Application and application of tRF-21 as colorectal cancer diagnostic marker

Abstract

The invention belongs to the technical field of biological detection and diagnosis, and particularly relates to application and application of tRF-21 as a colorectal cancer diagnostic marker, especially a colorectal cancer KRAS G12C gene mutation diagnostic marker. The invention provides application of tRF-21 in serum as a detection marker for colorectal cancer, especially KRAS G12C, KRAS G12C mutation can be accurately detected by detecting the expression level of tRF-21 in serum, so that diagnosis and screening of colorectal cancer based on a liquid biopsy technology are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection and diagnosis, and particularly relates to the use and application of tRF-21 as a diagnostic marker for colorectal cancer, especially for the diagnosis of KRAS G12C gene mutation in colorectal cancer. Background Art

[0002] Colorectal cancer is the third leading cause of cancer-related deaths globally, and most patients with colorectal cancer are diagnosed at an advanced stage once diagnosed. Therefore, novel biomarkers with high sensitivity and specificity for early diagnosis and prognosis are currently considered the best strategy to improve the treatment effect of colorectal cancer.

[0003] Studies have shown that KRAS is one of the common oncogenes in humans, which is involved in key processes such as cell proliferation, differentiation, and survival. Gene mutations of KRAS are one of the most common genetic alterations in colorectal cancer, and G12C is the most common mutation type. This mutation leads to an increase in the GTPase activity of the KRAS protein, making it in an active state, and then continuously activating downstream signaling pathways (such as the MAPK and PI3K pathways), promoting cell proliferation and survival. Therefore, the KRAS G12C mutation is closely related to the malignancy, prognosis, and drug treatment sensitivity of tumors.

[0004] Clinically, KRAS mutations have become important molecular markers for guiding tumor treatment strategies. Currently, the commonly used methods for detecting KRAS G12C mutations in clinical practice usually require tissue biopsies to obtain tissue samples, and usually need to obtain tumor tissues through surgical operations or punctures, etc. This is an invasive process for patients and may bring complications such as pain, infection, and bleeding. In addition, due to the possible heterogeneity between different regions of the tumor, the information obtained from a local tissue sample may not comprehensively reflect the characteristics of the entire tumor. Moreover, tissue biopsies usually take a certain amount of time to complete, including surgical waiting time, laboratory processing, and pathological analysis, etc., which may lead to delays in diagnosis and treatment decisions.

[0005] Liquid biopsy is a non-invasive method for tumor diagnosis and monitoring. By analyzing tumor-related markers in body fluids (such as serum), such as exosomes and circulating tumor RNA (ctRNA), etc., to obtain tumor information. Compared with traditional tissue biopsies, liquid biopsy has the following advantages:

[0006] Non-invasiveness: Liquid biopsy can collect routine body fluid samples such as blood, urine, and saliva, without the need for invasive surgeries or tissue biopsies, reducing patient discomfort and risks, and providing a more convenient way of sample collection;

[0007] Systemic: Liquid biopsy can provide systemic tumor information, not limited to a specific tumor site. It can detect circulating tumor markers throughout the body, reflecting the systemic characteristics and changes of tumors.

[0008] Dynamic monitoring: Since liquid biopsy can repeatedly collect and analyze samples, it can achieve dynamic monitoring of tumors. It can be used to detect residual lesions after treatment, monitor tumor recurrence and metastasis, and evaluate treatment effects and drug resistance, etc.

[0009] Consideration of spatial heterogeneity: Liquid biopsy can overcome the limitations of local heterogeneity in tissue biopsy because circulating tumor markers can reflect the genetic and expression characteristics of tumors throughout the body.

[0010] Providing individualized treatment strategies: Liquid biopsy can help select the best treatment plan and formulate individualized treatment strategies based on the molecular characteristics and variability of tumors.

[0011] In summary, as a non-invasive method for tumor diagnosis and monitoring, liquid biopsy has the advantages of non-invasiveness, systemic nature, dynamic monitoring, and individualized treatment, providing new opportunities and options for the diagnosis and treatment of cancer patients. Therefore, there is an expectation in this field to develop a simple and non-invasive method for detecting colorectal cancer, especially the KRAS G12C gene mutation, based on liquid biopsy technology, which is of great significance for the formulation of clinical diagnosis and treatment strategies. Summary of the Invention

[0012] For this reason, the technical problem to be solved by the present invention is to provide the use of tRF-21 as a diagnostic marker for colorectal cancer, especially for the diagnosis of KRAS G12C gene mutation in colorectal cancer, which can detect the KRAS G12C mutation by detecting the expression level of tRF-21 in serum, thus solving the defects of traditional tissue biopsy methods such as invasiveness, local heterogeneity, and time lag.

[0013] The second technical problem to be solved by the present invention is to provide the application of tRF-21 as a diagnostic marker for colorectal cancer, especially for the diagnosis of KRAS G12C gene mutation in colorectal cancer, and further provide a rapid, non-invasive, and systemic method for diagnosing and detecting colorectal cancer based on liquid biopsy technology.

[0014] To solve the above technical problems, the present invention relates to the use of tRF-21 as a diagnostic marker for colorectal cancer.

[0015] Specifically, the colorectal cancer includes the KRAS G12C gene mutation.

[0016] Specifically, the tRF-21 is derived from serum.

[0017] The present invention also discloses the use of tRF-21 as a diagnostic marker for KRAS G12C gene mutation.

[0018] The present invention also discloses the use of a substance and / or reagent for detecting the level of tRF-21 in the preparation of a product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of colorectal cancer.

[0019] The present invention also discloses the use of a substance and / or reagent for detecting the expression level of tRF-21 in the preparation of a product for detecting the correlation and / or mutation expression level of KRAS G12C mutation.

[0020] The present invention also discloses a product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of colorectal cancer, and the product includes a reagent for detecting the correlation and / or mutation level of KRAS G12C mutation based on RT-qPCR method.

[0021] Specifically, for the product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of colorectal cancer, the product includes a plasma kit.

[0022] Specifically, for the product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of colorectal cancer, the reagent includes a stem-loop fluorescence quantitative PCR kit.

[0023] Specifically, for the optional stem-loop fluorescence quantitative PCR kit of the present invention, the stem-loop primer of Thermo Fisher Scientific company can be used for reverse transcription, and its detection system is as shown in Table 1 below, and the PCR reaction program is as shown in Table 2 below.

[0024] Table 1 Stem-loop fluorescence quantitative PCR system

[0025] Reagent Volume (μL) AMV 0.5 5×AMV Buffer 2 dNTP Mix (2.5 mM) 4 DEPC 1.5 Stem-loop RT Primer 1 RNA 1 Total 10

[0026] Table 2 Reaction program

[0027] Temperature (°C) Time (min) 16 30 42 30 85 5

[0028] After the above reverse transcription is completed, quantitative detection is carried out using the corresponding qPCR probe. Specifically, the q-PCR system is as shown in Table 3 below, and the qPCR reaction program is as shown in Table 4 below.

[0029] Table 3 q-PCR system

[0030] Reagent Volume (μL) <![CDATA[ddH2O]]> 14.77 10×buffer 2 <![CDATA[MgCl2]]> 1.2 dNTP (10 mM) 0.4 rTaq 0.3 TM Probe 0.33 cDNA 1 Total 20

[0031] Table 4 The qPCR reaction program is as follows:

[0032]

[0033] The present invention also discloses a system for diagnosing or assisting in the diagnosis, screening or assisting in the screening of colorectal cancer, comprising:

[0034] a detection device for determining the KRAS G12C mutation correlation and / or mutation level in a biological sample;

[0035] a judgment device for diagnosing or assisting in the diagnosis, screening or assisting in the screening of whether a person to be tested has colorectal cancer based on whether KRAS G12C is mutated or the mutation expression level in the biological sample.

[0036] The present invention provides the use of tRF-21 in serum as a detection biomarker for colorectal cancer, especially for KRAS G12C, which can accurately detect the KRAS G12C mutation by detecting the expression level of tRF-21 in serum, effectively solving the problems of difficult early diagnosis and treatment selection for highly lethal colorectal cancer, thereby realizing the diagnosis and screening of colorectal cancer based on liquid biopsy technology and solving the defects such as invasiveness, local heterogeneity and time lag of traditional tissue biopsy methods.

[0037] The present invention further provides the application of tRF-21 in serum as a detection biomarker for colorectal cancer, especially for KRAS G12C, and further provides a rapid, non-invasive and systemic method for diagnosing and detecting colorectal cancer based on liquid biopsy technology, which is of great significance for the diagnosis and treatment of colorectal cancer.

[0038] Experiments of the present invention have confirmed that the tRNA-derived fragment tRF-21-VBY9PYKHD (tRF-21) plays an important role in cancer, participates in processes such as tumorigenesis, development and treatment resistance, has a tumor-suppressing effect, and has the clinical application potential as a potential tumor biomarker. Moreover, the upregulation of tRF-21 is closely related to the occurrence, development and prognosis of colorectal cancer; and the upregulation of tRF-21 is considered to be a promoting factor for the proliferation, invasion and metastasis of colorectal cancer cells, and tRF-21 participates in a series of key biological processes, such as cell cycle regulation, epithelial-mesenchymal transition, angiogenesis and anti-apoptosis, by regulating the expression of multiple target genes. In addition, the upregulation of tRF-21 is related to the poor prognosis of colorectal cancer patients. The research finds that the expression level of tRF-21 is closely related to clinicopathological features such as the pathological type, grade and lymph node metastasis of colorectal cancer; in addition, the expression level of tRF-21 is also related to the survival rate and recurrence rate of colorectal cancer patients, and the low expression of tRF-21 is often associated with poor prognosis and increased recurrence risk.

[0039] This method establishes a liquid biopsy method for detecting KRAS G12C mutations in colorectal cancer by detecting tRF-21 in patients' sera. This method has the following advantages:

[0040] Non-invasive: The detection of tRF-21 can be performed by collecting the patient's blood sample without the need for invasive surgery or tissue biopsy. This non-invasive detection method is more convenient and acceptable for patients.

[0041] KRAS mutation specificity: The upregulation of tRF-21 has a high correlation with KRAS mutations in colorectal cancer. Therefore, by detecting the expression level of tRF-21, the presence or absence of KRAS mutations can be indirectly inferred.

[0042] High sensitivity: The upregulation of tRF-21 is prevalent in colorectal cancer and is associated with KRAS mutations. Therefore, the detection of tRF-21 can provide high-sensitivity screening and diagnosis of KRAS mutations in colorectal cancer.

[0043] Dynamic monitoring: Due to the characteristics of liquid biopsy, the detection of tRF-21 can achieve dynamic monitoring of KRAS mutations in colorectal cancer. By repeatedly collecting samples, the changes in KRAS mutations can be monitored in a timely manner, including residual lesions, recurrence, and metastasis after treatment.

[0044] Potential prognostic value: Some studies have shown that the upregulation of tRF-21 is associated with poor prognosis in colorectal cancer patients. Therefore, the detection of tRF-21 may help evaluate the prognostic risk of patients and provide a reference for the formulation of individualized treatment plans. Brief Description of the Drawings

[0045] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, wherein,

[0046] Figure 1 Shows the results of comparing the levels of tRF-21 in the sera of healthy volunteers, sera of colorectal cancer patients with other mutations, and sera of colorectal cancer patients with KRAS G12C mutations in Example 1;

[0047] Figure 2 Shows the results of survival analysis (survival curves) of colorectal cancer patients with KRAS G12C mutations with low expression of tRF-21 in serum and colorectal cancer patients with other mutation types with high expression of tRF-21 in serum in Example 2;

[0048] Figure 3 Shows the results based on miRNA target prediction software in Example 3. Detailed Description of the Specific Embodiments

[0049] Example 1

[0050] In this example, serum samples of colorectal cancer patients were collected, and miRNAs in the serum were extracted by appropriate technical means (such as TRIzol method). The specific method is as follows.

[0051] Separate plasma: Collect anticoagulated blood samples of normal people and colorectal cancer patients (EDTA anticoagulant), centrifuge the blood samples at 3000 rpm for 10 minutes, carefully aspirate the supernatant plasma (avoid aspirating blood cells) into a 1.5 mL EP tube, and store the plasma in an ultra-low temperature refrigerator at -80°C.

[0052] Extraction of plasma small RNAs, the specific steps include:

[0053] (1) Aspirate 100 μL of plasma into a new 1.5 mL EP tube, add 900 μL of TRIzol, shake on a shaker for 30 s until completely homogenized, and invert to mix evenly;

[0054] (2) Let it stand at room temperature for 5 minutes to allow the sample to lyse fully. Add 200 μL of chloroform, shake vigorously on a vortex oscillator for 15 s to mix the solution evenly, and then let it stand at room temperature for 5 minutes; Centrifuge at 12000 rpm at 4°C for 15 minutes. After centrifugation, the sample will be divided into three layers: yellow organic phase, white intermediate layer, and colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the upper aqueous phase to a new 1.5 mL EP tube;

[0055] (3) Measure the volume when transferring the aqueous phase, and then slowly add an equal volume of isopropanol (for example: add 500 μL of isopropanol to 500 μL of the transfer solution, and slowly invert to mix evenly;

[0056] (4) Precipitate the obtained solution at -20°C overnight (precipitate for at least 1 h);

[0057] (5) Centrifuge the overnight-precipitated mixture at 12000 rpm at 4°C for 20 minutes;

[0058] (6) Discard the supernatant, resuspend the precipitate with 75% DEPC ethanol, and then centrifuge at 12000 rpm at 4°C for 20 minutes;

[0059] (7) Discard the supernatant, invert the EP tube to dry. Dissolve the precipitate with 20 μL of DEPC water, and store the RNA in an ultra-low temperature refrigerator at -80°C.

[0060] In this example, the stem-loop RT-qPCR method described above was used to measure the expression level of tRF-21. The levels of tRF-21 in the sera of healthy volunteers, sera of other mutant colorectal cancer patients, and sera of colorectal cancer patients with KRAS G12C mutation were compared. The results are shown in the appendix Figure 1 .

[0061] The results showed that the level of tRF-21 in the sera of colorectal cancer patients with KRAS G12C mutation was significantly decreased / increased compared with that of healthy individuals and colorectal cancer patients with other mutant types. It was indicated that the detection of serum tRF-21 could preferably reflect the mutation status of KRAS G12C in colorectal cancer patients.

[0062] Example 2

[0063] In this example, the correlation between the expression level of tRF-21 and the KRAS G12C mutation was analyzed, and the presence or absence of the KRAS G12C mutation in colorectal cancer patients was determined according to the expression level of tRF-21.

[0064] In this example, colorectal cancer patients with KRAS G12C mutation with low expression of tRF-21 in serum were selected and compared with colorectal cancer patients with other mutant types with high expression of tRF-21 in serum for survival analysis.

[0065] In this example, the KRAS G12C mutation was diagnosed by histopathological examination. The survival time of the patients was defined as the date of cancer diagnosis to the date of the last follow-up or death. Whether the patients died and when they died were determined according to the hospitalization and outpatient records, the patient's family members or follow-up calls. The data were all analyzed using GraphPad Prism software. Independent sample t-tests were performed for between-group comparisons, and repeated measures ANOVA was performed for comparisons at each time point within the group; the Log-Rank test was used for survival analysis, and P<0.05 was considered statistically significant. The results are shown in the appendix Figure 2 .

[0066] The results indicated that high expression of tRF-21 in serum was significantly correlated with poor prognosis of colorectal cancer patients.

[0067] Example 3

[0068] In this embodiment, the miRNA target prediction software miRanda v3.3a was used to verify the relationship between tRF-21 and the KRAS transcript NM_001369786.1. The Score Threshold was set to 120, and the Energy Threshold was set to -1.000000 kcal / mol. The binding with the lowest Energy Threshold, that is, the lowest binding free energy, was selected. The specific operations and results are as shown in the appendix Figure 3 as follows.

[0069] The results showed that there were potential binding sites between tRF-21 and KRAS, and the binding free energy was -19.389999 kCal / Mol, which was relatively stable, suggesting the regulatory correlation between tRF-21 and KRAS.

[0070] In summary, the above embodiments of the present invention verified that the tRNA-derived fragment tRF-21-VBY9PYKHD (tRF-21) plays an important role in cancer. It is involved in processes such as tumorigenesis, development, and treatment resistance, has tumor-suppressing effects, and has the potential for clinical application as a potential tumor marker. Moreover, the upregulation of tRF-21 is closely related to the occurrence, development, and prognosis of colorectal cancer. The upregulation of tRF-21 is considered to be a promoting factor for the proliferation, invasion, and metastasis of colorectal cancer cells. tRF-21 is involved in a series of key biological processes, such as cell cycle regulation, epithelial-mesenchymal transition, angiogenesis, and anti-apoptosis, by regulating the expression of multiple target genes.

[0071] In addition, the upregulation of tRF-21 is associated with poor prognosis in patients with colorectal cancer. The study found that the expression level of tRF-21 was closely related to clinicopathological features such as the pathological type, grade, and lymph node metastasis of colorectal cancer. In addition, the expression level of tRF-21 was also related to the survival rate and recurrence rate of patients with colorectal cancer. The low expression of tRF-21 was often associated with poor prognosis and an increased risk of recurrence.

[0072] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of tRF-21 as a diagnostic marker for colorectal cancer.

2. The use according to claim 1, wherein The colorectal cancer includes KRAS G12C gene mutation.

3. The use according to claim 1 or 2, characterized in that, The tRF-21 is derived from serum.

4. Use of tRF-21 as a diagnostic marker for KRAS G12C gene mutation.

5. Use of a substance and / or reagent for detecting the level of tRF-21 in the preparation of a product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of colorectal cancer.

6. Use of a substance and / or reagent for detecting the expression level of tRF-21 in the preparation of a product for detecting the correlation and / or expression level of KRAS G12C mutation.

7. A product for the diagnosis or auxiliary diagnosis, screening or auxiliary screening of colorectal cancer, characterized in that, The product includes a reagent for detecting the correlation and / or mutation level of KRAS G12C mutation based on RT-qPCR method.

8. The product for colorectal cancer diagnosis or auxiliary diagnosis, screening or auxiliary screening according to claim 7, characterized in that, The product includes a plasma kit.

9. The product for colorectal cancer diagnosis or auxiliary diagnosis, screening or auxiliary screening according to claim 7 or 8, characterized in that, The reagent includes a stem-loop fluorescence quantitative PCR kit.

10. A system for diagnosing or assisting in the diagnosis, screening or assisting in the screening of colorectal cancer, characterized in that, Comprising: A detection device for determining the correlation and / or mutation level of KRAS G12C mutation in a biological sample; A judgment device for diagnosing or assisting in the diagnosis, screening or assisting in the screening of whether a person to be tested has colorectal cancer based on whether KRAS G12C in the biological sample is mutated or the mutation expression level.

Images