Use of snord54, snord41 as diagnostic markers for non-small cell lung cancer

Abstract

The application relates to application of SNORD54 and SNORD41 as diagnostic markers of non-small cell lung cancer, and belongs to the technical field of medical biological detection. In the application scheme, the differential expression of serum exosome snoRNAs in NSCLC patients and healthy individuals is discussed, and the curative effect of the differentially expressed exosome snoRNAs in the diagnosis of NSCLC is evaluated. The results of the application show that SNROD54 and SNORD41 in tumor-derived serum exosomes can be used as new biomarkers of NSCLC, can significantly improve the AUC value of traditional tumor markers, and can make up for the deficiency of the prior art in early diagnosis of NSCLC, and has important significance for early diagnosis of NSCLC.

Description

Technical Field

[0001] This application relates to the application of SNORD54 and SNORD41 as diagnostic markers for non-small cell lung cancer, and belongs to the field of medical biodetection technology. Background Technology

[0002] Epidemiological statistics for cancer in 2022 show that lung cancer has become the second leading cause of new cancer cases, yet it remains the leading cause of cancer death. Non-small cell lung cancer (NSCLC), the most common histological subtype of lung cancer, accounts for approximately 85% of all lung cancers. Despite significant advancements in diagnosis and treatment in recent years, the five-year survival rate for NSCLC patients remains low. A key factor contributing to this poor prognosis is that clinical diagnosis is often at an advanced stage, depriving patients of the opportunity for radical treatment. Therefore, achieving early detection and diagnosis of NSCLC remains a major challenge in clinical practice.

[0003] Small nucleolar RNAs (snoRNAs) are a class of non-coding RNAs approximately 60–300 nucleotides in length, primarily located in the nucleolar region of the cell nucleus. Their classic molecular function is to guide the chemical modification of ribosomal RNA (rRNA), mainly including two types: 2'-O-methylation (Nm) modification guided by C / D box snoRNAs and pseudouridineization (ψ) modification guided by H / ACA box snoRNAs. They bind to a core group of proteins called ribonucleoproteins, forming stable functional granules. Increasing evidence suggests that snoRNAs play a crucial role in tumorigenesis and development, and aberrant expression has been observed in various cancers, some of which are cancer type-specific. For example, SNORD113-1 expression in hepatocellular carcinoma (HCC) is significantly lower than in adjacent normal tissues, and its downregulation is significantly associated with patient survival. Upregulated SNORD12C / SNORD78 in tumors promotes the translational activity of downstream cancer-related target proteins and promotes tumorigenesis by enhancing methylation at specific sites on 28 srRNA.

[0004] Exosomes are extracellular vesicles ranging from 30 to 150 nanometers in diameter, enclosed in a lipid bilayer membrane, and released from almost all cell types via the endosomal pathway. These vesicles play a crucial messenger role in intercellular communication, transporting various bioactive molecules, including proteins, lipids, RNA, and DNA, from their parent cells to recipient cells, influencing a wide range of physiological and pathological processes. Due to their stability, accessibility in body fluids (such as blood and urine), and cell-specific abundance, increasing research indicates that exosomes are promising biomarkers for disease diagnosis, particularly cancer diagnosis.

[0005] Exosomes contain snoRNAs, and multiple studies using high-throughput sequencing and other technologies have identified snoRNAs in exosomes from various cell sources. These snoRNAs are encapsulated within a lipid bilayer of the exosome and protected by ribonucleoproteins, enabling them to remain stably in circulating body fluids such as blood and urine, resisting degradation by RNases. Furthermore, exosomes can be indirectly obtained from body fluids such as serum, plasma, and urine. These characteristics make the detection of snoRNA biomarkers completely non-invasive or minimally invasive. A study on patients with pulmonary nodules found that the levels of snoRNAs U78 and U37 in serum exosomes were significantly higher in patients with malignant nodules than in patients with benign nodules. Four snoRNAs (SNORD99, SNORD22, SNORD26, and SNORA50C) identified from urinary exosomes can serve as diagnostic biomarkers for ccRCC, with a diagnostic model accuracy of up to 0.811.

[0006] The high mortality rate of NSCLC is directly attributed to the lack of early diagnosis, resulting in patients losing the opportunity for a cure at the time of diagnosis. Improving the sensitivity of early screening and developing highly specific biomarkers are key directions for improving prognosis. CEA and CYFRA21-1, as traditional NSCLC biomarkers, have significant limitations in clinical application due to their low sensitivity, high histology dependence, insufficient efficacy in early diagnosis, and significant nonspecific interference. Combined detection and the search for novel, promising circulating tumor biomarkers are necessary approaches to improve this situation.

[0007] Sequencing revealed that snoRNAs are enriched in exosomes. Exosomes provide a physical protection mechanism for snoRNAs by encapsulating them, ensuring their stable presence in the circulatory system. Furthermore, snoRNAs have been shown to be dysregulated in tumors and specifically expressed in different tumor types. snoRNAs play a crucial role in tumorigenesis and progression by regulating rRNA modification, signaling pathway activity, cell death mechanisms, and the immune microenvironment. Numerous studies have reported the stable presence of free snoRNAs dependent on ribonucleoprotein complexes in body fluids for cancer diagnosis. However, research on snoRNAs derived from exosomes as circulating tumor diagnostic biomarkers remains relatively scarce.

[0008] Therefore, it is necessary to provide a detection biomarker based on snoRNA in exosomes for the diagnosis of non-small cell lung cancer, so as to achieve non-invasive or minimally invasive detection. Summary of the Invention

[0009] To address the aforementioned issues, this application provides the use of SNORD54 and SNORD41 as diagnostic biomarkers for non-small cell lung cancer (NSCLC). The proposed method is based on data from a database and the differentially expressed SNORD54 and SNORD41 identified through FFPE sample detection. The expression of these two genes was then detected in serum exosomes from lung cancer patients and healthy individuals. The results indicate that SNORD54 and SNORD41 in tumor-derived serum exosomes can serve as novel biomarkers for NSCLC.

[0010] This application provides the application of exosomal snoRNA markers in the preparation of diagnostic reagents for non-small cell lung cancer, characterized in that the exosomal snoRNA markers include one or both of SNORD54 and SNORD41.

[0011] Optionally, the biomarkers used in the non-small cell lung cancer diagnostic reagent may also include one or both of CEA and CYFRA21-1.

[0012] Optionally, the sample taken during the diagnostic process may be from one or more of serum, plasma, and blood.

[0013] This application provides the application of exosomal snoRNA markers in the preparation of diagnostic kits for non-small cell lung cancer, characterized in that the exosomal snoRNA markers include one or both of SNORD54 and SNORD41.

[0014] Optionally, the biomarkers used in the non-small cell lung cancer diagnostic kit may also include one or both of CEA and CYFRA21-1.

[0015] Optionally, the sample taken during the diagnostic process may be from one or more of serum, plasma, and blood.

[0016] This application provides a diagnostic kit for non-small cell lung cancer, characterized in that the kit includes a detection reagent that specifically detects one or both of SNORD54 and SNORD41 in biological samples.

[0017] Optionally, the kit may also include a detection reagent for specifically detecting one or both of CEA and CYFRA21-1 in biological samples.

[0018] Optionally, the biological sample may be derived from one or more of serum, plasma, and blood.

[0019] This application provides the application of exosomal snoRNA markers in screening drugs for non-small cell lung cancer, characterized in that the exosomal snoRNA markers include one or both of SNORD54 and SNORD41.

[0020] The beneficial effects of this application include, but are not limited to:

[0021] 1. Based on the application of SNORD54 and SNORD41 as diagnostic biomarkers for non-small cell lung cancer (NSCLC) in this application, SNORD54 and SNORD41 were identified in the study subjects using a database. It was demonstrated that these two snoRNAs were significantly overexpressed in tumor tissues and serum exosomes of cancer patients compared to healthy volunteers, indicating their potential role in the development and progression of NSCLC. Furthermore, it was confirmed that these two serum exosomal snoRNAs can serve as promising tumor biomarkers for the diagnosis of NSCLC, especially early-stage NSCLC.

[0022] 2. Based on the application of SNORD54 and SNORD41 as diagnostic markers for non-small cell lung cancer in this application, these two snoRNAs can be stably enriched in exosomes, which is the structural basis for their use as circulating tumor markers. This has been confirmed in the specific scheme of this application through snoRNA enrichment experiments, RNase degradation experiments, and exosome storage time experiments.

[0023] 3. Based on the application of SNORD54 and SNORD41 as diagnostic markers for non-small cell lung cancer in this application, ROC curve analysis confirmed that they each have significantly high diagnostic efficacy. The AUCs of serum exosomes SNORD54 and SNORD41 in diagnosing NSCLC and early NSCLC were 0.8641, 0.8557 and 0.8746, 0.8311, respectively, which are close to the values ​​in tissues. In addition, in the diagnosis of NSCLC, the AUC of serum SNORD54 or SNORD41 combined with CEA and CYFRA21-1 reached 0.9524 (sensitivity 91.23%, specificity 88.7%) and 0.9404 (sensitivity 84.4%, specificity 95.16%), respectively. In the diagnosis of early NSCLC, the AUC of these two exosomal snoRNAs combined with CEA and CYFRA21-1 reached 0.9043 (sensitivity 88%, specificity 80) and 0.8721 (sensitivity 70.73%, specificity 93.55), respectively.

[0024] 4. Based on the application of SNORD54 and SNORD41 as diagnostic biomarkers for non-small cell lung cancer in this application, the use of biomarkers provided by the scheme of this application can achieve very considerable diagnostic efficacy and significantly improve the AUC value of traditional tumor markers, thereby making up for their shortcomings in the early diagnosis of NSCLC. The biomarkers provided by the scheme of this application are of great significance for the early diagnosis of NSCLC. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0026] Figure 1 This is a heatmap of differential snoRNA expression between tumor tissues and normal tissues in the SnoRNA database involved in Example 1 of this application (A: LUAD, B: LUSC).

[0027] Figure 2 This is a volcano diagram (A: LUAD, B: LUSC) showing the differential expression of snoRNAs between tumor tissues and normal tissues in the SnoRNA database involved in Example 1 of this application.

[0028] Figure 3 The results of TCGA validation of differential expression of SNORD54 involved in Example 1 of this application (A: tumor tissue, B: early tumor tissue).

[0029] Figure 4 The TCGA validation results of differential expression of SNORD41 involved in Example 1 of this application (A: tumor tissue, B: early tumor tissue).

[0030] Figure 5 The AUC results of SNORD54 in the database involved in Example 1 of this application (A: tumor tissue, B: early tumor tissue);

[0031] Figure 6 The AUC results of SNORD41 in the database involved in Example 1 of this application (A: tumor tissue, B: early tumor tissue);

[0032] Figure 7 The results of relative expression levels of paraffin-embedded FFPE tissue in tumor samples and adjacent tumor samples involved in Example 1 of this application (A: SNORD54, B: SNORD41).

[0033] Figure 8 The results of relative expression levels of paraffin-embedded FFPE tissue in early tumor samples and early adjacent tumor samples involved in Example 1 of this application (A: SNORD54, B: SNORD41).

[0034] Figure 9 The AUC results of SNORD54 and SNORD41 in the paraffin-embedded tumor tissue involved in Example 1 of this application are shown (A: SNORD54, B: SNORD41).

[0035] Figure 10The AUC results of SNROD54 and SNORD41 in paraffin-embedded tissue of an early tumor involved in Example 1 of this application are shown (A: SNORD54, B: SNORD41).

[0036] Figure 11 The images show the transmission electron microscope (A) and particle size detection results (B) of serum exosomes involved in Example 1 of this application;

[0037] Figure 12 The image shows the detection results of the exosome markers CD63, CD81 and TSG101 involved in Example 1 of this application;

[0038] Figure 13 The levels of SNORD54 and SNORD41 in the exosome-free supernatant (EDS) and exosomes (EXO) involved in Example 1 of this application;

[0039] Figure 14 The expression levels of SNORD54 and SNORD41 in the exosomes involved in Example 1 of this application after treatment with RNaseA;

[0040] Figure 15 The levels of SNORD54 (A) and SNORD41 (B) in exosomes at different time points (0, 6, 12, 18, and 24 hours) during the room temperature incubation experiment involved in Example 1 of this application;

[0041] Figure 16 The serum exosome SNORD54 involved in Example 1 of this application was significantly highly expressed in the serum exosomes of NSCLC (A) patients and early-stage NSCLC (B) patients;

[0042] Figure 17 The serum exosome SNORD41 involved in Example 1 of this application was significantly highly expressed in the serum exosomes of NSCLC (A) patients and early-stage NSCLC (B) patients;

[0043] Figure 18 In Example 1 of this application, the serum exosomes of SNORD54 were significantly elevated in patients with different TNM stages.

[0044] Figure 19 In Example 1 of this application, the serum exosomes of SNORD41 involved were significantly elevated in patients with different TNM stages;

[0045] Figure 20 In Example 2 of this application, serum exosome SNORD54 significantly improves the diagnostic efficacy of CEA in NSCLC (A: SNORD54, B: CEA, C: SNORD54+CEA).

[0046] Figure 21 In the SNORD54 study cohort involving serum exosomes of this application, the expression of CEA and CYFRA21-1 in the serum of NSCLC patients was significantly increased compared with that in healthy individuals.

[0047] Figure 22 In Example 2 of this application, serum exosome SNORD54 significantly improves the diagnostic efficacy of CYFRA21-1 and the combined AUC value of the three in NSCLC (A: CYFRA21-1, B: SNORD54+CYFRA21-1, C: SNORD54+CEA+CYFRA21-1).

[0048] Figure 23 In Example 2 of this application, serum exosome SNORD54 significantly improves the diagnostic efficacy of CEA in early-stage NSCLC (A: SNORD54, B: CEA, C: SNORD54+CEA).

[0049] Figure 24 In Example 2 of this application, serum exosome SNORD54 significantly improves the diagnostic efficacy of CYFRA21-1 and the combined AUC value of the three in early-stage NSCLC (A: CYFRA21-1, B: SNORD54+CYFRA21-1, C: SNORD54+CEA+CYFRA21-1).

[0050] Figure 25 In Example 2 of this application, in the serum exosome SNORD54 study cohort, the expression of CEA in the serum of early NSCLC patients was significantly increased compared with that of healthy individuals.

[0051] Figure 26 In Example 2 of this application, in the serum exosome SNORD54 study cohort, the expression of CYFRA21-1 in the serum of early NSCLC patients was significantly increased compared with that of healthy individuals.

[0052] Figure 27 Example 3 of this application relates to the serum exosome SNORD41 and its combined diagnostic efficacy in NSCLC (A: SNORD41, B: CEA, C: SNORD41+CEA, D: CYFRA21-1, E: SNORD41+CYFRA21-1, F: SNORD41+CEA+CYFRA21-1).

[0053] Figure 28 In the serum exosome SNORD41 study cohort involved in Example 3 of this application, the expression of CEA and CYFRA21-1 in the serum of NSCLC patients was significantly increased compared with that of healthy individuals.

[0054] Figure 29 Example 3 of this application describes the serum exosome SNORD41 and its combined diagnostic efficacy in early-stage NSCLC (A: SNORD41, B: CEA, C: SNORD41+CEA, D: CYFRA21-1, E: SNORD41+CYFRA21-1, F: SNORD41+CEA+CYFRA21-1).

[0055] Figure 30 In the SNORD41 study cohort involving serum exosomes involved in Example 3 of this application, the expression of CEA and CYFRA21-1 in the serum of early NSCLC patients was significantly increased compared with that of healthy individuals.

[0056] Figure 31 In the shared population of the SNORD54 and SNORD41 cohorts involved in Example 4 of this application, the expression levels of exosomal SNORD54, exosomal SNORD41, CEA and CYFRA21-1 in NSCLC patients were significantly increased compared with healthy individuals.

[0057] Figure 32 In the shared population of the SNORD54 and SNORD41 cohorts involved in Example 4 of this application, compared with healthy individuals, the expression levels of exosomal SNORD54, exosomal SNORD41, CEA and CYFRA21-1 in early NSCLC patients were significantly increased.

[0058] Figure 33 This application describes the diagnostic efficacy of serum exosomes SNORD54+SNORD41 and their combination with CEA and CYFRA21-1 in NSCLC in Example 4 of this application (A: SNORD54+SNORD41, B: CEA, C: SNORD54+SNORD41+CEA, D: CYFRA21-1, E: SNORD54+SNORD41+CYFRA21-1, F: SNORD54+SNORD41+CEA+CYFRA21-1).

[0059] Figure 34 This application describes the diagnostic efficacy of serum exosomes SNORD54+SNORD41 and their combination with CEA and CYFRA21-1 in early-stage NSCLC in Example 4 (A: SNORD54+SNORD41, B: CEA, C: SNORD54+SNORD41+CEA, D: CYFRA21-1, E: SNORD54+SNORD41+CYFRA21-1, F: SNORD54+SNORD41+CEA+CYFRA21-1).

[0060] In the image labels, "normal" and "HD" both represent healthy individuals, "LC" and "NSCLC" both represent non-small cell lung cancer, and "early" and "early-stage" both represent early-stage lung cancer. Detailed Implementation

[0061] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0062] Example 1

[0063] The experimental methods include the following:

[0064] 1) Patient and Sample Preparation

[0065] This study recruited 163 healthy donors and 129 non-small cell lung cancer (NSCLC) patients at the Affiliated Taian Central Hospital of Qingdao University from May to September 2023. After serum sample collection, the samples were first centrifuged at 2000×g for 10 minutes to remove red blood cells, white blood cells, and platelets, followed by centrifugation at 12000×g for 10 minutes at 4°C to remove cell debris. The supernatant was retained and stored at -80°C until ultracentrifugation. Tumor staging was assessed according to the 8th edition of the Tumor-Lymph Node-Metastasis (TNM) staging criteria for lung cancer developed by the International Association for the Study of Lung Cancer (IASLC). Healthy volunteers had not received any anti-tumor treatment and did not have other endocrine, immune, or metabolic diseases prior to peripheral blood collection. In addition, paraffin-embedded (FFPE) NSCLC tissue and corresponding adjacent normal tissue samples were collected from a total of 48 NSCLC patients.

[0066] 2) Database source

[0067] The snoRNA expression data were downloaded from the SNORic database, which is based on literature and contains tumor tissue and adjacent normal tissue samples from 46 patients with lung adenocarcinoma (LUAD) and 45 patients with lung squamous cell carcinoma (LUSC). Clinically relevant information for 989 cases of non-small cell lung cancer (NSCLC) was obtained from the TCGA database (http: / / cancergenome.nih.gov), including 784 early-stage cases.

[0068] 3) Serum exosome isolation

[0069] Serum exosomes were isolated and collected using ultracentrifugation: serum was centrifuged at 4°C and 10,000 × g for 30 minutes to remove large vesicles, followed by ultracentrifugation (Beckman Coulter, Brea, California, USA) at 4°C and 100,000 × g for 2 hours to precipitate exosomes. After washing with phosphate-buffered saline (PBS), exosomes were collected again by centrifugation at 4°C and 100,000 × g for 2 hours and verified by transmission electron microscopy, qNano assay, and Western blotting.

[0070] Electron microscopy verification: For transmission electron microscopy, a 15 mL vesicle sample was placed on a copper grid, and the vesicles were fixed with 50 mL of 1% glutaraldehyde for 5 minutes, followed by rinsing the copper grid with ddH2O for 2 minutes. Next, the copper grid was placed in 50 mL of uranyl oxalate solution (pH 7, 5 minutes) and 50 μL of methylcellulose UA solution (10 minutes). Residual liquid on the copper grid was blotted dry with filter paper. Finally, the copper grid was air-dried for 5 to 10 minutes. The vesicles on the copper grid were observed using a FEI Tecnai G2 Spirit transmission electron microscope (Thermo Fisher Scientific, Massachusetts, USA).

[0071] qNano uses the principle of adjustable resistance pulse sensing: isolated microvesicles are diluted with phosphate-buffered saline (PBS). The diameter of the microvesicles is measured using a light scattering particle size analyzer (TRPS) and the qNano platform (Izon Science Ltd, Christchurch, New Zealand). The results are analyzed using the Izon Control Suite v.3.3.2.2000 (Izon Science Ltd).

[0072] Western blotting: Cells and exosomes were lysed on ice using RIPA lysis buffer for 30 min, followed by centrifugation at 12000×g for 15 min to obtain protein extracts. Protein concentration was determined using a BCA kit. Exosome and cellular proteins were separated by SDS-PAGE, and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with 5% skim milk for 2 h, incubated overnight at 4°C with primary antibodies: TSG101, CD63, and CD81 (1:1000; Cell Signaling Technology, USA), and then incubated at room temperature with HRP-labeled secondary antibody for 1 h. Protein bands were visualized using ECL reagent (Bio-Rad, USA).

[0073] 4) RNA extraction and real-time PCR

[0074] Total exosomal RNA was extracted using 500 µl of TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CAR, USA) and then reverse transcribed into cDNA using the Mix-X miRNA First-Strand Synthesis Kit according to the manufacturer's instructions. The expression level of exosomal snoRNA was detected using a LightCycle 480 system (Roche, Basel, Switzerland). The reaction mixture consisted of 2 ml of cDNA template, 0.8 ml of upstream primer and 0.8 ml of downstream primer, 6.4 ml of RNase-free water, and 10 ml of SYBR-Green. U6 was used as an internal control. Relative gene expression levels were calculated using the δCT (CTsnoRNA-CTU6) method. The primer sequences involved for SNORD54, SNORD41, and U6 are listed in Table 1 below.

[0075] Table 1 Primer sequences involved in SNORD54, SNORD41, and U6

[0076]

[0077] 5) Statistical analysis

[0078] Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, USA) and SPSS 25.0 software (IBM, Eningen, Germany). The normality of the data was assessed using the Kolmogorov–Smirnov test. Normally distributed numerical variables were analyzed using parametric tests, while non-normally distributed variables were assessed using the Mann–Whitney test. Comparisons between two or more groups were performed using one-way ANOVA or Kruskal-Walis one-way ANOVA. Numerical data are expressed as median and interquartile range. Diagnostic efficacy was assessed using receiver operating characteristic (ROC) curves. A p-value <0.05 was considered statistically significant, and all tests were set to two-tailed tests.

[0079] The experimental results are as follows:

[0080] 1) Differentially identified snoRNAs from NSCLC databases

[0081] To screen for differentially expressed snoRNAs, normalized snoRNA expression data from 989 non-small cell lung cancer (NSCLC) patients were downloaded from the TCGA and SNORic databases. Paired data (paired tumors and adjacent tissues) were from 46 LUAD and 45 LUSC patients. Using the paired data, differential snoRNA expression between NSCLC patients and healthy donors was identified and visualized in a heatmap. Figure 1 ) and volcano map ( Figure 2 The results were presented in a heatmap based on the following criteria: adjusted p-value < 0.05 and absolute logarithmic fold change > 2.00. Significantly differentially expressed snoRNAs were listed in the heatmap. SnoRNAs significantly dysregulated in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were selected for further validation. First, 24 paired FFPE samples were used to analyze their expression by qRT-PCR. Unexpectedly, most were excluded due to low primer specificity and expression levels, as well as the lack of difference in expression between cancerous and adjacent tissues. Ultimately, SNORD54 (https: / / www.ncbi.nlm.nih.gov / datasets / gene / 26795 / ) and SNORD41 (https: / / www.ncbi.nlm.nih.gov / datasets / gene / 26810 / ) were selected as candidate genes.

[0082] Next, TCGA was used to perform large-sample validation of the differential expression of SNORD54 and SNORD41. Figures 3-6 As shown, compared with normal tissue (n=91), the levels of these two snoRNAs were significantly increased in tumor tissue (n=989) and early tumor tissue (n=784), with their respective areas under the curve (AUC) being 0.7477, 0.8065, 0.8608 and 0.8795, respectively.

[0083] 2) SNORD54 and SNORD41 are upregulated in non-small cell lung cancer and early-stage non-small cell lung cancer tissues.

[0084] To further identify differential expression of SNORD54 and SNORD41 in non-small cell lung cancer (NSCLC), these two snoRNAs were validated in paraffin-embedded (FFPE) tissues, including 48 NSCLC cases and their paired adjacent normal tissues.

[0085] Consistently, compared with adjacent normal tissues, the expression of SNORD54 and SNORD41 was significantly increased in NSCLC (p<0.0001, p<0.0001, respectively) and in 34 cases of early-stage NSCLC (p<0.0001, p=0.0004, respectively), with corresponding areas under the curve (AUC) of 0.6461, 0.7680, 0.6583, and 0.7344, respectively. Figures 7-10 ).

[0086] 3) Identification of isolated serum exosomes

[0087] Serum exosomes from healthy donors and NSCLC patients were separated by ultracentrifugation and verified by transmission electron microscopy, qNano chromatography, and Western blotting. Figure 11 As shown, transmission electron microscopy revealed a typical exosome-like circular morphology with a diameter of 50–150 nm, consistent with the results from qNano. CD63, CD81, and TSG101, common exosome markers, were significantly enriched in exosomes but not detected in cells. Figure 12 These results demonstrate that exosomes were successfully separated by ultracentrifugation.

[0088] 4) Characterization of SNORD54 and SNORD41 in serum

[0089] To characterize snoRNAs in exosomes, the levels of SNORD54 and SNORD41 in exosome desquamated supernatant (EDS) and exosomes were measured.

[0090] The results showed that the expression of these two snoRNAs in exosomes was significantly higher than that of EDS ( Figure 13 Furthermore, the levels of these two snoRNAs in exosomes did not change significantly after RNase A treatment. Figure 14 This indicates that the expression of snoRNAs in exosomes is stable.

[0091] These results indicate that SNORD54 and SNORD41 are contained within exosomes, preventing snoRNAs from being degraded by enzymes. In room temperature incubation experiments ( Figure 15 When exosomes were incubated at different time points (0, 6, 12, 18, and 24 hours), the levels of the two snoRNAs did not change significantly.

[0092] 5) SNORD54 and SNORD41 are upregulated in serum exosomes of patients with non-small cell lung cancer (NSCLC) and early-stage NSCLC.

[0093] Next, exosome samples were collected from 115 lung cancer patients, including 51 early-stage patients, and 116 healthy individuals to verify the expression of SNORD54.

[0094] like Figure 16 As shown, SNORD54 expression in serum exosomes of lung cancer patients and early-stage lung cancer patients was significantly higher than that in healthy individuals. Similarly, differential expression of SNORD41 was detected in exosome samples from 124 lung cancer patients, 42 early-stage lung cancer patients, and 111 healthy individuals. The results showed that serum exosome SNORD41 was also significantly highly expressed in lung cancer patients and early-stage lung cancer patients. Figure 17Furthermore, compared to healthy donors, both exosomal snoRNAs were significantly elevated at different TNM stages. Figure 18 and Figure 19 ).

[0095] Example 2: Serum exosome SNORD54 as an effective biomarker for the early diagnosis of non-small cell lung cancer.

[0096] To evaluate the performance of exosomal SNORD54 in the diagnosis of non-small cell lung cancer (NSCLC), the AUC value was calculated using receiver operating characteristic (ROC) curves. Figure 20 As shown in Figure A, the area under the curve (AUC) is 0.8641, with a sensitivity of 75.9% and a specificity of 86.1%. Carcinoembryonic antigen (CEA) is a broad-spectrum prognostic marker for tumors and is recommended for the diagnosis of NSCLC. Figure 20 B, Figure 20 C and Figure 21 As shown in Figure A, SNORD54 significantly increased the AUC value of CEA from 0.7989 to 0.9349. Cytokeratin 19 fragment CYFRA21-1 is another established circulating tumor marker with an AUC value of 0.7855. Figure 22 China A and Figure 21 (B). The combined detection of SNORD54 and CYFRA21-1 showed a higher AUC value of 0.9324 ( Figure 22 (B) As expected, the combined detection of the three biomarkers increased the AUC to 0.9524, and improved the sensitivity and specificity to 91.23% and 88.7%, respectively. Figure 22 (C)

[0097] Similarly, in a comparison between early-stage NSCLC patients and healthy controls, the ROC curve of exosomal SNORD54 showed good diagnostic efficacy, with an AUC value of 0.8746, a sensitivity of 80.39%, and a specificity of 86.96%. Figure 23 Similarly, the combined detection of CEA and serum exosome SNORD54 (AUC=0.8877) significantly improved the diagnostic efficacy of early NSCLC compared to CEA detection alone (AUC=0.6441). Figure 23 B, Figure 23 C and Figure 25 The combined detection of SNORD54 and CYFRA21-1 (AUC=0.8927) was also superior to CYFRA21-1 detection alone (AUC=0.6126). Figure 24 China A Figure 24 China B and Figure 26 ).

[0098] Importantly, the combined detection of the above three biomarkers achieved the highest AUC value of 0.9043, with sensitivity and specificity correspondingly improved to 88% and 80%, respectively. Figure 24 (C) In summary, serum exosome SNORD54 holds promise as a non-invasive circulating biomarker to enhance the diagnostic efficacy of established biomarkers such as CEA and CYFRA21-1 in NSCLC and its early stages.

[0099] Example 3: Serum exosome SNORD41 as an effective biomarker for the early diagnosis of non-small cell lung cancer.

[0100] Following the method used to evaluate whether serum exosome SNORD54 can serve as a good diagnostic marker for circulating tumors, the diagnostic efficacy of serum exosome SNORD41 for NSCLC and early-stage NSCLC was tested. Figure 27 and Figure 28 As shown, the AUC of serum exosome SNROD41 for NSCLC was 0.8557, and when combined with CEA or CYFRA21-1, it significantly improved diagnostic efficacy, from 0.8174 to 0.9204 and from 0.8096 to 0.9330, respectively. When all three were used together to diagnose NSCLC, the AUC reached 0.9404, with a sensitivity of 84.4% and a specificity of 95.16%.

[0101] In addition, such as Figure 29 and Figure 30 As shown, the AUC of serum exosomal SNORD41 was 0.8311, significantly higher than that of CEA (0.6559) and CYFRA21-1 (0.6069) for early diagnosis of NSCLC. When exosomal SNORD41 was combined with these traditional tumor markers, the AUC increased to 0.8609 and 0.8674, respectively. However, the highest AUC value was obtained when SNORD41, CEA, and CYFRA21-1 were combined for the diagnosis of early NSCLC. Figure 29 (F, AUC=0.8721, sensitivity 70.73, 93.55).

[0102] The above results demonstrate that serum exosome SNORD41 can serve as a valuable diagnostic marker for NSCLC and early-stage NSCLC.

[0103] Example 4: The binding of serum exosomes SNORD54 and SNORD41 as an effective biomarker for the diagnosis of non-small cell lung cancer (NSCLC) and early-stage NSCLC.

[0104] Next, the diagnostic efficacy of serum exosomes SNORD54 and SNORD41 in combination for NSCLC and early-stage NSCLC was evaluated.

[0105] First, common samples were selected from the SNORD54 and SNORD41 cohorts, which included 77 healthy individuals, 98 NSCLC patients, and 41 early-stage NSCLC patients. Figure 31 and Figure 32 Serum exosomal SNORD54, SNORD41, CEA, and CYFRA21-1 were significantly elevated in NSCLC patients and early-stage NSCLC patients compared to healthy individuals. A series of ROC curves were then analyzed to evaluate the diagnostic value of these two serum exosomal snoRNAs.

[0106] like Figure 33 As shown, the AUC of serum exosome SNORD54 and SNORD41 combined for the diagnosis of NSCLC was 0.8667, with a sensitivity of 75.51 and a specificity of 87.01. Furthermore, the combination of serum exosome SNORD54+41 with CEA or CYFRA21-1 significantly improved diagnostic efficacy, from 0.8192 to 0.9373 and from 0.7808 to 0.9372, respectively.

[0107] The highest area under the curve obtained by all the above combinations of markers is 0.9495 ( Figure 33 The sensitivity and specificity of the assay were increased to 89.58% and 92.91%, respectively.

[0108] In addition, such as Figure 34 As shown, the combined AUC of serum exosomes SNORD54 and SNORD41 was 0.8806, significantly higher than that of CEA (0.6622) and CYFRA21-1 (0.5583) for early diagnosis of NSCLC. When serum SNORD54 + SNORD41 were combined with them for diagnosis, the AUC increased to 0.8922 and 0.8867, respectively. The highest AUC value was obtained when SNORD54, SNORD41, CEA, and CYFRA21-1 were combined for the diagnosis of early NSCLC. Figure 34 (In the middle F, AUC=0.8989).

[0109] In summary, serum exosomes SNORD54 and SNROD41 can serve as effective diagnostic markers for NSCLC, especially early-stage NSCLC. The combined diagnosis of serum exosomes SNORD54 and SNROD41 can significantly improve the diagnostic efficacy for NSCLC and early-stage NSCLC, and they are valuable circulating tumor markers.

[0110] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. The application of a serum exosome snoRNA marker detection reagent in the preparation of a diagnostic reagent for serum samples of non-small cell lung cancer, characterized in that, The detection reagent for serum exosomal snoRNA markers includes a detection reagent for specifically detecting the levels of exosomal SNORD54 and SNORD41 markers in serum, and the serum sample diagnostic reagent for non-small cell lung cancer also includes a detection reagent for specifically detecting the levels of CEA and CYFRA21-1 proteins in serum.

2. The application of a serum exosome snoRNA marker detection reagent in the preparation of a diagnostic kit for serum samples of non-small cell lung cancer, characterized in that, The detection reagent for serum exosomal snoRNA markers includes a detection reagent for specifically detecting the levels of exosomal SNORD54 and SNORD41 markers in serum, and the serum sample diagnostic kit for non-small cell lung cancer also includes a detection reagent for specifically detecting the levels of CEA and CYFRA21-1 proteins in serum.

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