Hematological marker and kit for colorectal cancer diagnosis

Abstract

The invention discloses a hematological marker and a kit for colorectal cancer diagnosis, and belongs to the technical field of colorectal cancer detection. The hematological marker is a serum exosome tsRNA-Glu-i-0315, and the sequence of the serum exosome tsRNA-Glu-i-0315 is as shown in SEQ ID NO. 1 (sequence identifier number 1). The kit for early diagnosis or progress early warning of colorectal cancer comprises a fluorescent PCR reaction system, the fluorescent PCR reaction system comprises a forward primer aiming at the tsRNA-Glu-i-0315 and a reverse primer aiming at the tsRNA-Glu-i-0315, and the forward primer and the reverse primer are used for detecting the tsRNA-Glu-i-0315. 1, and the sequence of a forward primer aiming at the tsRNA-Glu-i-0315 is as shown in SEQ ID NO. 2. By detecting tsRNA-Glu-i-0315, it is proposed for the first time that compared with healthy volunteers, the expression level of tsRNA-Glu-i-0315 in serum exosomes of colorectal cancer patients is remarkably reduced, ROC curve analysis shows that AUC is 0.789, the sensitivity is 86.8%, and the specificity is 62.5%.

Description

Technical Field

[0001] The present invention belongs to the technical field of colorectal cancer detection, and in particular relates to a hematological marker and a kit for diagnosing colorectal cancer. Background Art

[0002] Colorectal cancer is a common malignant tumor with a long cancer transformation process. Currently, most patients are diagnosed in the late stage, which is not conducive to the prognosis and survival of patients. Effective early screening methods are expected to reduce the incidence and mortality. Therefore, it is urgent to explore a tumor marker with good sensitivity and specificity to provide an objective basis for the early diagnosis and treatment of colorectal cancer.

[0003] Exosomes are nanovesicles with a diameter of 30-200nm and wrapped by a lipid bilayer. They can be secreted by a variety of cells, mainly from hematopoietic cells such as reticulocytes, B lymphocytes and T lymphocytes, and can also be produced by non-hematopoietic cells, such as intestinal epithelial cells and tumor cells. Exosomes can be secreted at different stages of the disease, and can be stably present in body fluids after secretion, while carrying molecules of their parent cells, including DNA, different types of RNA, proteins, lipids and metabolites. Exosomes secreted by tumor cells, namely tumor-derived exosomes (TDEs), carry tumor-derived molecules, which can not only participate in the information exchange between tumor cells as part of cell communication, but also judge the progression, treatment, prognosis and recurrence of cancer. In addition, the lipid bilayer structure ensures the stability of exosomes, so that the molecules they carry can be stored for several weeks at room temperature without being degraded and destroyed. Current colorectal cancer screening and diagnosis methods, such as serological screening of common tumor markers, are not ideal in sensitivity; fecal immunochemical testing is easy to operate but has low sensitivity and is easily affected by diet, drugs, etc.; rectal examination and colonoscopy are both invasive methods with poor patient compliance and high technical requirements for operators, which are not suitable for large-scale screening. Compared with traditional tissue biopsy, liquid biopsy is less invasive, less risky, has high specificity and sensitivity, and can perform dynamic detection, which is a new direction for cancer screening. Summary of the invention

[0004] In view of the problems of low sensitivity, high operation difficulty and low specificity of the existing colorectal cancer screening and diagnosis methods, the present invention provides a hematological marker and a kit for the diagnosis of colorectal cancer, which are easy and fast to operate, improve the sensitivity and accuracy of detection, and have good application value in the diagnosis of colorectal cancer and early warning of progression.

[0005] The present invention is achieved through the following technical solutions: A hematological marker for diagnosing colorectal cancer, wherein the hematological marker is serum exosome tsRNA-Glu-i-0315, and the sequence of serum exosome tsRNA-Glu-i-0315 is shown in SEQ ID NO.1.

[0006] The present invention also discloses a kit for early diagnosis and early warning of progression of colorectal cancer, which is used to detect the hematological markers described in claim 1; The kit for early diagnosis or early warning of colorectal cancer comprises a fluorescent PCR reaction system; the fluorescent PCR reaction system comprises a forward primer for tsRNA-Glu-i-0315 and a reverse primer for tsRNA-Glu-i-0315; The sequence of the forward primer for tsRNA-Glu-i-0315 is shown in SEQ ID NO.2.

[0007] Furthermore, the fluorescent PCR reaction system also includes SYBR Green mixed solution and water.

[0008] Furthermore, the SYBR Green mixed solution includes PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ .

[0009] Furthermore, it also includes a reverse transcription reaction system and an internal reference system; The internal reference system includes a forward primer of internal reference U6 and a reverse primer of internal reference U6; The sequence of the forward primer of the internal reference U6 is shown in SEQ ID NO.4; The sequence of the reverse primer of the internal reference U6 is shown in SEQ ID NO.5.

[0010] Furthermore, the internal reference system also includes SYBR Green mixed solution and water.

[0011] Furthermore, the reverse transcription reaction system includes polyadenylic acid polymerase, reverse transcriptase mixture, reverse transcription buffer and water.

[0012] Beneficial Effects The present invention detected tsRNA-Glu-i-0315 and found that the expression level of tsRNA-Glu-i-0315 in the serum exosomes of colorectal cancer patients was significantly reduced: the present invention proposed for the first time that compared with healthy volunteers, the expression level of tsRNA-Glu-i-0315 in the serum exosomes of colorectal cancer patients was significantly reduced, and ROC curve analysis showed that the AUC was 0.789, the sensitivity was 86.8%, and the specificity was 62.5%, providing a new method for the diagnosis of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the expression level of serum exosome tsRNA-Glu-i-0315 in colorectal cancer patients and healthy volunteers; Figure 2 ROC curve of tsRNA-Glu-i-0315 in diagnosing colorectal cancer patients. DETAILED DESCRIPTION

[0014] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the implementation cases are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0015] Example 1 The hematological marker for early diagnosis or progression warning of colorectal cancer is serum exosome tsRNA-Glu-i-0315. The sequence of serum exosome tsRNA-Glu-i-0315 is shown in SEQ ID NO.1 in Table 1. Studies have shown that compared with healthy volunteers, the expression level of tsRNA-Glu-i-0315 in serum exosomes of colorectal cancer patients is significantly reduced; compared with healthy volunteers, the expression level of tsRNA-Glu-i-0315 in serum exosomes of early colorectal cancer patients is significantly reduced.

[0016] A kit for early diagnosis or early warning of colorectal cancer, the kit is used to detect the above-mentioned hematological markers, the kit for early diagnosis or early warning of colorectal cancer comprises a fluorescent PCR reaction system, a reverse transcription reaction system and an internal reference system; the fluorescent PCR reaction system comprises a forward primer for tsRNA-Glu-i-0315 and a reverse primer for tsRNA-Glu-i-0315, a SYBR Green mixed solution and water, the sequence of the forward primer for tsRNA-Glu-i-0315 is shown in SEQ ID NO.2 in Table 1, the sequence of the reverse primer for tsRNA-Glu-i-0315 is shown in SEQ ID NO.3 in Table 1, the SYBR Green mixed solution comprises a PCR reaction buffer, a SYBR Green I dye, ROX, dNTPs, Mg 2+ The internal reference system includes a forward primer of internal reference U6, a reverse primer of internal reference U6, a SYBR Green mixed solution and water, the sequence of the forward primer of internal reference U6 is shown in SEQ ID NO.4 in Table 1, and the sequence of the reverse primer of internal reference U6 is shown in SEQ ID NO.5 in Table 1. The reverse transcription reaction system includes polyadenylic acid polymerase, a reverse transcriptase mixed solution, a reverse transcription buffer and water.

[0017] Table 1 Sequences related to the kit for early diagnosis or early warning of progression of colorectal cancer Example 2 Application of a kit for early diagnosis or early warning of progression of colorectal cancer in diagnosing early colorectal cancer and early warning of progression (1) Experimental design Serum samples were collected from patients with colorectal cancer who were diagnosed with colorectal cancer in the Affiliated Tumor Hospital of Shandong First Medical University and healthy volunteers, and exosomes were separated and enriched, and exosomal RNA was extracted. The expression of tsRNA-Glu-i-0315 was detected by qPCR, and the expression difference between colorectal cancer patients and healthy volunteers was statistically analyzed, and its clinical diagnostic efficacy was analyzed.

[0018] (2) Patient samples involved in the experiment From July to December 2021, 174 colorectal cancer patients and 157 healthy volunteers were admitted to the Affiliated Tumor Hospital of Shandong First Medical University. Among them, 81 patients with early colorectal cancer were included. The enrolled patients were classified by TNM by pathological methods. The clinical data included in this study mainly included age, gender, UICC tumor stage (eighth edition), smoking and drinking history, hypertension and diabetes history. Before surgery, chemotherapy and radiotherapy, about 3 ml of peripheral blood samples were collected from patients and placed in a vacuum blood collection tube containing a coagulant. After standing for blood coagulation, the blood was centrifuged at 3000g for 10 minutes at room temperature. The separated serum was centrifuged at 12,000g for 30 minutes at 4°C, the supernatant was taken and then centrifuged at 100,000g for 2 hours at 4°C, the supernatant was discarded, 500 μL of Trizol reagent was added to the exosome precipitate, lysed at 4°C for 30 minutes, and then transferred to a clean enzyme-free EP tube and frozen at -80°C for later use.

[0019] (3) Determination of exosomal tsRNA-Glu-i-0315 levels in serum The qPCR method was used to analyze the expression level of exosomal tsRNA-Glu-i-0315 in the serum of colorectal cancer patients and healthy volunteers. The specific steps are as follows: 1) Extraction of exosomes.

[0020] 2) Extraction of total exosome RNA The operation was carried out in a biosafety cabinet. The frozen exosome samples after pretreatment were thawed at room temperature, 100 μL of chloroform was added, and the mixture was shaken vigorously for 30 seconds. After standing for 3 minutes, it was centrifuged at 12,000 g for 15 minutes at 4°C (the mixture was separated into a lower pink phenol-chloroform liquid phase, a middle layer of white flocs, and an upper layer of colorless aqueous phase, and RNA was distributed in the upper aqueous phase); 250 μL of the upper aqueous phase was pipetted into a clean enzyme-free EP tube, an equal volume of isopropanol was added, and the mixture was shaken for 30 seconds. After standing for 10 minutes, it was centrifuged at 12,000 g for 10 minutes at 4°C, and the liquid was discarded; 750 μL of 75% ethanol solution was added, and the mixture was gently inverted and mixed, and centrifuged at 7,500 g for 5 minutes at 4°C; the supernatant was discarded as much as possible, and the white RNA precipitate at the bottom of the tube was retained. The EP tube was opened and inverted on a new filter paper, and dried naturally for 10 to 15 minutes. Add 10 μL RNase-free water and mix thoroughly with a pipette to completely dissolve the RNA. The dissolved RNA sample can be stored at -80°C for later use. Use Nanodrop ultra-micro spectrophotometer to detect RNA concentration and purity.

[0021] 3) Reverse transcription reaction Reverse transcription reaction system: 3.75μL Total RNA, 1.25μL RT Enzyme Mix, 5μL RT Reaction Solution; Reverse transcription reaction conditions: 37°C for 60 min, 85°C for 15 min, and storage at 4°C.

[0022] 4) Preparation before real-time fluorescence PCR amplification Prepare primer solution: The primer reagent is a dry powder. Centrifuge at 3000 rpm for 60 seconds, then slowly open the lid, add an appropriate amount of nuclease-free double-distilled water to make the final concentration 10 μM, close the lid and shake thoroughly to dissolve and mix; 5) Fluorescence PCR amplification reaction The fluorescence PCR reaction system is shown in Table 2 below: Table 2 Fluorescence PCR reaction system Each component in the fluorescent PCR reaction system is added to the corresponding amplification reaction plate in turn, and the sample cDNA is added. To avoid reagent contamination, a transparent sealing film should be attached immediately after the sample is added (Note: To avoid exposure contamination during the operation, the system preparation and sample addition should be carried out in a biosafety cabinet), centrifuged at 800g for 3 minutes at room temperature, mixed, and then loaded on the machine. Use the LightCycler 480 RT-PCR instrument to set the fluorescent PCR reaction conditions in Table 3 below. In this reaction, each sample is set to one replicate, and the average is taken to reduce the error. The experiment is repeated three times.

[0023] Table 3 Fluorescence PCR reaction conditions 6) Amplification reaction of internal reference system The U6 gene was used as the internal reference gene. The internal reference system included 10 μL SYBR Green mixed solution, 0.8 μL internal reference U6 forward primer, 0.8 μL internal reference U6 reverse primer, and 6.4 μL enzyme-free water. The reaction conditions of the internal reference system included 95°C pre-denaturation for 30 seconds, 95°C denaturation for 5 seconds, 60°C annealing for 20 seconds, and 65°C extension for 15 seconds, for a total of 40 cycles.

[0024] (4) Data statistical analysis Draw the fluorescence amplification curve and melting curve to obtain the Ct value, and use ΔCt to analyze the real-time fluorescence quantitative PCR reaction results. The ΔCt value represents the relative expression of the target RNA. The larger the ΔCt value, the weaker the expression of the target RNA. .

[0025] Prism 9.0 was used to analyze the qPCR data. tThe test was expressed as mean ± standard deviation. When the test was not satisfied, the Mann-Whitney test was used to express the median ± interquartile range. SPSS26.0 was used to calculate the ROC curve to evaluate the clinical diagnostic efficacy of tsRNA-Glu-i-0315. P The difference was considered statistically significant when the p<0.05; all tests were two-tailed. (5) Summary of clinical pathological characteristics of colorectal cancer patients and their relationship with tsRNA-Glu-i-0315 expression levels The results in Table 4 analyzed the clinical characteristics of colorectal cancer patients, including age, gender, history of smoking and drinking, history of hypertension, history of diabetes, tumor location, etc. The expression level of tsRNA-Glu-i-0315 was related to tumor location, but not to age, gender, drinking, smoking, diabetes, hypertension, metastasis and TNM stage.

[0026] Table 4 Summary of clinical pathological characteristics of colorectal cancer patients and their relationship with tsRNA-Glu-i-0315 expression levels * : Did not participate in hypothesis testing.

[0027] (6) Difference in expression levels of serum exosomal tsRNA-Glu-i-0315 between colorectal cancer patients and healthy volunteers The qPCR method was used to verify the level of serum exosome tsRNA-Glu-i-0315 in colorectal cancer patients and healthy volunteers; the expression level of serum exosome tsRNA-Glu-i-0315 in colorectal cancer patients and healthy volunteers is shown in the figure Figure 1 As shown, compared with healthy volunteers, the expression level of tsRNA-Glu-i-0315 in colorectal cancer patients was significantly reduced, and the difference was statistically significant ( P <0.0001). .

[0028] (7) Analysis of the diagnostic efficacy of serum exosomal tsRNA-Glu-i-0315 for colorectal cancer The ROC curve was constructed to evaluate the clinical diagnostic efficacy of tsRNA-Glu-i-0315 for colorectal cancer. Figure 2 As shown, the results showed that the AUC was 0.789, the sensitivity was 86.8%, and the specificity was 62.5%.

Claims

1. A hematological marker for the diagnosis of colorectal cancer, characterized in that: The hematological marker is serum exosome tsRNA-Glu-i-0315, and the sequence of serum exosome tsRNA-Glu-i-0315 is shown in SEQ ID NO.

1.

2. A kit for early diagnosis and progression warning of colorectal cancer, characterized in that: Used to detect the hematological markers according to claim 1; The kit for early diagnosis or early warning of colorectal cancer comprises a fluorescent PCR reaction system; the fluorescent PCR reaction system comprises a forward primer for tsRNA-Glu-i-0315 and a reverse primer for tsRNA-Glu-i-0315; The sequence of the forward primer for tsRNA-Glu-i-0315 is shown in SEQ ID NO.

2.

3. The kit for early diagnosis and progression warning of colorectal cancer according to claim 2, characterized in that: The fluorescent PCR reaction system also includes SYBR Green mixed solution and water.

4. The kit for early diagnosis and progression warning of colorectal cancer according to claim 3, characterized in that: The SYBR Green mixed solution includes PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ .

5. The kit for early diagnosis and progression warning of colorectal cancer according to claim 2, characterized in that: It also includes a reverse transcription reaction system and an internal reference system; The internal reference system includes a forward primer of internal reference U6 and a reverse primer of internal reference U6; The sequence of the forward primer of the internal reference U6 is shown in SEQ ID NO.4; The sequence of the reverse primer of the internal reference U6 is shown in SEQ ID NO.

5.

6. The kit for early diagnosis and progression warning of colorectal cancer according to claim 5, characterized in that: The internal reference system also includes SYBR Green mixed solution and water.

7. The kit for early diagnosis and progression warning of colorectal cancer according to claim 5, characterized in that: The reverse transcription reaction system comprises polyadenylic acid polymerase, reverse transcriptase mixed solution, reverse transcription buffer and water.

Images