Application of tRF-Val-TAC-022 in diagnosis of lymph node metastasis of lung cancer

Abstract

The invention relates to the technical field of molecular biology, and discloses an application of a biomarker tRF-Val-TAC-022 in preparation of a lung cancer lymph node metastasis diagnosis product. The expression level of the biomarker tRF-Val-TAC-022 in serum of a patient suffering from lung cancer lymph node metastasis is remarkably higher than that of a lung cancer patient without lymph node metastasis, the biomarker tRF-Val-TAC-022 can be used as a biomarker for diagnosis of lung cancer lymph node metastasis and is high in sensitivity and specificity, and early diagnosis of lung cancer lymph node metastasis can be simply and quickly performed by using the molecule.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to the application of a biomarker tRF-Val-TAC-022 in the diagnosis of lung cancer lymph node metastasis. Background Art

[0002] Lung tumors refer to abnormal masses or growths in the lungs, including benign and malignant tumors, inflammatory lesions, infectious lesions, and vascular lesions. Malignant tumors, including lung adenocarcinoma, squamous cell carcinoma, large cell lung cancer, and small cell lung cancer, are among the most common malignant tumors worldwide with high morbidity and mortality. While recent advances in lung cancer treatment have been made, such as the widespread adoption of low-dose CT screening, which has significantly increased the window for detecting lung tumors and enabled earlier surgical intervention, significantly improving the five-year survival rate, this presents new challenges. For patients with early-stage lung cancer, less invasive surgical procedures and targeted lymph node dissection are often used. However, some patients develop lymph node metastases despite relatively small tumors, requiring more extensive surgical resection and more detailed lymph node dissection. Therefore, early diagnosis of lymph node metastasis can allow for preemptive modification of surgical approaches, personalized treatment plans, and even advance surgical planning, benefiting patients. However, patients with small lung tumors and lymph node metastasis may not experience obvious symptoms in the early stages, and specific diagnostic methods for lung cancer lymph node metastasis are currently lacking. Therefore, the search for biomarkers for early diagnosis of lung cancer lymph node metastasis (i.e., diagnosing whether lung tumor patients have lymph node metastasis) has become an urgent need for the diagnosis and treatment of lung cancer lymph node metastasis.

[0003] Transfer RNA-derived fragments (tRFs) are a class of non-coding RNAs (ncRNAs) less than 50 nt in length, formed by cleavage of transfer RNA (tRNA). Research has shown that tRFs are not products of random tRNA degradation, but rather are produced through precise cleavage by specific RNA cleavage enzymes. Their abnormal expression is often highly correlated with specific disease pathologies. They are also highly conserved, stable, and have more diverse regulatory pathways than miRNAs. These characteristics hold great promise for the development and application of tRFs in disease diagnosis and treatment. Summary of the Invention

[0004] The present invention provides a biomarker tRF-Val-TAC-022. The expression level of the biomarker tRF-Val-TAC-022 in the serum of lung cancer patients with positive lymph node metastasis is significantly higher than that in lung cancer patients with negative lymph node metastasis. The biomarker tRF-Val-TAC-022 can be used as a biomarker for the early diagnosis of lung cancer lymph node metastasis. The molecule can be used to simply and quickly perform early diagnosis of lung cancer lymph node metastasis.

[0005] In a first aspect of the present invention, a preparation for detecting the expression level of tRF-Val-TAC-022 is provided for use in preparing a product for diagnosing or predicting lung cancer lymph node metastasis. The nucleotide sequence of the tRF-Val-TAC-022 is shown in SEQ ID NO: 1, which is AAGCCCCAGTGGAACCA.

[0006] Alternatively, the preparation includes specific primers for detecting tRF-Val-TAC-022, wherein the primers include an upstream primer and a downstream primer.

[0007] As an alternative, the test sample for detecting the expression level of tRF-Val-TAC-022 is a serum sample.

[0008] As an optional option, the method for using the lung cancer lymph node metastasis diagnosis or prediction product includes: detecting the expression level of tRF-Val-TAC-022 in the serum of the subject; comparing the tRF-Val-TAC-022 expression level with a preset value; if the tRF-Val-TAC-022 expression level is higher than the preset value, diagnosing or predicting that the subject is likely to have lung cancer lymph node metastasis.

[0009] Alternatively, the subject is a lung tumor patient.

[0010] As an option, the product includes reagents, test strips, kits, gene chips or high-throughput sequencing platforms.

[0011] As an optional solution, the kit includes an RNA tissue extraction reagent, a reverse transcription reagent, a real-time fluorescence quantitative PCR reagent, and a primer capable of amplifying tRF-Val-TAC-022.

[0012] As an optional solution, the method of using the kit includes extracting RNA from the subject's serum, preparing cDNA, real-time fluorescence quantitative PCR amplification of tRF-Val-TAC-022, determining the expression level of tRF-Val-TAC-022 and comparing it with a preset value.

[0013] A second aspect of the present invention provides an auxiliary diagnosis device for lung cancer lymph node metastasis, the device comprising: A data input module, for inputting the expression level of tRF-Val-TAC-022 in the serum of a subject, wherein the nucleotide sequence of tRF-Val-TAC-022 is as shown in SEQ ID NO: 1, which is AAGCCCCAGTGGAACCA; a data processing module, for comparing the expression level of tRF-Val-TAC-022 with a preset value; The result output module is used to output the comparison result. If the expression level of tRF-Val-TAC-022 is higher than the preset value, the result indicating a high possibility of lung cancer lymph node metastasis is output.

[0014] In a third aspect of the present invention, an electronic device for auxiliary diagnosis of lung cancer lymph node metastasis is provided, the electronic device comprising: one or more processors and a storage device, wherein the storage device stores one or more programs; when the one or more programs are executed by the one or more processors, the one or more processors can implement the data processing process in the above-mentioned lung cancer lymph node metastasis auxiliary diagnosis device.

[0015] The present invention's tRF-Val-TAC-022 can be used to prepare diagnostic products for lung cancer lymph node metastasis and is a biomarker for the early diagnosis of lung cancer lymph node metastasis. The present invention demonstrates that this marker is upregulated in the serum of patients with lung cancer lymph node metastasis and can be used as an aid in the diagnosis of lung cancer lymph node metastasis. By measuring the expression level of tRF-Val-TAC-022 in serum, clinicians can quickly and accurately identify early-stage lung cancer and assist in selecting appropriate treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the position of tRF-Val-TAC-022 in its source tRNA.

[0017] Figure 2 This is the amplification curve of tRF-Val-TAC-022 detected by real-time fluorescence quantitative PCR.

[0018] Figure 3 This is the melting curve of tRF-Val-TAC-022 detected by real-time fluorescence quantitative PCR.

[0019] Figure 4 This is a graph showing the difference in expression levels of tRF-Val-TAC-022 in patients with positive and negative lung cancer lymph node metastasis.

[0020] Figure 5 This figure verifies the difference in expression levels of tRF-Val-TAC-022 in patients with positive and negative lung cancer lymph node metastasis.

[0021] Figure 6 Figure 3 is the ROC curve and area under the curve (AUC) graph for testing the diagnostic efficacy of tRF-Val-TAC-022. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] In this invention, the term "tRNA derived fragments (tRFs)" refers to a class of non-coding RNAs less than 50 nt in length formed by tRNA cleavage. In the past, tRFs were considered to be molecular junk produced after tRNA translation and had no biological function. In recent years, it has been discovered that tRFs are not products of random tRNA degradation, but are instead produced by precise cleavage by specific RNA cleavage enzymes. Their abnormal expression is often highly correlated with the pathological state of specific diseases, and therefore has received widespread attention. Currently, tRFs have been reported to be dysregulated in diseases such as cancer, neurodegenerative diseases, acquired metabolic diseases, and pathological stress injuries. However, to date, the role of most tRFs remains unclear.

[0024] The terms "tRF", "tRF(s)", "tRNA-derived fragment", and "transfer RNA-derived fragment" have the same meaning and can be used interchangeably to refer to a non-protein-encoding RNA fragment formed by tRNA cleavage and generally less than 50 nt in length.

[0025] The term "nuclease-free water" refers to water that has been completely free of nucleases and is free of contamination by nonspecific endonucleases, exonucleases, and ribonucleases. It is commonly used to dissolve DNA / RNA and prepare reaction systems containing nucleic acids.

[0026] The present invention provides a biomarker, tRF-Val-TAC-022, a transfer RNA-derived fragment with the nucleotide sequence AAGCCCCAGTGGAACCA (SEQ ID NO: 1). tRF-Val-TAC-022 is produced by degradation of the 3' end of tRNA-Val-TAC located on chromosome 10. The present invention has discovered and confirmed that its expression level is significantly higher in patients with lung cancer lymph node metastasis than in patients without lymph node metastasis, making it a promising molecular marker for the diagnosis of lung cancer lymph node metastasis.

[0027] The present invention also provides the use of the biomarker tRF-Val-TAC-022 or a reagent for detecting the expression level of the biomarker tRF-Val-TAC-022 in the preparation of a product for diagnosing or predicting lung cancer lymph node metastasis.

[0028] Based on the above biomarkers, the present invention also provides a method for predicting or diagnosing lung cancer lymph node metastasis, which is actually a method for using the above lung cancer lymph node metastasis diagnosis or prediction product. The method includes: detecting the expression level of tRF-Val-TAC-022 in the serum of the subject; comparing the tRF-Val-TAC-022 expression level with a preset value. If the tRF-Val-TAC-022 expression level is higher than the preset value, diagnosing or predicting that the subject is likely to have lung cancer lymph node metastasis.

[0029] The expression level, i.e., the statistically significant value of expression, should be expressed in the same numerical format as the preset value to facilitate comparison. More specifically, the term "expression level" refers to "normalized expression," which is the difference between the RT-qPCR cycle number and the internal reference gene U6.

[0030] Regarding the preset value, in some schemes, the preset value is obtained by statistical data processing from patient samples with known negative lung cancer lymph node metastasis, that is, the expression level of tRF-Val-TAC-022 detected in patients with negative lung cancer lymph node metastasis is used as a reference, and the expression level value that is higher than the reference value by a certain degree or has a statistically significant difference is used as the preset value.

[0031] In some embodiments, the products include lung cancer lymph node metastasis diagnostic reagents, lung cancer lymph node metastasis diagnostic test strips, lung cancer lymph node metastasis diagnostic kits, lung cancer lymph node metastasis diagnostic gene chips, or lung cancer lymph node metastasis diagnostic high-throughput sequencing platforms.

[0032] The present invention further provides a lung cancer auxiliary diagnosis device, which includes: The data input module is used to input the expression level of tRF-Val-TAC-022 in the serum of the subject.

[0033] The data processing module is used to compare the expression level of tRF-Val-TAC-022 with a preset value.

[0034] The result output module is used to output the comparison result. If the expression level of tRF-Val-TAC-022 is higher than the preset value, a result indicating a high possibility of lung cancer lymph node metastasis is output; otherwise, a result indicating a high possibility of lung cancer without lymph node metastasis is output.

[0035] In the present invention, all lung cancer patients and their samples were obtained from Jiangsu Cancer Hospital and have passed the hospital's ethical approval.

[0036] Example 1

[0037] This example provides a method for detecting the expression level of tRF-Val-TAC-022 in the serum of lung cancer patients. Specifically, it includes: (1) Collect serum samples: Take 2-5 ml of blood (40 lung cancer patients with lymph node metastasis (i.e., lung cancer lymph node metastasis positive) and 40 lung cancer patients without lymph node metastasis (i.e., lung cancer lymph node metastasis negative)), centrifuge at 4°C for 2 min, take the supernatant, and soak it in an enzyme-free centrifuge tube containing 1-2 mL of RNA preservation solution; (2) Lysis: Add 1-2 mL of RNA extraction reagent TRIzol to the centrifuge tube, mix the serum sample thoroughly, and let it stand at room temperature for 5 minutes; (3) Chloroform extraction: Add 0.2 mL of chloroform to every 1 mL of homogenate sample, cover the tube, shake vigorously for 15 seconds, place at room temperature for 3 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. The sample will then separate into three layers: a pink organic phase at the bottom, a middle layer, and a colorless aqueous phase at the top. RNA is mainly in the aqueous phase. Transfer the aqueous phase to a new centrifuge tube to avoid contact with the middle layer. (4) Isopropanol precipitation: Add an equal volume of isopropanol to the upper layer of water, pre-cooled at 4°C, to a new 1.5 mL centrifuge tube. Mix thoroughly by inverting the tube. Let it stand at 20°C for 30 min to fully precipitate the RNA. Centrifuge at 12,000 rpm at 4°C for 10 min. (5) Ethanol washing: Carefully discard the supernatant, retain the precipitate at the bottom of the test tube, add 1 mL of 75% ethanol solution pre-cooled at 4°C, invert evenly, and place at room temperature for 5 minutes. Centrifuge at 7500 rpm at 4°C for 5 minutes. (6) RNA dissolution: Carefully discard the supernatant, retain the precipitate at the bottom of the test tube, invert the centrifuge tube on the operating table for 10 minutes, dry it thoroughly, and add 10-15 μL of nuclease-free water to fully dissolve the precipitate, and place it in a -80°C deep freezer for later use; (7) RNA concentration determination: After melting the RNA solution on ice, the extracted RNA concentration was detected using a Nano-drop instrument; (8) Preparation of cDNA: The RNA samples were extracted and cDNA was prepared using the riboSCRIPT Reverse Transcription Kit (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.). The reaction system was prepared according to Table 1, and cDNA was prepared. The total amount of RNA template was 100 μg (because the concentration of extracted RNA was different, the volume in the table was marked as X, determined according to the concentration of extracted RNA); the total amount of RNA + RNase-free water was 5 μL; the reverse transcription reaction conditions were as follows: 42°C for 60 min, 70°C for 10 min; Table 1 Reaction system for cDNA preparation

[0038] (9) Real-time fluorescence quantitative PCR (RT-qPCR) detection: Vazyme ChamQ Universal SYBRqPCR Master Mix fluorescence quantitative kit (purchased from Nanjing Novozymes Biotechnology Co., Ltd., catalog number Q711-02) was used for amplification, and the reverse transcribed cDNA was used as a template for fluorescence quantitative PCR amplification; the QuantStudioTM6Flex system was used to perform RT-qPCR experiments on the prepared cDNA, and the upstream and downstream primers of tRF-Val-TAC-022, Bulge LoopTM miRNA qRT PCR Primer, were purchased from Guangzhou Ruibo Biotechnology Co., Ltd.; the RT-qPCR reaction system is shown in Table 2, and the RT-qPCR reaction conditions are shown in Table 3; Table 2 RT-qPCR reaction system

[0039] Table 3 RT-qPCR reaction conditions

[0040] RT-qPCR result analysis: RT-qPCR amplification curve and melting curve are as follows: Figure 2 and Figure 3 shown. Figure 2 The amplification curve is S-shaped, with four obvious periods. The difference between the CT values ​​of each group of duplicate wells is less than 0.5 CT values, which meets the amplification curve standard. Figure 3 The melting curve is used to determine whether the amplified product is single. A single peak in the melting curve indicates a single amplified product. The Tm value indicates the melting temperature of the primer itself, and the standard value is 80℃~90℃.

[0041] (10) Obtaining standardized expression Using the U6 gene as an internal reference, the difference in cycle number between tRF and U6 genes, ΔCT, was obtained by RT-qPCR and used for subsequent comparison of gene expression levels among different tissues.

[0042] (11) Comparison of tRF expression in serum of patients with positive and negative lung cancer lymph node metastasis The above RT-qPCR assay revealed the difference in tRF-Val-TAC-022 expression levels in the serum of patients with positive and negative lung cancer lymph node metastasis. Figure 4 As shown in the figure, it can be seen that tRF-Val-TAC-022 is significantly overexpressed in the serum of patients with positive lung cancer lymph node metastasis, and the difference is statistically significant (P < 0.0001).

[0043] The above results indicate that tRF-Val-TAC-022 can be used as a molecular marker for the diagnosis of lung cancer lymph node metastasis.

[0044] Example 2 This example provides the validation of the biomarker tRF-Val-TAC-022 in the diagnosis of lung cancer lymph node metastasis.

[0045] The serum levels of tRF-Val-TAC-022 were detected in another 60 lung cancer patients, including 30 patients with lung cancer with lymph node metastasis and 30 patients without lymph node metastasis. Figure 5 The results showed that tRF-Val-TAC-022 was significantly expressed in the serum of patients with positive lung cancer lymph node metastasis, and the difference was statistically significant (P < 0.001). At the same time, the ROC curve was generated, and the results were as follows: Figure 6 As shown, the AUC value is 0.89 (the closer the AUC is to 1, the better the diagnostic efficacy). It can be concluded that tRF-Val-TAC-022 can be used as a biomarker for the diagnosis of lung cancer lymph node metastasis, with a sensitivity of 80% and a specificity of 90%.

[0046] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. Use of a preparation for detecting the expression level of tRF-Val-TAC-022 in the preparation of a product for diagnosing or predicting lung cancer lymph node metastasis, wherein the nucleotide sequence of the tRF-Val-TAC-022 is shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the preparation comprises specific primers for detecting tRF-Val-TAC-022, and the specific primers comprise an upstream primer and a downstream primer.

3. The use according to claim 1, characterized in that The test sample used to detect the expression level of tRF-Val-TAC-022 is a serum sample.

4. The use according to claim 1, characterized in that The method for using the lung cancer lymph node metastasis diagnosis or prediction product includes: detecting the expression level of tRF-Val-TAC-022 in the serum of a subject; comparing the expression level of tRF-Val-TAC-022 with a preset value; if the expression level of tRF-Val-TAC-022 is higher than the preset value, diagnosing or predicting that the subject has a high possibility of suffering from lung cancer lymph node metastasis.

5. The use according to claim 4, characterized in that The subject is a lung tumor patient.

6. The use according to claim 1, wherein the product comprises a reagent, a test paper, a kit, a gene chip or a high-throughput sequencing platform.

7. The use according to claim 6, characterized in that The kit comprises an RNA tissue extraction reagent, a reverse transcription reagent, a real-time fluorescence quantitative PCR reagent, and a primer capable of amplifying tRF-Val-TAC-022.

8. The use according to claim 7, characterized in that The method of using the kit includes extracting RNA from the serum of a subject, preparing cDNA, amplifying tRF-Val-TAC-022 by real-time fluorescence quantitative PCR, determining the expression level of tRF-Val-TAC-022, and comparing the expression level with a preset value.

9. A lung cancer lymph node metastasis auxiliary diagnosis device, characterized in that: The device comprises: A data input module, for inputting the expression level of tRF-Val-TAC-022 in the serum of a subject, wherein the nucleotide sequence of tRF-Val-TAC-022 is as shown in SEQ ID NO: 1, which is AAGCCCCAGTGGAACCA; a data processing module, for comparing the expression level of tRF-Val-TAC-022 with a preset value; The result output module is used to output the comparison result. If the expression level of tRF-Val-TAC-022 is higher than the preset value, the result indicating a high possibility of lung cancer lymph node metastasis is output.

10. An electronic device for auxiliary diagnosis of lung cancer lymph node metastasis, characterized in that: The electronic device includes: one or more processors and a storage device, on which one or more programs are stored; when the one or more programs are executed by the one or more processors, the one or more processors can implement the data processing process in the lung cancer lymph node metastasis auxiliary diagnosis device of claim 9.

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