33 overexpression alone (G3) moderately inhibited tumor growth; PD-1 antibody alone (G2) had a clear tumor-suppressive effect. Most importantly, the combination therapy of TRIM33 overexpression and PD-1 antibody (G4) exhibited a significant synergistic effect, with its tumor-suppressive effect significantly superior to either single-agent treatment group (p<0.01). This in vivo experiment directly confirms that upregulating TRIM33 in combination with a PD-1 inhibitor can synergistically enhance the anti-tumor effect, providing a novel and feasible combination therapy regimen for overcoming immunotherapy resistance in clinical practice.

[0061] Example 8: Validation of plasma miR-BART8-3p expression level as a predictive biomarker for immunotherapy efficacy in patients with advanced, metastatic nasopharyngeal carcinoma (stage IVB). Pretreatment plasma samples were collected from 50 patients with metastatic nasopharyngeal carcinoma (stage IVB) who received first-line PD-1 inhibitor therapy. Patients were divided into an objective response group (ORR, n=38) and a non-response group (Non-ORR, n=12) according to RECIST 1.1 criteria. The expression of miR-BART8-3p in plasma was detected using quantitative immunofluorescence PCR (specific primer and probe sequences and detection system are described in patent CN111334579A).

[0062] The median miR-BART8-3p expression in the ORR group (500.00 copies / mL) was significantly lower than that in the Non-ORR group (113095.18 copies / mL), a statistically significant difference. Receiver operating characteristic (ROC) curves showed that the area under the curve (AUC) for miR-BART8-3p predicting objective response to immunotherapy was 0.848 (95% CI: 0.728–0.967). Using 3531 copies / mL as the optimal cutoff value, the predictive sensitivity and specificity were 83.3% and 89.5%, respectively. This example demonstrates that detecting miR-BART8-3p expression in plasma can be used to prepare products for predicting the efficacy of PD-1 inhibitors in nasopharyngeal carcinoma patients (such as primers and probes for detecting miR-BART8-3p). The "reference value" in this example is 3531 copies / mL. Based on the above data, those skilled in the art can prepare kits containing specific primers, probes, instructions for use, and the aforementioned judgment criteria (copies / mL) for detecting miR-BART8-3p, to assist in clinical decision-making. This predictive method involves detecting the expression level of miR-BART8-3p in the patient's tumor plasma and comparing it to a reference value (e.g., 3531 copies / mL) to assess the patient's likelihood of benefiting from PD-1 inhibitor therapy. A value higher than this reference value suggests a lower likelihood of benefit from PD-1 inhibitor therapy, while a value lower than this reference value suggests a higher likelihood of benefit, thus providing a basis for individualized clinical treatment.

[0063] In summary, this invention systematically reveals a novel mechanism by which EBV miR-BART8-3p upregulates PD-L1 expression by targeting TRIM33. This discovery is independent of the known metastatic function of miR-BART8-3p, expanding our understanding of its oncogenic function. TRIM33 modulators or miR-BART8-3p inhibitors targeting this pathway provide a novel treatment strategy for immunotherapy in nasopharyngeal carcinoma patients, especially those resistant to immunotherapy. Furthermore, reagents for detecting TRIM33 and / or miR-BART8-3p can be used to prepare products for predicting the efficacy of immunotherapy. The targets, treatment strategies, and predictive tools provided by this invention offer a novel solution to overcoming the clinical challenge of immunotherapy resistance in nasopharyngeal carcinoma.

[0064] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this utility model are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The use of a regulator of TRIM33 protein in the preparation of a drug for treating EB virus-positive nasopharyngeal carcinoma, characterized in that: The regulator downregulates PD-L1 expression by upregulating the expression of TRIM33 protein in tumor cells.

2. The application according to claim 1, characterized in that: The regulator is an agonist that overexpresses the TRIM33 gene, and the agonist upregulates the expression of TRIM33 protein by infecting tumor cells.

3. The application according to claim 1 or 2, characterized in that: The drug, when used in combination with immune checkpoint inhibitors, is used to enhance the efficacy of immunotherapy for EBV-positive nasopharyngeal carcinoma.

4. The application according to claim 3, characterized in that... The immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

5. The use of an inhibitor of miR-BART8-3p encoded by EB virus in the preparation of a medicament for treating EB virus-positive nasopharyngeal carcinoma, characterized in that... The inhibitor upregulates TRIM33 protein and downregulates PD-L1 expression by inhibiting miR-BART8-3p.

6. The application according to claim 5, characterized in that... The drug, when used in combination with immune checkpoint inhibitors, is used to enhance the efficacy of immunotherapy for EBV-positive nasopharyngeal carcinoma.

7. The application according to claim 6, characterized in that: The immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

8. A pharmaceutical composition for treating EBV-positive nasopharyngeal carcinoma, characterized in that... :include: (1) TRIM33 agonists or miR-BART8-3p inhibitors; (2) PD-1 / PD-L1 inhibitors.

9. The use of miR-BART8-3p and / or TRIM33 as biomarkers in the preparation of products for detecting or predicting the efficacy of PD-1 inhibitor therapy in EBV-positive nasopharyngeal carcinoma.

10. Application of reagents for detecting miR-BART8-3p and / or TRIM33 expression levels in the preparation of products for predicting the efficacy of PD-1 inhibitors in EB virus-positive nasopharyngeal carcinoma.

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