Biomarker tRF-Arg-CCG-019 and its application in the preparation of lung cancer diagnostic products

Abstract

The present application discloses a biomarker tRF-Arg-CCG-019 and its application in the preparation of a lung cancer diagnosis product, belonging to the field of molecular biology technology. The nucleotide sequence of the biomarker tRF-Arg-CCG-019 is AGGTAATCCGGTGCGCCGG (SEQ ID NO.1), and its expression level in lung cancer tissue (malignant tumor tissue) is significantly higher than that in adjacent tissues. It can be used as a biomarker for early diagnosis of lung cancer, with a sensitivity of 88% and a specificity of 92.9%. The molecule can be used to simply and quickly diagnose lung cancer in the early stage.

Description

Technical Field

[0001] The present application belongs to the field of molecular biology technology, and specifically relates to a biomarker tRF-Arg-CCG-019 and its application in the preparation of lung cancer diagnostic products. Background Art

[0002] Lung tumors refer to abnormal masses or growths in the lungs, including benign tumor tissue, malignant tumor tissue, inflammatory lesions, infectious lesions, and vascular lesions. Among them, malignant tumor tissue, namely lung cancer, includes lung adenocarcinoma, squamous cell lung carcinoma, large cell lung cancer, and small cell lung cancer. It is the second most common malignant tumor in the world and the first in mortality, and has become a huge medical burden in the world. Although progress has been made in the treatment of lung cancer in recent years, the treatment of lung cancer still faces many challenges. The most difficult problem is that about 75% of patients are already in the late stage at the time of diagnosis and have missed the best time for treatment. If it can be diagnosed at an early stage, it will effectively improve the treatment effect of lung cancer. However, malignant tumor tissue may not have obvious symptoms in the early stage, and there is still a lack of specific molecular diagnostic methods for lung cancer. Therefore, finding biomarkers for early diagnosis of lung cancer (diagnosing whether the tumor tissue is benign or malignant) has become an urgent need for the diagnosis and treatment of lung cancer.

[0003] tRNA derived fragments (tRFs) are a type of non-coding RNAs (ncRNAs) less than 50 nt in length formed by the cleavage of transfer RNA (tRNA). Studies have shown that tRFs are not the product of random degradation of tRNA, but are produced by precise cleavage of specific RNA cleavage enzymes. Their abnormal expression is often highly correlated with the pathological state of specific diseases, and they are highly conservative, stable, and more diverse in regulation than miRNA. These characteristics make tRFs have broad prospects in the development and application of disease diagnosis and treatment methods. Summary of the invention

[0004] 1. Purpose of the Invention

[0005] The purpose of the present application is to provide a biomarker tRF-Arg-CCG-019 and its application in the preparation of lung cancer diagnostic products. The nucleotide sequence of the biomarker tRF-Arg-CCG-019 is shown in SEQ ID NO.1. The expression level in lung cancer tissue (malignant tumor tissue) is significantly higher than that in adjacent cancer tissue. It can be used as a biomarker for early diagnosis of lung cancer. The molecule can be used to simply and quickly perform early diagnosis of lung cancer.

[0006] 2. Technical solution

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted in this application is as follows:

[0008] The present application provides a biomarker tRF-Arg-CCG-019, whose nucleotide sequence is: AGGTAATCCGGTGCGCCGG (SEQ ID NO.1). tRF-Arg-CCG-019 is produced by degradation of the 3' end of tRNA1-Arg-CCG-16 located on chromosome 16. Its expression level in lung cancer tissue is significantly higher than that in adjacent cancer tissue or benign tumor tissue, and can be used as a molecular marker for the diagnosis of lung cancer.

[0009] The present application also provides the use of the above-mentioned biomarker tRF-Arg-CCG-019 in the preparation of lung cancer diagnostic products.

[0010] The present application also provides the use of a reagent for detecting the expression level of the above-mentioned biomarker tRF-Arg-CCG-019 in the preparation of a lung cancer diagnosis product.

[0011] Furthermore, the expression level of the above-mentioned biomarker tRF-Arg-CCG-019 refers to the expression level of tRF-Arg-CCG-019 in lung tissue.

[0012] Furthermore, the above application includes: detecting the expression level of tRF-Arg-CCG-019 in the lung tissue of the subject; comparing the tRF-Arg-CCG-019 expression level with a preset value, and if it is higher than the preset value, the possibility of having lung cancer is high.

[0013] Furthermore, the above-mentioned reagent for detecting the expression level of the biomarker tRF-Arg-CCG-019 includes a real-time fluorescence quantitative PCR detection reagent, and the expression level of the biomarker tRF-Arg-CCG-019 is detected by the real-time fluorescence quantitative PCR method.

[0014] Furthermore, the above-mentioned real-time fluorescence quantitative PCR detection reagent includes primers for amplifying tRF-Arg-CCG-019.

[0015] Furthermore, the above-mentioned lung cancer diagnosis products include: lung cancer diagnostic reagents, lung cancer diagnostic test strips, lung cancer diagnostic kits, lung cancer diagnostic gene chips or lung cancer diagnostic high-throughput sequencing platforms.

[0016] Furthermore, the above-mentioned lung cancer diagnosis product is a lung cancer diagnosis kit, which includes the above-mentioned reagent for detecting the expression level of the biomarker tRF-Arg-CCG-019.

[0017] Furthermore, the lung cancer diagnosis kit is a real-time fluorescence quantitative PCR detection kit, which includes the real-time fluorescence quantitative PCR detection reagent.

[0018] Furthermore, the above-mentioned lung cancer diagnostic product is a high-throughput sequencing platform, and the expression level of tRF-Arg-CCG-019 is obtained through high-throughput sequencing.

[0019] The present application also provides a lung cancer diagnosis kit, which includes the above-mentioned reagent for detecting the expression level of the biomarker tRF-Arg-CCG-019.

[0020] Furthermore, the lung cancer diagnosis kit comprises: a tissue RNA extraction reagent, a reverse transcription reagent, a real-time fluorescence quantitative PCR reagent, and a primer for amplifying tRF-Arg-CCG-019.

[0021] The present application also provides an application of the above-mentioned lung cancer diagnostic kit, which includes the following steps: extraction of tumor tissue RNA, preparation of cDNA, real-time fluorescence quantitative PCR amplification of tRF-Arg-CCG-019, and comparison of the expression level of tRF-Arg-CCG-019 with a preset value.

[0022] Furthermore, the preparation of cDNA includes preparing cDNA from the extracted RNA sample using a riboSCRIPT Reverse Transcription Kit.

[0023] Furthermore, the real-time fluorescence quantitative PCR amplification of tRF-Arg-CCG-019 includes: using VazymeChamQ Universal SYBR qPCR Master Mix fluorescence quantitative kit to perform fluorescence quantitative PCR amplification with reverse transcribed cDNA as a template.

[0024] The present application also provides a lung cancer auxiliary diagnosis device, which comprises:

[0025] A data input module, used to input the expression level of tRF-Arg-CCG-019 in the lung tumor tissue of the subject;

[0026] A data processing module, for comparing the expression level of tRF-Arg-CCG-019 with a preset value;

[0027] The result output module is used to output the comparison result. If the expression level of tRF-Arg-CCG-019 is higher than the preset value, the possibility of lung cancer is high; otherwise, the possibility of non-lung cancer is high.

[0028] The present application also provides an electronic device for auxiliary diagnosis of lung cancer, comprising: one or more processors; a storage device on which one or more programs are stored, and when the one or more programs are executed by one or more processors, the one or more processors implement the process in the above-mentioned lung cancer auxiliary diagnosis device.

[0029] The present application also provides a computer storage medium on which a computer program is stored, wherein when the program is executed by a processor, the process in the above-mentioned lung cancer auxiliary diagnosis device is implemented.

[0030] The present application also provides a method for detecting the expression level of tRF-Arg-CCG-019 in lung tumor tissue for non-diagnostic purposes, the method comprising: extraction of RNA from lung cancer tissue, preparation of cDNA and real-time fluorescence quantitative PCR amplification of tRF-Arg-CCG-019.

[0031] Furthermore, the preparation of cDNA includes preparing cDNA from the extracted RNA sample using a riboSCRIPT Reverse Transcription Kit.

[0032] Furthermore, the real-time fluorescence quantitative PCR amplification of tRF-Arg-CCG-019 includes: using VazymeChamQ Universal SYBR qPCR Master Mix fluorescence quantitative kit to perform fluorescence quantitative PCR amplification with reverse transcribed cDNA as a template.

[0033] The present application also provides a method for molecular diagnosis of lung cancer, which comprises the following steps:

[0034] S1: Collect lung tumor tissue from subjects and extract total RNA from the tissue;

[0035] S2: pre-treat total RNA and reverse transcribe it into cDNA;

[0036] S3: Detect the expression level of tRF-Arg-CCG-019 in the tumor tissue of the subjects;

[0037] S4: Compare the tRF-Arg-CCG-019 expression level with the preset value, and diagnose lung cancer if it is higher than the preset value.

[0038] Furthermore, the reverse transcription into cDNA includes preparing cDNA from the extracted RNA sample using a riboSCRIPT Reverse Transcription Kit.

[0039] Furthermore, the above-mentioned detection of the expression level of tRF-Arg-CCG-019 in the tumor tissue of the subject was performed using the VazymeChamQ Universal SYBR qPCR Master Mix fluorescence quantitative kit, and the reverse transcribed cDNA was used as a template for fluorescence quantitative PCR amplification to obtain the expression level of tRF-Arg-CCG-019.

[0040] 3. Beneficial effects

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] (1) The present application discloses a biomarker tRF-Arg-CCG-019 and its application in the preparation of a lung cancer diagnosis product. The sequence of tRF-Arg-CCG-019 is AGGTAATCCGGTGCGCCGG (SEQ ID NO. 1). This fragment is upregulated in lung cancer tissue and can be used as a biomarker to assist in the diagnosis of lung cancer.

[0043] (2) The application discloses the biomarker tRF-Arg-CCG-019 and its application in the preparation of lung cancer diagnostic products, which are used to quickly and accurately detect the expression level of tRF-Arg-CCG-019 in tissues, which can help clinicians accurately identify early-stage lung cancer and provide assistance in the selection of appropriate clinical treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the position of tRF-Arg-CCG-019 in its source tRNA.

[0045] Figure 2 It is the amplification curve diagram of tRF-Arg-CCG-019 detected by real-time fluorescence quantitative PCR.

[0046] Figure 3 It is the melting curve diagram of tRF-Arg-CCG-019 detected by real-time fluorescence quantitative PCR.

[0047] Figure 4 The expression level of tRF-Arg-CCG-019 in lung cancer tissues and adjacent normal tissues is different.

[0048] Figure 5 It is the ROC curve and area under the curve (AUC) for testing the diagnostic efficacy of tRF-Arg-CCG-019. DETAILED DESCRIPTION

[0049] The present application is further described below in conjunction with specific embodiments.

[0050] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0052] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0053] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0054] 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" expressly includes only A, only B, only C, and combinations of each thereof.

[0055] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0056] As used in this application, the term "non-coding RNA (ncRNA)" refers to RNA that does not encode proteins. In the human genome, most of it is non-coding RNA. Only 2% of the transcripts produced in the human genome are coding RNA, and the remaining 98% are non-coding RNA, which are functional RNA molecules that cannot be translated into proteins. Among non-coding RNA, molecules with regulatory effects are mainly divided into two categories according to their size: short-chain non-coding RNA (including tRF, siRNA, miRNA, piRNA) and long-chain non-coding RNA (Longnon coding RNA, LncRNA). These ncRNAs are widely involved in almost all physiological and pathological activities of the human body, including participating in, regulating or mediating the occurrence and development of many tumors.

[0057] As used in this application, 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 the product of random degradation of tRNA, but are produced under the precise cleavage of 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.

[0058] As used in this application, the terms "tRF", "tRF(s)", "tRNA-derived fragment", and "transfer RNA-derived fragment" have the same meaning and can be used interchangeably. They all refer to an RNA fragment that is formed by tRNA cleavage, does not encode protein, and is generally less than 50 nt in length.

[0059] As used in this application, the term "nuclease-free water" refers to water from which nucleases are completely removed and which is not contaminated by nonspecific endonucleases, exonucleases, and ribonucleases, and is commonly used to dissolve DNA / RNA and configure reaction systems containing nucleic acids.

[0060] As used in this application, the term "expression level" refers to "normalized expression amount", and "normalized expression amount" refers to the difference between the RT-qPCR cycle number and the internal reference gene U6.

[0061] In this application, all lung cancer patients were from Jiangsu Cancer Hospital and had passed the hospital's ethical approval.

[0062] Example 1

[0063] This example provides the biomarker tRF-Arg-CCG-019.

[0064] The biomarker tRF-Arg-CCG-019 is derived from tRNA1-Arg-CCG-16 ( Figure 1 ) is produced after the 3' end of the peptide is degraded, and its nucleotide sequence is: AGGTAATCCGGTGCGCCGG (SEQ ID NO.1).

[0065] Example 2

[0066] This embodiment provides the detection of the expression level of tRF-Arg-CCG-019 in lung cancer tissue and adjacent normal tissue. Specifically includes:

[0067] (1) Collect tissue samples: Take 10-20 mg of lung cancer tissue or adjacent normal tissue (a total of 100 lung cancer tissues and 42 adjacent normal tissues) and immerse them in an enzyme-free centrifuge tube containing 1-2 mL of RNA preservation solution;

[0068] (2) Lysing tissue: Add 1-2 mL of RNA extraction reagent TRIzol into a centrifuge tube, grind the tissue sample thoroughly into a homogenate, and let it stand at room temperature for 5 minutes;

[0069] (3) Chloroform extraction: add 0.2 mL of chloroform to every 1 mL of homogenate sample, cover the tube, shake vigorously for 15 seconds, leave at room temperature for 3 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes. At this time, the sample will 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.

[0070] (4) Isopropanol precipitation: Add an equal volume of isopropanol precooled at 4°C to a new 1.5 mL centrifuge tube. Mix by inverting the tube, and let stand at 20°C for 30 min to fully precipitate the RNA. Centrifuge at 4°C, 12,000 rpm, for 10 min.

[0071] (5) Ethanol washing: Carefully discard the supernatant, retain the precipitate at the bottom of the test tube, add 1 mL of 75% ethanol solution precooled at 4°C, invert evenly, and place at room temperature for 5 min. Centrifuge at 7500 rpm at 4°C for 5 min.

[0072] (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, fully dry, 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;

[0073] (7) RNA concentration determination: After melting the RNA solution on ice, the extracted RNA concentration was detected using a Nano-drop instrument;

[0074] (8) Preparation of cDNA: The extracted RNA samples were used to prepare cDNA using the riboSCRIPT Reverse Transcription Kit (purchased from Guangzhou Ribo 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 was marked as X here, 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;

[0075] Table 1 Reaction system for cDNA preparation

[0076]

[0077] (9) Real-time fluorescence quantitative PCR (RT-qPCR) detection: Vazyme ChamQ Universal SYBRqPCR Master Mix fluorescence quantitative kit (purchased from Nanjing Novozyme 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-Arg-CCG-019 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;

[0078] Table 2 RT-qPCR reaction system

[0079]

[0080] Table 3 RT-qPCR reaction conditions

[0081]

[0082] RT-qPCR result analysis:

[0083] The amplification curve and melting curve of RT-qPCR are as follows Figure 2 and Figure 3 As shown;

[0084] Figure 2The amplification curve is S-shaped, with four distinct periods. The difference between the CT values ​​of each group of duplicate wells is less than 0.5 CT value, which meets the amplification curve standard.

[0085] Figure 3 The melting curve is used to determine whether the amplified product is single. A single peak in the melting curve indicates that the amplified product is single. The Tm value indicates the melting temperature of the primer itself, and the standard value is 80℃~90℃.

[0086] (10) Obtaining standardized expression: Using the U6 gene as an internal reference, the difference in the cycle number between tRF-Arg-CCG-019 and the U6 gene was obtained by RT-qPCR, and used for subsequent comparison of gene expression levels between different tissues;

[0087] (11) Comparison of tRF expression in lung cancer tissues and adjacent normal tissues

[0088] The above RT-qPCR detection revealed the difference in the expression levels of tRF-Arg-CCG-019 in lung cancer tissues and adjacent normal tissues. Figure 4 As shown; it can be seen that tRF-Arg-CCG-019 is significantly overexpressed in lung cancer tissues, and the difference is statistically significant (P < 0.0001).

[0089] The above results indicate that tRF-Arg-CCG-019 can be used as a molecular marker for the diagnosis of lung cancer.

[0090] Example 3

[0091] This example provides the validation of the biomarker tRF-Arg-CCG-019 in the diagnosis of lung cancer.

[0092] The tRF-Arg-CCG-019 level in lung cancer tissues of 100 lung cancer patients was detected and the ROC curve was prepared. The results are as follows Figure 5 As shown, the AUC value is 0.9457 (the closer the AUC is to 1, the better the diagnostic efficacy). It can be considered that tRF-Arg-CCG-019 can be used as a biomarker for lung cancer diagnosis. Its sensitivity as a lung cancer marker is 88% and its specificity is 92.9%.

[0093] Example 4

[0094] This embodiment provides a lung cancer auxiliary diagnosis device, which includes:

[0095] A data input module, used to input the expression level of tRF-Arg-CCG-019 in the lung tumor tissue of the subject; the detection of the expression level of tRF-Arg-CCG-019 can refer to Example 2;

[0096] A data processing module, for comparing the expression level of tRF-Arg-CCG-019 with a preset value;

[0097] The result output module is used to output the comparison result. If the expression level of tRF-Arg-CCG-019 is higher than the preset value, the possibility of lung cancer is high; otherwise, the possibility of non-lung cancer is high.

Claims

1. Use of a reagent for detecting the expression level of the biomarker tRF-Arg-CCG-019 in the preparation of a lung cancer diagnosis product, characterized in that: The nucleotide sequence of tRF-Arg-CCG-019 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The application includes: detecting the expression level of tRF-Arg-CCG-019 in the lung tissue of the subject; comparing the tRF-Arg-CCG-019 expression level with a preset value, and if the expression level is higher than the preset value, the possibility of having lung cancer is high.

3. The use according to claim 2, characterized in that: The reagent for detecting the expression level of the biomarker tRF-Arg-CCG-019 includes a real-time fluorescence quantitative PCR detection reagent, and the expression level of the biomarker tRF-Arg-CCG-019 is detected by a real-time fluorescence quantitative PCR method.

4. The use according to claim 3, characterized in that: The real-time fluorescence quantitative PCR detection reagent includes primers for amplifying tRF-Arg-CCG-019.

5. A lung cancer auxiliary diagnosis device, characterized in that: The device comprises: A data input module, used to input the expression level of tRF-Arg-CCG-019 in the lung tumor tissue of the subject, wherein the nucleotide sequence of tRF-Arg-CCG-019 is shown in SEQ ID NO.1; A data processing module, for comparing the expression level of tRF-Arg-CCG-019 with a preset value; The result output module is used to output the comparison results. If the expression level of tRF-Arg-CCG-019 is higher than the preset value, the possibility of lung cancer is high.

6. An electronic device for auxiliary diagnosis of lung cancer, characterized in that: The electronic device includes: one or more processors; a storage device on which one or more programs are stored, and when the one or more programs are executed by the one or more processors, the one or more processors implement the process in the lung cancer auxiliary diagnosis device of claim 5.

7. A computer storage medium, characterized in that: The computer storage medium stores a computer program, wherein when the program is executed by the processor, the process in the lung cancer auxiliary diagnosis device of claim 5 is implemented.

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