Application of SORD in treatment and prognosis evaluation of adenoid cystic carcinoma

Abstract

The invention relates to application of SORD in treatment and prognosis evaluation of adenoid cystic carcinoma. The key effect of SORD in adenoid cystic carcinoma is systematically revealed, the high expression of SORD is independently related to poor prognosis of a patient, malignant progression of tumors is driven by promoting cell proliferation, clone formation, migration and invasion, and the potential mechanism of SORD relates to activation of cell cycle pathways and metabolic reprogramming. The invention finds and defines that SORD can be used as an independent prognostic marker for ACC diagnosis, treatment and prognostic evaluation, and solves the problem of insufficient prognostic prediction precision of the existing clinical staging system; risk stratification of ACC patients is facilitated, intensive treatment and close monitoring are carried out on the patients with high SORD expression in advance, and a direction is provided for solving the dilemma that ACC lacks effective treatment means. The molecular pathogenesis of ACC is enriched, the limitation of traditional research is broken through, metabolic enzyme and tumor progression are closely linked, and a new thought is developed for research of ACC and even other cancers.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to application of SORD in treatment and prognosis evaluation of adenoid cystic carcinoma. BACKGROUND

[0002] Adenoid cystic carcinoma (ACC) is a malignant tumor derived from salivary glands and other exocrine glands, which is particularly prone to occur in oral salivary glands. ACC has unique biological behaviors, including slow growth but high invasiveness, early invasion of nerves (perineural invasion, PNI), high rate of distant metastasis and late recurrence tendency. At present, there are limited systemic treatment methods for advanced, recurrent or metastatic ACC, and the clinical efficacy is poor. For example, the objective response rates of drugs targeting VEGF such as lenvatinib and rivoceranib are only 12-16% and 1.7% respectively, indicating that ACC is generally resistant to existing targeted drugs. Therefore, finding new biomarkers and therapeutic targets is an urgent need to improve the survival rate of ACC patients and improve the prognosis

[0003] Sorbitol dehydrogenase (SORD) is a key enzyme in the polyol metabolic pathway, which catalyzes the reversible conversion between sorbitol and fructose, accompanied by the conversion of NAD+ / NADH. SORD is highly expressed in the liver and participates in the regulation of blood glucose homeostasis, and its abnormal activity is closely related to diabetic complications. However, the expression pattern, biological function and clinical significance of SORD in ACC and other malignant tumors are not clear. SUMMARY

[0004] The purpose of the present application is to address the problems existing in the prior art in the treatment of adenoid cystic carcinoma, to determine the expression level of SORD in ACC tissue and its relationship with the clinical pathological characteristics and prognosis of patients, to verify the feasibility of SORD as an independent prognostic biomarker for ACC, to clarify the role of SORD in ACC cell proliferation, migration, invasion and other processes, and to provide new strategies for ACC diagnosis, prognosis evaluation and treatment targeting SORD, aiming to provide a new direction and basis for risk stratification and targeted treatment strategies for this refractory malignant tumor.

[0005] To solve the above technical problems, the present application is realized by the following technical scheme.

[0006] The present application provides, in a first aspect, use of a SORD inhibitor in the preparation of a medicament for preventing and / or treating adenoid cystic carcinoma.

[0007] Preferably, the SORD inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the SORD gene.

[0008] Preferably, the SORD inhibitor is selected from siRNA designed based on the SORD gene, and the siRNA is selected from one or more of si#1, si#2, and si#3. The si#1 sequence is shown in SEQ ID NO:1 (5'-TTGTATCGGCCCATCTTGCTT-3'), the si#2 sequence is shown in SEQ ID NO:2 (5'-TGACCTTCTACTTTCCTGGTT-3'), and the si#3 sequence is shown in SEQ ID NO:3 (5'-TTGCAGTATCGAAACACGCTT-3').

[0009] Preferably, the adenoid cystic carcinoma is oral adenoid cystic carcinoma.

[0010] A second aspect of the present invention provides the use of a reagent for detecting SORD expression levels in the preparation of products for the auxiliary diagnosis and / or prognostic assessment of adenoid cystic carcinoma.

[0011] Preferably, the reagent for detecting SORD expression level includes primers for detecting SORD gene expression level and / or reagents for detecting SORD protein content.

[0012] Preferably, the primers for detecting SORD gene expression levels are selected from the following primer pairs:

[0013] The upstream sequence of the primer pair is shown in SEQ ID NO: 4 (5'-TTTCAAAGGTTGGAAAACAAAGAACCA-3'), and the downstream sequence is shown in SEQ ID NO: 5 (5'-TGCTTATAGAACCGGCAGAGG-3').

[0014] Preferably, the reagent for detecting SORD protein content is selected from anti-SORD antibody; for example, it can be selected from AB_2647748 (ThermoFisher RRID).

[0015] Preferably, the adenoid cystic carcinoma is oral adenoid cystic carcinoma.

[0016] A third aspect of the present invention provides a kit for the auxiliary diagnosis and / or prognostic assessment of adenoid cystic carcinoma, comprising reagents for detecting SORD expression levels.

[0017] Preferably, the reagent for detecting SORD expression level includes primers for detecting SORD gene expression level and / or reagents for detecting SORD protein content.

[0018] Preferably, the primers for detecting SORD gene expression levels are selected from the following primer pairs:

[0019] The upstream sequence of the primer pair is shown in SEQ ID NO: 4, and the downstream sequence is shown in SEQ ID NO: 5.

[0020] Preferably, the reagent for detecting SORD protein content is selected from anti-SORD antibody.

[0021] Preferably, the kit further includes one or more of PCR enzyme, PCR buffer, dNTPs, and fluorescent substrate.

[0022] Preferably, the fluorescent substrate is selected from Syber Green or fluorescently labeled probes.

[0023] Preferably, the adenoid cystic carcinoma is oral adenoid cystic carcinoma.

[0024] A fourth aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of adenoid cystic carcinoma, comprising a SORD inhibitor and pharmaceutically acceptable excipients.

[0025] Preferably, the SORD inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the SORD gene.

[0026] Preferably, the SORD inhibitor is selected from siRNA designed based on the SORD gene, and the siRNA is selected from one or more of si#1, si#2, and si#3, wherein the si#1 sequence is shown in SEQ ID NO:1, the si#2 sequence is shown in SEQ ID NO:2, and the si#3 sequence is shown in SEQ ID NO:3.

[0027] Preferably, the adenoid cystic carcinoma is oral adenoid cystic carcinoma.

[0028] Preferably, the pharmaceutically acceptable pharmaceutical excipient is selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants, and colorants.

[0029] It should be understood that, unless otherwise specified, in the context of this invention, SORD includes SORD nucleotides and the SORD protein encoded by those nucleotides. SORD inhibitors refer to substances capable of specifically downregulating SORD expression levels and / or the transcriptional level of its mature mRNA and / or the expression level or activity of SORD protein. For example, methods such as antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA erasers, target masking, and / or multi-target methods can be used to downregulate SORD expression levels and / or activity; any method that can reduce SORD levels and / or activity is acceptable. Primers and / or primer pairs refer to PCR primers used to synthesize the SORD gene cDNA strand in PCR, thereby detecting the expression level of the SORD gene mRNA. In addition to the primers and / or primers listed in this invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the SORD gene sequence using conventional methods and techniques in the field, including but not limited to molecular biology, and screening the designed primers and / or primer pairs using conventional experimental methods, as long as they can specifically detect the SORD expression level; conventional reagents and methods in the field can also be used to detect the SORD protein expression level; the same applies to other genes / proteins.

[0030] Adenoid cystic carcinoma, especially oral adenoid cystic carcinoma, is a malignant tumor with unique clinicopathological features, characterized by slow growth, high neurotropic invasiveness, high distant metastasis rate, and late recurrence. Patients often face a very high risk of local recurrence after initial surgical treatment due to nerve invasion or positive surgical margins. More importantly, even with good control of the primary lesion, fatal distant metastases can still occur years later, most commonly in the lungs. Currently, systemic therapy for advanced or metastatic ACC has limited efficacy, lacking an effective prognostic assessment system and therapeutic targets. Systemic treatment regimens primarily based on platinum-based, anthracycline-based, or VEGF-targeting drugs generally have objective response rates below 20%, indicating minimal efficacy.

[0031] In recent years, the role of metabolic reprogramming in tumor progression has received increasing attention. Sorbitol dehydrogenase, as a key enzyme in the polyol metabolic pathway, directly affects the intracellular NAD+ / NADH ratio, and may thus regulate the energy metabolism and redox balance of the entire cell. Although the role of SORD in diabetic complications has been extensively studied, its expression pattern, biological function, and clinical significance in malignant tumors such as ACC are almost entirely unknown.

[0032] This invention, through extensive research, systematically elucidates the evidence that SORD is upregulated in ACC, promotes malignant tumor progression, and is closely related to poor prognosis. It reveals the potential mechanism by which SORD exerts its effects through the regulation of cell cycle and metabolic pathways, and demonstrates the great potential of SORD as a prognostic biomarker and novel therapeutic target for ACC, providing new scientific basis and strategic directions for the precision diagnosis and treatment of ACC.

[0033] This invention first analyzed pan-cancer data from public databases such as TCGA, finding that SORD expression was significantly upregulated in various cancer tissues, including thyroid cancer and cholangiocarcinoma. More importantly, in ACC clinical tissue samples, immunohistochemical (IHC) results directly showed that SORD protein expression levels in ACC tumor tissues were significantly higher than in adjacent normal tissues, validating high SORD expression in ACC at the protein level. Survival analysis, the gold standard for evaluating biomarker value, clearly demonstrated in multiple independent ACC patient cohorts (GSE153002, PRJNA287156, and PMTD34552580) that patients with high SORD expression had significantly shorter overall survival, recurrence-free survival, and distant metastasis-free survival than those with low SORD expression. This indicates that high SORD expression is closely associated with a more aggressive clinical course and poorer prognosis in ACC. To exclude interference from traditional factors such as age, sex, and tumor stage, this invention performed univariate and multivariate Cox regression analyses. The results confirmed that, regardless of other clinical variables, SORD was an independent risk factor for predicting OS, LRRFS, and DMFS in ACC patients. This conclusion elevates SORD from a simple correlation indicator to an independent prognostic biomarker with strong clinical application potential, which can be used to identify high-risk patients in the early stages of diagnosis, thereby enabling more aggressive treatment and follow-up strategies. To demonstrate that SORD is not only a "bystander" but also a "participant," this invention used siRNA to target and silence the SORD gene, finding that knocking down SORD significantly inhibited the proliferation, colony formation, migration, and invasion of ACC cells. These functional experiments collectively demonstrate that SORD is a key functional gene essential for maintaining the core malignant phenotypes of ACC cells, such as proliferation, self-renewal, migration, and invasion, positioning it as a potential therapeutic target. GSEA analysis based on ACC transcriptome data revealed that samples with high SORD expression showed significant enrichment of gene sets closely related to cell cycle progression, such as the E2F target, G2M checkpoint, and MYC target V1. This suggests that SORD may promote tumor growth by driving cell cycle circulation and accelerating cell division. Gene set variation analysis (GSVA) ​​revealed a positive correlation between SORD expression and the activity of multiple metabolic pathways, including ascorbic acid and aldose metabolism, drug metabolism-cytochrome P450, and the citric acid cycle (TCA cycle). This suggests that ACC cells with high SORD expression may exhibit unique metabolic dependence, and their pro-cancer effect may be related to the remodeling of the intracellular metabolic network, providing energy and raw materials for rapid proliferation. Mutation spectrum analysis of the ACC cohort showed differences in the mutation frequencies of classic ACC driver genes such as MYB and NOTCH1 in the SORD-high expression group.This suggests that SORD may have a synergistic effect with the specific genetic mutation background of ACC, jointly driving the malignant evolution of the disease.

[0034] In summary, this invention systematically reveals the crucial role of SORD in adenoid cystic carcinoma (ACC): it is an oncogene-associated protein that is significantly upregulated in ACC, and its high expression is independently associated with poor patient prognosis. SORD drives malignant progression by promoting cell proliferation, colony formation, migration, and invasion, with its potential mechanisms involving activation of cell cycle pathways and metabolic reprogramming. This invention identifies and clarifies that SORD can serve as an independent prognostic biomarker for the diagnosis, treatment, and prognostic assessment of ACC, addressing the problem of insufficient prognostic prediction accuracy in existing clinical staging systems. It facilitates risk stratification of ACC patients, enabling early intensive treatment and close monitoring of patients with high SORD expression, providing direction for addressing the lack of effective treatments for ACC. This invention enriches the understanding of the molecular pathogenesis of ACC, overcomes the limitations of traditional research, and closely links metabolic enzymes with tumor progression, opening new avenues for research on ACC and other cancers. Attached Figure Description

[0035] Figure 1 Box plots of SORD expression levels based on TCGA and other cancer datasets.

[0036] Figure 2 This is a schematic diagram showing the results of pairwise comparisons of SORD expression levels in different types of cancer.

[0037] Figure 3 This is a schematic diagram of the tissue-specific distribution of SORD expression in human tumor tissue.

[0038] Figure 4 Heatmaps showing the three-dimensional structure of SORD protein and its subcellular localization in the cytoplasm, mitochondria, and extracellular matrix.

[0039] Figure 5 This is a schematic diagram showing the correlation analysis results between SORD expression and overall survival (OS) in various types of cancer.

[0040] Figure 6 This is a schematic diagram showing the prognostic correlation analysis results of SORD in the GSE153002, PRJNA287156, and PMID34552580 cohorts.

[0041] Figure 7 This is a schematic diagram illustrating the correlation between SORD expression and clinical variables (such as age, gender, stage, etc.) and OS, LRRFS, and DMFS in a univariate analysis.

[0042] Figure 8This diagram illustrates the correlation between SORD expression and clinical variables (such as age, sex, stage, etc.) and OS, LRRFS, and DMFS in a multivariate analysis.

[0043] Figure 9 This is a schematic diagram showing the results of immunohistochemical detection of SORD expression in ACC tissue.

[0044] Figure 10 This is a schematic diagram showing the efficiency results of WB testing si#1-3 on SORD knockdown.

[0045] Figure 11 This is a schematic diagram illustrating the effect of inhibiting SORD on cell proliferation.

[0046] Figure 12 A schematic diagram illustrating the effect of inhibiting SORD on cell colony formation.

[0047] Figure 13 A schematic diagram illustrating the effect of inhibiting SORD on cell migration.

[0048] Figure 14 This is a schematic diagram illustrating the effect of inhibiting SORD on cell invasion.

[0049] Figure 15 This is a schematic diagram of the GSEA enrichment analysis results for the PRJNA287156 dataset.

[0050] Figure 16 The GSEA enrichment score curve is shown.

[0051] Figure 17 This is a schematic diagram showing the results of variational analysis of SORD expression levels and gene sets for multiple metabolic pathways.

[0052] Figure 18 This diagram illustrates the differences in SORD expression levels in GSVA scores across ascorbic acid and aldose metabolism, drug metabolism, foreign body metabolism, and citric acid cycling.

[0053] Figure 19 This is a schematic diagram showing the correlation analysis results between SORD expression levels and ascorbic acid and aldose metabolism, drug metabolism, foreign substance metabolism, and citric acid cycle.

[0054] Figure 20 Heatmaps showing the frequencies of common gene mutations in the SORD low-expression and high-expression groups.

[0055] Figure 21 This is a schematic diagram showing the relative expression levels of different genes in the SORD low-expression and high-expression groups under mutated and non-mutated states. Detailed Implementation

[0056] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0057] Unless otherwise specified, the cell lines listed in this invention, including the SACC-LM cell line, were purchased from the ATCC cell bank and cultured according to existing techniques. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). The cells were then cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available.

[0058] In this invention, informed consent was obtained from all patients using clinical samples, and the relevant procedures and methods were approved by the ethics committee, complying with medical ethics requirements and the Good Clinical Practice (GCP) guidelines for drug clinical trials. All experimental procedures adhered to the Declaration of Helsinki. The experimental methods used in this invention, such as bioinformatics analysis, molecular biology experiments, cell biology experiments, and immunohistochemistry, are all conventional methods and techniques in the field. Bioinformatics analysis was performed using R software version 4.3, with the "limma" package used for differential analysis and the "survival" and "survminer" packages used for KM survival curve plotting. Representative results from biological experimental replicates are presented in the contextual figures, and data are displayed as mean ± SD and mean ± SEM as specified in the figures. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 8.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance were used to compare the differences in means between two or more groups. *p < 0.05 was considered a significant difference.

[0059] Example 1

[0060] To investigate the expression and prognosis of SORD in human cancers, we systematically analyzed the expression of SORD in various cancers and its relationship with patient prognosis using public databases such as TCGA. Specifically, we obtained RNA-seq data of 21 cancer tissues (pan-cancer) from the TCGA database using the "TCGA plot R" package, and obtained the expression level of SORD mRNA in each sample. Clinical data was integrated from the UCSC Xena platform, and the samples were divided into normal and tumor groups for differential SORD expression analysis. Data are expressed as log2(TPM+1). After data processing, we finally obtained SORD expression data for 19 paired cancer types, which were then divided into normal and tumor groups for differential expression analysis. Data are expressed as log2(TPM+1), with red and blue representing tumor and normal tissues, respectively. The differential expression analysis showed that compared with normal tissues, SORD expression was significantly upregulated in various cancer tissues, including thyroid cancer (THCA) (see [link to relevant documentation]). Figure 1 For paired samples, SORD expression was observed to be significantly upregulated in cancers such as cholangiocarcinoma (CHOL) compared to paired normal tissue (see [link to relevant documentation]). Figure 2 This suggests that the upregulation of SORD expression may be associated with the occurrence of various cancers and has the potential to be studied as a cancer biomarker.

[0061] Subsequently, protein expression data of SORD in cell lines were obtained from the Human Protein Atlas (HPA) database, and the distribution specificity of SORD in human tumor tissues, SORD protein structure, and the Human Protein Atlas (HPA) were analyzed. Figure 3 The tissue-specific distribution of SORD expression in human tumor tissues is shown, with red highlights indicating the main affected organs and tissues. The results show that SORD is enriched in tumors of organs such as salivary glands and lungs. Figure 4 The protein structure of SORD and a heatmap of its subcellular localization in the cytoplasm, mitochondria, and extracellular matrix are shown. Green indicates detected components, and gray indicates undetected components, indicating that SORD is mainly localized in the cytoplasm. The correlation between SORD expression levels and overall survival (OS) in various cancers was analyzed, showing hazard ratios (HR) and 95% confidence intervals (CI). A p-value less than 0.05 was considered statistically significant. The results showed that upregulation of SORD expression was significantly associated with a higher hazard ratio (HR) for OS in various cancers (see [link to study]). Figure 5 ).

[0062] To identify biomarkers associated with ACC, transcriptomic data and corresponding survival information (including transcriptomic, mutation, and clinical data) for GSE153002, PRJNA287156, and PMID34552580 were obtained from the GEO database. In four independent cohorts (GSE153002, PRJNA287156, and PMID34552580), patients were divided into high-SORD expression groups and low-SORD expression groups based on SORD expression. Kaplan-Meier survival analysis was performed using the Survival (version 3.7.0) and Survminer (version 0.5.0) software packages. The log-rank test was used to compare survival differences between groups to assess the impact of SORD gene expression on ACC prognosis, investigating the influencing factors on overall survival (OS), recurrence-free survival (LRRFS), and distant metastasis-free survival (DMFS) in ACC. For the GSE153002 cohort, the difference in overall survival (OS) between the low-SORD expression group (n=17) and the high-SORD expression group (n=12) was shown (p<0.014, Log-rank test), and the number of high- and low-risk patients and follow-up time (months) were indicated. For the PRJNA287156 cohort, SORD... The study compared the overall survival (OS) between the low expression group (n=8) and the high expression group (n=19) (p=0.022, Log-rank test), and noted the number of high- and low-risk patients and the follow-up time (months). For the PMD34552580_LRRFS cohort, the study compared the recurrence-free survival (LRRFS) between the low SORD expression group (n=42) and the high expression group (n=3) (p=0.025, Log-rank test), and noted the number of high- and low-risk patients and the follow-up time (months). For the PMD34552580_DMFS cohort, the study compared the distant metastasis-free survival (DMFS) between the low SORD expression group (n=32) and the high expression group (n=24) (p=0.024, Log-rank test), and noted the number of high- and low-risk patients and the follow-up time (months). The GSE153002 dataset showed that patients with low SORD expression had significantly better overall survival than those with high SORD expression (p=0.014, Log-rank test). The PRJNA287156 dataset further confirmed that the low SORD expression group had better overall survival than the high SORD expression group (p=0.022). In the PMD34552580 dataset, the low SORD expression group had better local recurrence-free survival (LRRFS) than the high SORD expression group (p=0.025); simultaneously, the low SORD expression group also had better distant metastasis-free survival than the high SORD expression group (p=0.024). Therefore, high SORD expression is associated with significantly poor prognosis; the high SORD expression group had worse overall survival, LRRFS, and DMFS (see [link to relevant documentation]). Figure 6 ).

[0063] To further assess the prognostic potential of SORD, univariate and multivariate Cox regression analyses were performed on the treated cohort to investigate the correlation between SORD expression levels and common clinical variables (age, sex, stage, etc.) and overall survival (OS), low-risk response rate (LRRFS), and deep vein stenosis (DMFS). Data are expressed as hazard ratios (HR) and 95% confidence intervals (CI), with p-values ​​less than 0.05 considered statistically significant. Univariate Cox regression analysis results showed that SORD was significant in predicting OS, prognostic fibrosis index (PFI), and diabetic renal syndrome (DSS) (p < 0.05) (see [link to relevant documentation]). Figure 7 Multivariate Cox regression analysis was performed using clinicopathological factors with p < 0.05. After adjusting for clinical variables such as age, sex, and stage, SORD was found to be an independent prognostic factor for ACC patients with OS, PFS, and DSS as endpoints (see [link to analysis]). Figure 8 The above results indicate that SORD is an independent prognostic factor for OS, PFI, and DSS in ACC, and the higher its expression level, the worse the patient's prognosis. In conclusion, based on the above results, SORD can be identified as an independent prognostic risk factor for OS, PFI, and DSS in ACC.

[0064] Considering the potential for population differences and systemic bias in public datasets, we collected tissue specimens from 20 ACC patients for immunohistochemical analysis to verify the prognostic robustness of SORD. The inclusion criteria for patients were as follows: (1) pathologically confirmed ACC; (2) no treatment had been received prior to tissue specimen collection. The specific steps of immunohistochemistry are as follows:

[0065] (1) The prostate cancer tissue was sliced ​​to a thickness of 4μm using a pathological tissue slicer, spread on a non-slip glass slide, and dried at 65℃ for 2 hours for later use.

[0066] (2) Dewaxing: Immerse the glass slide in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → ddH2O I for 3 min → ddH2O II for 3 min.

[0067] (3) Blocking peroxidase: Soak in 3% hydrogen peroxide for 10 min, then wash with PBS for 5 min.

[0068] (4) High-pressure antigen retrieval: Prepare EDTA retrieval solution and add it to the pressure cooker. Place the glass slide in the cooker, heat at 800W for 20 minutes, and then let it cool naturally.

[0069] (5) Wash the slide twice with PBS for 3 minutes each time. Use a small piece of paper to absorb the moisture around the tissue. Use a biochemical pen to draw circles 0.5 cm away from the tissue boundary.

[0070] (6) Oily tissue surface: Immerse the glass slide in 0.1% PBST, lift it up and down and soak for 3 minutes.

[0071] (7) Primary antibody incubation: Dilute the antibody (anti-SORD) with antibody diluent according to the instructions, incubate at 4°C overnight with 50uL, wash with PBS for 3min, and then wash with PBST for 3min.

[0072] (8) Secondary antibody incubation: 1 drop of DAKO secondary antibody per tissue, covering the tissue surface, incubated at 37°C for 1 hour, washed with PBS for 3 minutes, and then washed with PBST for 3 minutes.

[0073] (9) Prepare DAB chromogenic solution: Prepare the chromogenic solution according to the ratio. After shaking off the liquid on the slide, add freshly prepared DAB chromogenic solution to the circle. Control the chromogenic time under the microscope. The positive result is brownish-yellow. Soak the slide in PBS to stop the chromogenic process.

[0074] (10) Counterstaining cell nuclei: After terminating DAB staining, the slides were counterstained with hematoxylin for about 3 minutes, rinsed with running water, mounted with 20 μL of mounting medium, and observed under a microscope. SORD immunoreactivity was assessed based on the staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (0: <5%, 1: 5-25%, 2: 25-50%, 3: 50-75%, 4: >75%). The final IHC score was the product of the two.

[0075] Experimental results are as follows Figure 9 As shown in the figure. The results are consistent with the previous analysis, that is, SORD protein expression was significantly higher in ACC samples than in adjacent normal tissues.

[0076] Example 2

[0077] To more precisely elucidate the role of SORD in the biological behavior of ACC, a series of in vitro functional experiments were conducted. First, three SORD-targeting specific siRNAs were designed and transfected into SACC-LM cells. The knockdown efficiency was verified by Western blot. The specific steps are as follows:

[0078] (1) Using siRNA-mate (Genepharma), small interfering RNAs (siRNAs) targeting SORD (Tsingke) were transfected into SACC-LM cells (si#1 sequence as shown in SEQ ID NO: 1, 5'-TTGTATCGGCCCATCTTGCTT-3'; si#2 sequence as shown in SEQ ID NO: 2, 5'-TGACCTTCTACTTTCCTGGTT-3'; si#3 sequence as shown in SEQ ID NO: 3, 5'-TTGCAGTATCGAAACACGCTT-3'). Cells transfected with blank vector si-NC were set as a control and cultured under normal culture conditions. Cells in the logarithmic growth phase were digested, collected, and lysed with lysis buffer on ice for 1 h.

[0079] (2) Centrifuge at 15000×g at 4℃ for 15 min, take the supernatant and add loading buffer (to 1×), then incubate at 95℃ for 5 min.

[0080] (3) Take the sample prepared in step (2) and perform protein gel electrophoresis.

[0081] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200mA constant current transfer for 2h).

[0082] (5) After the transfer is completed, the PVDF membrane is removed and placed in the sealing solution and sealed at room temperature for 2 hours on a vertical shaker at 10 rpm.

[0083] (6) After sealing, clean the PVDF membrane, immerse it in the primary antibody (anti-SORD, AB_2647748 (ThermoFisherRRID)), and incubate it overnight on a vertical shaker at 10 rpm and 4°C.

[0084] (7) After the primary antibody incubation is complete, wash the PVDF membrane and then immerse it in the secondary antibody (HRP-linked anti-rabbit IgG) and incubate it at room temperature for 2 hours at 10 rpm on a vertical shaker.

[0085] (8) After the secondary antibody incubation is completed, the PVDF membrane is washed and the target protein is detected using a chemiluminescence analyzer (β-Tubulin is used as an internal control).

[0086] Test results as follows Figure 10As shown in the figure. The results showed that, compared with the blank vector si-NC group, the expression of SORD in SACC-LM cells transfected with si#1, si#2 or si#3 was significantly inhibited (****p<0.0001); then qRT-PCR was used to further verify the knockout efficiency of si#1-3 (the upstream sequence of the primer pair for detecting SORD level is shown in SEQ ID NO: 4 (5'-TTTCAAAGGTTGGAAAACAAAGAACCA-3'), and the downstream sequence is shown in SEQ ID NO: 5 (5'-TGCTTATAGAACCGGCAGAGG-3')), and the results were consistent with those of Western blot.

[0087] The SORD-knockdown prostate cancer cells constructed above were used to perform cell proliferation, colony formation, migration, and invasion assays. The specific steps for the cell proliferation assay are as follows:

[0088] (1) Transfect SORD-targeting siRNAs (si#2, si#3) into SACC-LM cells (constructed using the aforementioned method), and set up cells transfected with blank vector si-NC as a control.

[0089] (2) When the cells grow to the logarithmic phase, trypsin digest and count them. Select the appropriate cell density according to the doubling time of various cells and seed them into 96-well plates (3 replicates).

[0090] (3) The cells were cultured in an incubator at 37°C, and the cells were collected after 1 day, 2 days, 3 days, 4 days and 5 days of culture. 10 μL of CCK-8 was added to each well, and the culture plate was incubated in the incubator for 2 hours. The absorbance at 450 nm was measured to evaluate the cell proliferation status.

[0091] Experimental results are as follows Figure 11 As shown in the figure. The results showed that, compared with the control group (si-NC), inhibiting SORD expression with SORD inhibitor effectively suppressed the proliferation of prostate cancer cells.

[0092] The specific experimental steps for cell clone formation are as follows:

[0093] (1) Transfect SORD-targeting siRNAs (si#2, si#3) into SACC-LM cells (constructed using the aforementioned method), and set up cells transfected with blank vector si-NC as a control.

[0094] (2) When the cells grow to the logarithmic phase, digest them with trypsin and count them. Select an appropriate cell density (about 1000 cells / well) according to the doubling time of various cells. Seed them into a 6-well plate containing 2 mL of 37℃ pre-warmed culture medium and gently rotate it to disperse the cells evenly. Incubate the cells in a cell culture incubator at 37℃ with 5% CO2 and saturated humidity.

[0095] (3) When visible clones appear in the culture dish, stop the culture, discard the supernatant, carefully wash twice with PBS, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol and wash the residual methanol with water; cell clones can then be observed; under a microscope, a number of cells > 50 is considered a valid clone.

[0096] Test results as follows Figure 12 As shown in the figure. The results showed that, compared with the blank vector si-NC group, silencing the SORD gene with siRNA significantly reduced the clonogenic ability of prostate cancer cells and significantly inhibited the clonogenic formation of prostate cancer cells, with statistically significant differences.

[0097] The specific steps of the cell migration experiment are as follows:

[0098] The 8μm pore size chambers and 24-well plates of Corning Transwell Migration were used.

[0099] (1) Place the chamber in a 24-well plate and add 1 mL of basal culture medium to each well to moisten it.

[0100] (2) After trypsin digestion of SACC-LM cells (transfected with siRNA (si#2, si#3)) in the logarithmic growth phase, the cells were resuspended in basal medium to form a cell suspension, counted, and the cell density was adjusted to 5 × 10⁶ cells / year. 5 per mL.

[0101] (3) Remove and discard the basal culture medium in the transwell chamber and the 24-well plate. Add 200 μL of cell suspension to the upper chamber of the transwell chamber and 600 μL of complete culture medium (basal culture medium + 10% fetal bovine serum) to the lower chamber of the 24-well plate.

[0102] (4) The culture plate was placed in a CO2 incubator at 37°C and cultured for another 14 hours.

[0103] (5) Remove the chamber, rinse twice with PBS, fix with 4% paraformaldehyde in a 24-well plate for 20 min, and stain with crystal violet solution for 15 min.

[0104] (6) Carefully wipe away the cells in the upper layer of the microporous membrane of the chamber with a cotton swab and take a picture under an inverted microscope.

[0105] The results are as follows Figure 13 As shown in the figure. The results showed that treatment with SORD inhibitors si#2 or si#3 significantly inhibited the cell migration ability, and this difference was statistically significant compared with the untreated control group siNC.

[0106] The specific steps of the cell invasion experiment are as follows:

[0107] (1) One day before the experiment, put a tube of Matrigel matrix gel that had been dispensed from -20°C into a 4°C refrigerator overnight to melt it from a solid state to a liquid state.

[0108] (2) Prepare 10% Matrigel matrix gel on ice, take 50 μL to coat the upper chamber surface of the bottom membrane of the Transwell chamber, place it at 37°C, and wait for the matrix gel to solidify for 30 min.

[0109] (3) After trypsin digestion of SACC-LM cells (transfected with siRNA (si#2, si#3)) in the logarithmic growth phase, the cells were resuspended in basal medium to form a cell suspension, counted, and the cell density was adjusted to 1×10⁻⁶. 5 per mL.

[0110] (4) Remove and discard the basal culture medium from the small chamber and the 24-well plate. Add 200 μL of cell suspension to the upper chamber of the Transwell chamber and 600 μL of complete culture medium (basal culture medium + 10% fetal bovine serum) to the lower chamber of the 24-well plate.

[0111] (5) The culture plate was placed in a CO2 incubator at 37°C and cultured for another 48 hours.

[0112] (6) Remove the chamber, rinse twice with PBS, fix with 4% paraformaldehyde in a 24-well plate for 20 min, and stain with crystal violet solution for 15 min.

[0113] (7) Carefully wipe away the cells and matrix gel in the upper layer of the microporous membrane of the chamber with a cotton swab, and take a picture under an inverted microscope.

[0114] Experimental results are as follows Figure 14 As shown in the figure. The results showed that the number of cells migrating across the basement membrane was significantly reduced in the Transwell assay. The above experiments collectively demonstrate that SORD can promote the proliferation, colony formation, migration, and invasion of ACC cells, while inhibiting SORD expression can effectively suppress the progression of ACC.

[0115] Example 3

[0116] To investigate the potential molecular function of SORD and the biological processes it participates in, GSEA enrichment analysis was performed using the ClusterProfiler R package based on the correlation between SORD and mRNA in the PRJNA287156 dataset. Figures 15-16 The GSEA enrichment analysis results for the PRJNA287156 dataset are shown (see [link]). Figure 15 ) and GSEA enrichment score curve (see Figure 16 ).in Figure 15 The normalized enrichment scores (NES) and significance p-values ​​of SORD-related gene sets are displayed. The color gradient represents the p-value (0.00-0.06), and the size of the dots reflects the NES value (1.3-1.7). It shows that high SORD expression is associated with enrichment of multiple cancer-promoting pathways, including E2F target (NES=1.7), G2M checkpoint (NES=1.6), MYC target V1 (NES=1.5), and reactive oxygen species pathway. Figure 16 The enrichment trends of gene sets targeting E2F, G2M checkpoints, MYC target V1, reactive oxygen species pathway, adipogenesis, mitotic spindle, DNA repair, oxidative phosphorylation, and unfolded protein responses are shown. Green bands indicate high-expression regions of these genes in the sorted dataset, further confirming the positive enrichment of these gene sets in the high-expression sort. Based on these results, it can be concluded that SORD drives ACC progression by regulating pathways such as cell cycle, DNA repair, and oxidative stress.

[0117] The correlation between SORD expression levels and metabolic pathways in adenoid cystic carcinoma was then analyzed, and the results were as follows: Figures 17-19 As shown. Among them Figure 17 The heatmap shows the Gene Set Variation Analysis (GSVA) ​​scores of SORD expression (low / high) and various metabolic pathways (such as amino acid metabolism, carbohydrate metabolism, lipid metabolism, etc.). The color gradient represents the GSVA score, and the category is labeled as the metabolic pathway type. It shows that the SORD high expression group has upregulated scores in pathways such as amino acid, carbohydrate and lipid metabolism. Figure 18 Box plots showed the differences in GSVA scores between the low SORD expression group and the high SORD expression group in ascorbic acid and aldose metabolism, drug metabolism (cytochrome P450 and non-P450 enzymes), foreign substance metabolism (cytochrome P450), and the citric acid cycle (TCA cycle). *p<0.05 indicates statistical significance. The results showed that the high SORD expression group had significantly higher GSVA scores in ascorbic acid and aldose metabolism, drug metabolism (non-P450 enzymes), foreign substance metabolism (cytochrome P450), and the TCA cycle. Figure 19Scatter plots show the correlations between SORD expression and ascorbic acid and aldose metabolism, drug metabolism (non-P450 enzymes), foreign substance metabolism (cytochrome P450), and the citric acid cycle (TCA cycle). R-values ​​and p-values ​​indicate the strength and significance of the correlations. These results reveal that SORD reprograms the ACC metabolic microenvironment.

[0118] Studies have shown that high levels of somatic mutations and CNVs are associated with poorer patient survival. To clarify the molecular mechanism of SORD in ACC, a mutational genomics analysis was performed, and the experimental results are as follows: Figures 20-21 As shown. Among them Figure 20 The mutation spectrum heatmap shows the mutation frequency (0-47%) of common genes in the SORD low expression group and high expression group. The color gradient represents the mutation ratio, showing that the mutation frequency of genes such as MYB and NOTCH1 is increased in the SORD high expression group (p<0.05). Figure 21 Box plots comparing the relative expression levels of genes MYB, NOTCH1, ACTB, KDM6A, BCOR, KMT2D, CREBBP, PPP2R2B, ARID1A, EP300, POU3F3, KMT2C, MYH9, AHNAK, MYOM1, TP53, VCAN, MUC12, FGFR2, and ADNP in mutated and non-mutated states in the SORD low-expression and high-expression groups are shown. ns indicates no statistical significance. These results suggest that high SORD expression may synergistically promote the malignant phenotype of ACC with specific driver mutations.

[0119] In summary, this invention, through extensive research, has found that SORD is significantly highly expressed in ACC tissues, and its expression level is significantly negatively correlated with patient OS, LRRFS, and DMFS, clarifying the expression and prognostic value of SORD in ACC. Multivariate analysis confirms that SORD is an independent prognostic factor for ACC and can serve as a reliable biomarker for clinical prognostic assessment. A series of in vitro experiments demonstrate that knockdown of SORD significantly inhibits ACC cell proliferation, colony formation, migration, and invasion, suggesting that SORD is a key promoting factor for the malignant progression of ACC and revealing its functional role in the malignant behavior of ACC. Furthermore, GSEA and GSVA analyses indicate that SORD promotes ACC development through pathways such as cell cycle regulation and metabolic reprogramming. Mutation analysis provides direction for further research on the role of SORD in the ACC gene network, elucidating some of the molecular mechanisms of SORD's action.

[0120] In summary, this invention systematically reveals for the first time the expression, function, and clinical significance of SORD in adenoid cystic carcinoma (ACC), emphasizing its crucial role and filling a gap in ACC research. SORD overexpression promotes ACC cell proliferation and migration, is an independent risk factor for poor overall survival (OS), progression-free survival (PFI), and diabetic subarachnoid syndrome (DSS), and is closely associated with poor immunotherapy response. This invention clarifies that SORD can serve as a prognostic biomarker and therapeutic target for ACC. Inhibiting SORD or reducing its biological activity can significantly suppress the development of adenoid cystic carcinoma, prolong patient survival, and improve prognosis. Currently, ACC lacks effective targeted therapies and reliable prognostic biomarkers. This invention enriches the understanding of the relevant molecular mechanisms in the development and progression of adenoid cystic carcinoma, providing a new tool for ACC prognostic assessment and laying the experimental and theoretical foundation for developing SORD-targeted ACC therapeutics. It also proposes a gene therapy strategy targeting SORD, offering a new direction for precision treatment of ACC and possessing significant scientific value and clinical translational potential.

[0121] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. Application of SORD inhibitors in the preparation of drugs for the prevention and / or treatment of adenoid cystic carcinoma.

2. The application according to claim 1, characterized in that, The SORD inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the SORD gene.

3. The application according to claim 1, characterized in that, The adenoid cystic carcinoma mentioned is oral adenoid cystic carcinoma.

4. Application of reagents for detecting SORD expression levels in the preparation of products for the auxiliary diagnosis and / or prognostic assessment of oral adenoid cystic carcinoma.

5. The application according to claim 4, characterized in that, The reagents for detecting SORD expression levels include primers for detecting SORD gene expression levels and / or reagents for detecting SORD protein content.

6. The application according to claim 4, characterized in that, The primers used to detect SORD gene expression levels are selected from the following primer pairs: The upstream sequence of the primer pair is shown in SEQ ID NO: 4, and the downstream sequence is shown in SEQ ID NO:

5.

7. The application according to claim 4, characterized in that, The adenoid cystic carcinoma mentioned is oral adenoid cystic carcinoma.

8. A pharmaceutical composition for the prevention and / or treatment of adenoid cystic carcinoma, characterized in that, This includes SORD inhibitors and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The SORD inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the SORD gene.

10. The pharmaceutical composition according to claim 8, characterized in that, The adenoid cystic carcinoma mentioned is oral adenoid cystic carcinoma.

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