Marker tiRNA-gln-ttg-001 and applications thereof

Abstract

The application discloses a marker tiRNA-Gln-TTG-001 and application thereof, and belongs to the technical field of biotechnology and molecular diagnosis. The applicant finds that, compared with lung cancer patients without brain metastasis, tiRNA-Gln-TTG-001 is significantly highly expressed in the peripheral blood circulation of lung cancer patients with brain metastasis, which provides an important basis for the marker as a specific plasma marker for predicting, monitoring or diagnosing lung cancer brain metastasis. The new clinical cases are selected for verification, and the results show that tiRNA-Gln-TTG-001 can be used as a specific metastasis marker for lung cancer brain metastasis, which opens up a new non-invasive, convenient and specific detection way for early prediction, dynamic monitoring, diagnosis and efficacy evaluation of lung cancer brain metastasis.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and molecular diagnostics, specifically relating to the biomarker tiRNA-Gln-TTG-001 and its applications. Background Technology

[0002] As the second most common and deadliest malignant tumor worldwide, lung cancer poses a significant public health challenge. Brain metastasis is one of the most common distant metastases of lung cancer and a major cause of poor patient prognosis. Diagnosis and monitoring of lung cancer brain metastases primarily rely on imaging examinations (such as MRI), but these are not sensitive to micrometastases and cannot be performed frequently. Against this backdrop, the development of minimally invasive, highly sensitive, and easily implemented technologies for the early detection and monitoring of lung cancer brain metastases is particularly urgent.

[0003] In related technologies, for example, Chinese invention patent CN107589196A discloses a small molecule biomarker for predicting brain metastasis in lung cancer and its application in diagnosis, using a composition of lysophosphatidylcholine (18:0), 3-hydroxypropionic acid, and dihydrocholesterol as a small molecule biomarker for predicting brain metastasis in lung cancer; another example is Chinese invention patent CN116064793A, which discloses the ADGRA2 mutant gene and its application in lung cancer brain metastasis, using the mutant gene formed by the g.650_652insAAC mutation of the wild-type ADGRA2 gene as a small molecule biomarker for predicting brain metastasis in lung cancer. Molecular markers for brain metastasis; for example, Chinese invention patent CN119391861A describes the construction and application of a prognostic model for lung cancer brain metastasis based on a combination of plasma miRNA markers, which uses a combination of plasma miRNA markers composed of miR-31-5p, miR-219-5p, miR-1-3p, miR-130a-5p, miR-452-3p, miR-598-3p, miR-9-5p, miR-1246, miR-196a-5p, and miR-210-3p as a molecular marker for lung cancer brain metastasis.

[0004] tRNA-derived small RNAs (tsRNAs) are non-coding small RNAs derived from mature tRNA or precursor tRNA, widely distributed across various species. They are mainly divided into two categories: tRNA-derived RNA fragments (tRFs) and tRNA halves (tiRNAs). In recent years, with the rapid development of bioinformatics tools and high-throughput RNA sequencing technology, the biological functions of tsRNAs and their roles in the pathophysiology of various diseases have been extensively studied, making them a cutting-edge research area in the field of non-coding RNA. In related technologies, for example, Chinese invention patent CN118406751A describes tsRNA, kits, and their applications for predicting, diagnosing, or monitoring acute kidney injury in sepsis, using 5'Leader-LeuCAG-2-2, tRF3b-LeuTAG-2, or 3'tiRNA-49-LeuTAG-2 as molecular markers for predicting, diagnosing, or monitoring acute kidney injury in sepsis; another example is Chinese invention patent CN111424085A, which describes the application of tRNA-derived fragments in the preparation of breast cancer diagnostic reagents, using tRF-Arg-CCT-017 and tRF-Gly-CCC-00 One or more of tiRNA-Phe-GAA-003 are used as molecular markers for breast cancer diagnosis; for example, Chinese invention patent CN115141883A describes a tsRNA biomarker, kit and application for the diagnosis of depression, using tiRNA-Gly-GCC-001 as a molecular marker for the diagnosis of depression or for evaluating the efficacy of antidepressants; for example, Chinese invention patent CN117965718A describes a biomarker related to sepsis and its application, using tiRNA-1:34-Lys-CTT-5 as an applied molecular marker for the early diagnosis and / or prognostic assessment of sepsis.

[0005] Therefore, it is of great significance to explore whether there are tsRNAs (tRFs or tiRNAs) that can be used for the detection or monitoring of lung cancer brain metastases. Summary of the Invention

[0006] 1. Purpose of the invention

[0007] The purpose of this application is to provide tiRNA-Gln-TTG-001 and its application as a biomarker for predicting, monitoring, or diagnosing lung cancer brain metastases, as well as the application of reagents for detecting tiRNA-Gln-TTG-001 expression levels in the preparation of products for predicting, monitoring, or diagnosing lung cancer brain metastases. The applicant's research found that tiRNA-Gln-TTG-001 exhibits significantly higher expression in the peripheral blood circulation of lung cancer patients with brain metastases compared to lung cancer patients without brain metastases. This finding provides important evidence for its use as a specific plasma biomarker for predicting, monitoring, or diagnosing lung cancer brain metastases.

[0008] 2. Technical Solution To achieve the aforementioned objectives, the technical solution adopted in this application is as follows: Firstly, this application provides tiRNA-Gln-TTG-001, the nucleotide sequence of which is as follows: 5'-UAGGAUGGGGUGUGAUAGGUGGCACGGAGAAUUU-3' (SEQ ID NO.1). As further clarification of this application, the tiRNA-Gln-TTG-001 molecule belongs to a family of non-coding RNAs with a length of 34 nucleotides.

[0009] Secondly, this application provides the application of the aforementioned tiRNA-Gln-TTG-001 as a biomarker for predicting, monitoring, or diagnosing lung cancer brain metastases. As a further explanation, this application has demonstrated through clinical data studies that the expression level of tiRNA-Gln-TTG-001 in plasma is significantly correlated with the status of lung cancer brain metastases. Specifically, compared with lung cancer patients without brain metastases, tiRNA-Gln-TTG-001 exhibits significantly high expression in the peripheral blood circulation of lung cancer patients with brain metastases. Therefore, it can serve as a specific plasma biomarker for lung cancer brain metastases and has clinical application value as a plasma biomarker.

[0010] Furthermore, the aforementioned lung cancers include lung adenocarcinoma, etc.

[0011] Thirdly, this application also provides the application of reagents for detecting the expression level of tiRNA-Gln-TTG-001 in plasma in the preparation of products for predicting, monitoring, or diagnosing lung cancer brain metastases. As a further explanation of this application, tiRNA-Gln-TTG-001 can serve as a specific plasma biomarker for lung cancer brain metastases, exhibiting significantly high expression in the peripheral blood circulation of lung cancer patients with brain metastases. Therefore, the expression level of tiRNA-Gln-TTG-001 in plasma can be detected to predict, monitor, or diagnose lung cancer brain metastases. Knowing the nucleotide sequence of tiRNA-Gln-TTG-001, those skilled in the art can detect the expression level of tiRNA-Gln-TTG-001 using PCR technology, gene sequencing technology, etc.

[0012] Furthermore, the aforementioned lung cancers include lung adenocarcinoma, etc.

[0013] Furthermore, the above reagents include primers for amplifying the tiRNA-Gln-TTG-001 fragment.

[0014] Furthermore, the primers mentioned above include an upstream primer of tiRNA-Gln-TTG-001 and a downstream primer of tiRNA-Gln-TTG-001, the nucleotide sequences of which are as follows: upstream primer for tiRNA-Gln-TTG-001: 5'-GCCTAGGATGGGGTGTGATAG-3' (SEQ ID NO.2). tiRNA-Gln-TTG-001 downstream primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO.3).

[0015] Fourthly, this application also provides reagents for detecting the expression level of tiRNA-Gln-TTG-001, including an upstream primer and a downstream primer for tiRNA-Gln-TTG-001, the nucleotide sequences of which are as follows: upstream primer for tiRNA-Gln-TTG-001: 5'-GCCTAGGATGGGGTGTGATAG-3' (SEQ ID NO.2). tiRNA-Gln-TTG-001 downstream primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO.3).

[0016] Fifthly, this application also provides a kit for predicting, monitoring, or diagnosing brain metastases in lung cancer, the kit comprising reagents for detecting the expression level of tiRNA-Gln-TTG-001 in plasma.

[0017] Furthermore, the above reagents include primers for amplifying the tiRNA-Gln-TTG-001 fragment.

[0018] Furthermore, the primers mentioned above include an upstream primer of tiRNA-Gln-TTG-001 and a downstream primer of tiRNA-Gln-TTG-001, the nucleotide sequences of which are as follows: upstream primer for tiRNA-Gln-TTG-001: 5'-GCCTAGGATGGGGTGTGATAG-3' (SEQ ID NO.2). tiRNA-Gln-TTG-001 downstream primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO.3).

[0019] 3. Beneficial effects Compared with the prior art, the advantages of this application are as follows: This application provides the biomarker tiRNA-Gln-TTG-001 and its applications. Firstly, through a systematic experimental study of clinical cases, this application found that tiRNA-Gln-TTG-001 exhibits significantly high expression in the peripheral blood circulation of lung cancer patients with brain metastases. Specifically, the expression level of tiRNA-Gln-TTG-001 is significantly correlated with the brain metastasis status of lung cancer, and this association is independent of the T stage of the primary tumor. This finding indicates that tiRNA-Gln-TTG-001 has high metastasis predictive efficacy, providing important evidence for its use as a specific plasma biomarker for brain metastases. Subsequently, this application validated the biomarker using new clinical cases, and the results showed that tiRNA-Gln-TTG-001 can serve as a specific metastasis biomarker for lung cancer brain metastases (AUC of the ROC curve = 0.87). This opens up a new, non-invasive, convenient, and highly specific detection method for the early prediction, dynamic monitoring, diagnosis, and efficacy evaluation of lung cancer brain metastases. Attached Figure Description

[0020] Figure 1 This is a schematic diagram showing the concentration and purity of extracted plasma RNA as detected by a Nanodrop spectrophotometer.

[0021] Figure 2 This is the amplification curve of tiRNA-Gln-TTG-001 and cel-miR-39-3p detected by real-time quantitative PCR.

[0022] Figure 3 This is the melting curve of tiRNA-Gln-TTG-001 and cel-miR-39-3p detected by real-time quantitative PCR.

[0023] Figure 4This refers to the expression levels of tiRNA-Gln-TTG-001 in the plasma of lung cancer patients with brain metastases and those without.

[0024] Figure 5 The ROC curve and area under the curve (AUC) are used to validate tiRNA-Gln-TTG-001 as a biomarker for detecting brain metastases in lung cancer. Detailed Implementation

[0025] The present application will be further described below with reference to specific embodiments.

[0026] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0027] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0029] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0030] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0031] As used in this article, non-coding RNA specifically refers to RNA molecules that do not have protein-coding function. Genomic studies have shown that these non-coding RNAs constitute the dominant proportion in the human genome transcriptome. Specifically, transcripts with protein-coding function account for only 2% of the total transcripts in the human genome, while the remaining 98% belong to the category of non-coding RNA. It is noteworthy that although these non-coding RNA molecules do not participate in protein synthesis, they play important regulatory roles in various physiological and pathological processes, especially showing a significant correlation with the occurrence and development of various malignant tumors. Based on differences in molecular length, non-coding RNAs with regulatory functions can be divided into two main categories: short-chain non-coding RNAs and long-chain non-coding RNAs. Studies have shown that these non-coding RNA molecules play important roles in various physiological and pathological processes in the human body, particularly in multiple stages of tumor development, where they exert significant influence through participation, regulation, or mediation.

[0032] As used in this article, transfer RNA (tRNA) is a linker molecule involved in decoding mRNA and translating proteins. Recent studies have shown that tRNA can also serve as a major source of small non-coding RNAs (sncRNAs). These tRNA-derived ncRNAs are not products of random degradation but are produced through precise biogenetic processes. ncRNAs derived from tRNA are broadly classified into two categories: tiRNAs (or tRNA halves) and tRFs (tRNA-derived fragments), each with specific molecular size, nucleotide composition, physiological function, and biogenesis. tiRNAs are 5'- and 3'-tRNA halves produced by the specific cleavage of mature tRNA at the anticodon loop by angiogenin (ANG) under various stress conditions, with a length of approximately 29-50 nt. tRFs are approximately 16-28 nt in length and originate from mature tRNA or precursor tRNA.

[0033] As used in this paper, "external reference gene" is defined as an indicator parameter used to quantify the relative expression level of an external reference gene. Given the technical limitation of the lack of stable internal reference genes in plasma samples, this study adopts a strategy of adding external reference genes before RNA extraction. When performing gene expression analysis using real-time quantitative PCR, the external reference gene is used as a standardized reference, thereby effectively eliminating systematic errors introduced by sample volume and experimental operation, and ensuring the reliability and accuracy of gene expression detection data.

[0034] As used in this article, "nuclease-free water" specifically refers to a high-purity aqueous solution that has undergone strict nuclease removal treatment. Its key feature is that it is completely free of contaminants such as endonucleases, exonucleases, and ribonucleases with non-specific catalytic activity. This reagent has important application value in molecular biology experiments, and is especially suitable for the dissolution of nucleic acids and the preparation of various nucleic acid-containing reaction systems.

[0035] Example 1 This embodiment provides the detection of tiRNA-Gln-TTG-001 expression levels in plasma.

[0036] In this embodiment, real-time quantitative PCR (RT-qPCR) was used to detect the expression level of tiRNA-Gln-TTG-001 in plasma. Specifically, this included: (1) Extraction of RNA from plasma Plasma sample preparation: First, collect 1-2 mL of venous blood from the patient / sample and place it in an anticoagulant blood collection tube. Let it stand for at least 30 minutes to allow it to settle naturally. Then, place the blood collection tubes symmetrically in a centrifuge and centrifuge at 3000-3500 rpm for 10 minutes. After centrifugation, carefully aspirate the supernatant plasma using a micropipette and transfer it to a dedicated cryopreservation tube. Store the plasma in an ultra-low temperature environment of -80℃. The storage period for all samples should be controlled within 60 days.

[0037] Plasma lysis buffer preparation: First, transfer 250 μL of plasma sample to a 1.5 mL enzyme-free centrifuge tube, then add 750 μL of TRIzol™ LS lysis reagent (Thermo Fisher Scientific, catalog number: 12183025), mix thoroughly, and incubate at room temperature for 5 minutes to ensure complete dissociation of the nucleic acid-protein complex; for subsequent quantitative analysis, add 1 picomol of cel-miR-39-3p standard RNA (product code: miRB0000010) as an exogenous reference (exogenous reference gene) to the lysis system, and finally centrifuge at 12000 rpm for 10 minutes at 4°C. After centrifugation, collect the supernatant for subsequent experiments.

[0038] Chloroform extraction: Take 1 mL of plasma lysate, add 0.2 mL of chloroform, immediately seal the tube, and treat with vigorous shaking for 15 seconds, then let it stand at room temperature for 3 minutes. After completing the above operation, centrifuge the sample at 12000 rpm for 15 minutes at 4℃. At this time, a clear three-phase separation phenomenon can be observed: the lower layer is a pink organic phase, the middle layer is a transition layer, and the upper layer is a colorless and transparent aqueous phase, in which RNA is mainly enriched in the aqueous phase. Carefully transfer the upper aqueous phase to a new centrifuge tube using a pipette, taking special care to avoid contact with the intermediate phase during the operation.

[0039] RNA precipitation using isopropanol: Add an equal volume of isopropanol pre-cooled to 4°C to a fresh 1.5 mL centrifuge tube, mix thoroughly with the upper aqueous phase, and incubate at -20°C for 30 minutes to ensure complete RNA precipitation. Then, centrifuge at 12,000 rpm for 10 minutes at 4°C to complete the separation.

[0040] Ethanol washing: First, carefully remove the supernatant, ensuring that the precipitate at the bottom of the test tube is retained. Then, add 1 mL of 75% ethanol solution pre-cooled at 4°C, mix by inverting the tube, let it stand at room temperature for 5 minutes, and finally centrifuge at 7500 rpm for 5 minutes at 4°C.

[0041] RNA lysis: First, carefully remove the supernatant, ensuring that the precipitate at the bottom of the centrifuge tube is retained. Then, invert the centrifuge tube on the lab bench and let it stand for 10 minutes to allow it to dry completely. Next, add 10-15 µL of sterile nuclease-free water to completely dissolve the precipitate. Finally, transfer the dissolved product to an ultra-low temperature environment of -80℃ for storage.

[0042] RNA concentration detection: After thawing the RNA solution on ice, the extracted RNA samples were quantitatively analyzed using a Nano-drop spectrophotometer, and the absorbance values ​​at wavelengths of 260 nm and 280 nm were measured. The experimental results are as follows: Figure 1 As shown, the OD260 / OD280 ratio of the sample measured in this experiment was 2.08, and the RNA concentration was 405.4 ng / µL. The concentration of the extracted RNA was within the standard range of 25~500 ng / µL, and its OD260 / OD280 ratio was stably maintained in the ideal range of 1.8~2.1. These data fully confirm that the extracted RNA sample has high purity and concentration.

[0043] (2) Preparation of cDNA In this embodiment, the reverse transcription reaction system was carried out using the riboSCRIPT Reverse Transcription Kit (product number: C11027-2) produced by Guangzhou Ruibo Biotechnology Co., Ltd. The reaction system in Table 1 was continuously reacted at 42°C for 60 minutes, and then maintained at 70°C for 10 minutes to complete the entire reverse transcription reaction process.

[0044] Table 1. Reaction system for cDNA preparation

[0045] Note: Primer concentration range can be optimized between 200 nM and 800 nM.

[0046] (3) Real-time quantitative PCR In this embodiment, real-time quantitative PCR detection was performed using ChamQ Universal SYBR qPCR Master Mix (catalog number: Q711-02) provided by Nanjing Novizan Biotechnology Co., Ltd., with the cDNA sample synthesized in (2) as a template, and QuantStudio was used. TM Quantitative analysis was performed using the Flex real-time quantitative PCR system.

[0047] Specific upstream and downstream primers were designed for tiRNA-Gln-TTG-001 and cel-miR-39-3p, respectively. upstream primer for tiRNA-Gln-TTG-001: 5'-GCCTAGGATGGGGTGTGATAG-3' (SEQ ID NO.2). tiRNA-Gln-TTG-001 downstream primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO.3); upstream primer for cel-miR-39-3p: 5'-GCGCTCACCGGGTGTAAATC-3' (SEQ ID NO.4); cel-miR-39-3p downstream primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO.5).

[0048] Construct the RT-qPCR reaction system according to Table 2, and perform the RT-qPCR reaction according to the RT-qPCR reaction conditions in Table 3.

[0049] Table 2 RT-qPCR reaction system

[0050] Table 3 RT-qPCR reaction conditions

[0051] The amplification plot of the RT-qPCR reaction is as follows: Figure 2 As shown, the melting curve is as follows: Figure 3As shown, the results indicate that both the amplification and melting curves exhibit typical dynamic change characteristics. Specifically, amplification curve analysis shows that both cel-miR-39-3p (left group in the figure) and tiRNA-Gln-TTG-001 (right group in the figure) demonstrate a standard S-type amplification pattern, with the amplification process clearly divided into four characteristic stages. Notably, the CT value differences among the replicate samples were all controlled within 0.5 CT values, a result fully conforming to internationally recognized qPCR amplification quality evaluation standards. Melting curve analysis indicates that the amplification products are highly specific, with both curves exhibiting a single peak characteristic. The melting temperature of the cel-miR-39-3p primer (left peak in the figure) was 87℃, while the Tm value of the tiRNA-Gln-TTG-001 primer (right peak in the figure) was 90℃. Both values ​​are within the standard range of 80-90℃, fully validating the rationality of the primer design.

[0052] Example 2 This embodiment provides the difference in expression levels of tiRNA-Gln-TTG-001 in the plasma of lung cancer patients with brain metastases and those without brain metastases.

[0053] In this embodiment, the test queue (n=100, where: Met=40, Non-Met=60) consists of 100 lung cancer patients collected from Nanjing Medical University Cancer Hospital, of which 40 are lung cancer patients with brain metastases (Met) and 60 are lung cancer patients without brain metastases (Non-Met).

[0054] The expression level of tiRNA-Gln-TTG-001 was detected as described in Example 1. The results of tiRNA-Gln-TTG-001 expression level detection in the plasma of lung cancer patients with brain metastases and those without brain metastases are as follows: Figure 4 As shown, the difference in expression levels of tiRNA-Gln-TTG-001 in the plasma of lung cancer patients with brain metastases and those without metastases is visually presented. The brain metastasis group (Met) showed significant and specific high expression. This statistical difference indicates that tiRNA-Gln-TTG-001 has diagnostic value as a specific plasma biomarker for lung cancer brain metastases.

[0055] Example 3 This embodiment provides tiRNA-Gln-TTG-001 as a biomarker for detecting lung cancer brain metastases.

[0056] In this embodiment, the validation cohort (n=66) consists of 66 lung cancer patient samples collected from Xuzhou Central Hospital and Taixing People's Hospital between March 2021 and January 2022. The patients include those with brain metastases and those without brain metastases.

[0057] The expression level of tiRNA-Gln-TTG-001 was detected according to Example 1, and the ROC curve is shown below. Figure 5 As shown, its AUC (area under the curve) was 0.87, close to 1, indicating that there is a significant difference in the abundance of plasma tiRNA-Gln-TTG-001 between patients with brain metastases and those without. tiRNA-Gln-TTG-001 can serve as a biomarker for detecting brain metastases in lung cancer and has important clinical value in the specific diagnosis of lung cancer brain metastases.

Claims

1. tiRNA-Gln-TTG-001, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of a reagent for detecting the expression level of tiRNA-Gln-TTG-001 in plasma in the preparation of products for predicting, monitoring, or diagnosing lung cancer brain metastases, characterized in that... The nucleotide sequence of the tiRNA-Gln-TTG-001 is shown in SEQ ID NO.

1.

3. The application according to claim 2, characterized in that, The lung cancer mentioned includes lung adenocarcinoma.

4. The application according to claim 2 or 3, characterized in that, The reagents include primers for amplifying the tiRNA-Gln-TTG-001 fragment.

5. The application according to claim 4, characterized in that, The primers include an upstream primer tiRNA-Gln-TTG-001 and a downstream primer tiRNA-Gln-TTG-001, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

6. Reagents for detecting the expression level of tiRNA-Gln-TTG-001, including the upstream primer and the downstream primer of tiRNA-Gln-TTG-001, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

7. A kit for predicting, monitoring, or diagnosing lung cancer brain metastasis, characterized in that, The kit includes reagents for detecting the expression level of tiRNA-Gln-TTG-001 in plasma, the nucleotide sequence of which is shown in SEQ ID NO.

1.

8. The reagent kit according to claim 7, characterized in that, The reagents include primers for amplifying the tiRNA-Gln-TTG-001 fragment.

9. The reagent kit according to claim 8, characterized in that, The primers include an upstream primer tiRNA-Gln-TTG-001 and a downstream primer tiRNA-Gln-TTG-001, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

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