Hematological marker and kit for early diagnosis of lung cancer based on plasma exosome

Abstract

The invention discloses a blood marker and a kit for early diagnosis of lung cancer based on plasma exosome, and belongs to the technical field of gene detection. The hematological marker is a blood plasma exosome 5 'Leader-ValAAC-1-2, and the gene sequence of the blood plasma exosome 5' Leader-ValAAC-1-2 is as shown in SEQ ID NO. 1. The lung cancer early diagnosis kit based on the plasma exosome comprises a fluorescent PCR (Polymerase Chain Reaction) reaction system, wherein the fluorescent PCR reaction system comprises a forward primer for detecting the plasma exosome 5 'Leader-ValAAC-1-2 and a reverse primer aiming at the 5' Leader-ValAAC-1-2. Research finds that compared with benign pulmonary nodule patients, the expression level of 5 'Leader-ValAAC-1-2 in plasma exosomes of malignant pulmonary nodule groups is remarkably increased, ROC curve analysis shows that AUC is 0.914, sensitivity is 80.3%, specificity is 89.8%, and the kit has the advantages of being high in sensitivity and accuracy, capable of conducting preliminary diagnosis and early warning on early-stage lung cancer and good in application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a hematological marker and a kit for early diagnosis of lung cancer based on plasma exosomes. Background Art

[0002] Lung cancer is one of the main causes of death worldwide, and its incidence and mortality are affected by changes in population aging and the prevalence of risk factors. Pulmonary nodules can indicate either a benign process or a malignant process, which is related to factors such as the morphology, size, growth potential, smoking, and age of the nodules. Early detection of the benign and malignant nature of pulmonary nodules has significant value in reducing the mortality of lung cancer and improving the prognosis of patients.

[0003] Liquid biopsy technology is an important research direction and means for early screening, diagnosis, and efficacy monitoring of tumors at the present stage. One widely studied direction is related to exosomes. Exosomes can be secreted by tumor cells and contain abundant tumor-related molecules such as RNA, DNA, proteins, and metabolites, with obvious tissue specificity. In addition, the double-layer vesicle structure of exosomes can support their stable existence in blood, body fluids, etc. for a certain period of time. When exosomes participate in cell-to-cell communication as messenger molecules and promote the growth of cancer cells, the cancer cell-related molecular information carried by exosomes can be dynamically monitored for clinical assessment of the patient's condition at the early stage of the disease.

[0004] Current clinical screening and diagnostic methods such as imaging examinations can detect smaller lesion tissues, but the specificity is low, and the diagnosis needs to rely on tissue biopsy for assistance; the tumor markers commonly used in hematological screening still have unsatisfactory sensitivity; endoscopy technology belongs to an invasive method, which may cause damage and pain to patients, with poor patient compliance, and at the same time has high professional technical requirements for operators, and is not suitable for large-scale screening; although Chinese non-small cell lung cancer patients have different driver factor spectra, due to social and economic imbalances, the testing of multiple driver genes in rural areas is restricted. Detection of tumor markers in blood has the advantages of less trauma, higher specificity, and faster detection speed, and is a new research direction for cancer screening and diagnosis. Summary of the Invention

[0005] Aiming at the problems of low specificity and low sensitivity in the existing screening and diagnostic technologies, the present invention provides a hematological marker and a kit for early diagnosis of lung cancer based on plasma exosomes, which are convenient and fast to operate, can detect benign and malignant pulmonary nodules, improve the sensitivity and accuracy of detection, and have good application value in the early diagnosis of lung cancer.

[0006] The present invention is achieved through the following technical solutions: On the one hand, the present invention provides a hematological marker for early diagnosis of lung cancer based on plasma exosomes, and the hematological marker is plasma exosome 5’Leader-ValAAC-1-2; the gene sequence of the plasma exosome 5’Leader-ValAAC-1-2 is shown in SEQ ID NO.1; On the other hand, the present invention also provides a kit for detecting the plasma exosome 5’Leader-ValAAC-1-2; The kit for early diagnosis of lung cancer based on plasma exosomes includes a fluorescence PCR reaction system, and the fluorescence PCR reaction system includes a forward primer for detecting plasma exosome 5’Leader-ValAAC-1-2 and a reverse primer for 5’Leader-ValAAC-1-2; The sequence of the forward primer for detecting plasma exosome 5’Leader-ValAAC-1-2 is shown in SEQ ID NO.2; The sequence of the reverse primer for detecting plasma exosome 5’Leader-ValAAC-1-2 is shown in SEQ ID NO.3.

[0007] Furthermore, the fluorescence PCR reaction system further includes a SYBR Green mixture and water.

[0008] Furthermore, the kit for early diagnosis of lung cancer based on plasma exosomes further includes a reverse transcription reaction system and an internal reference system; The internal reference system includes a forward primer for internal reference U6 and a reverse primer for internal reference U6; The sequence of the forward primer for internal reference U6 is shown in SEQ ID NO.4; The sequence of the reverse primer for internal reference U6 is shown in SEQ ID NO.5.

[0009] Furthermore, the reverse transcription reaction system includes polyadenylate polymerase, reverse transcriptase mixture, reverse transcription buffer and water.

[0010] Furthermore, the amplification conditions for the fluorescence PCR reaction system during the fluorescence PCR reaction are 95°C for 30 s, 95°C for 5 s, 60°C for 20 s, 65°C for 15 s, for 40 cycles.

[0011] Compared with the prior art, the beneficial effects achieved by the present invention are: The early-stage lung cancer diagnostic kit provided by the present invention can detect 5’Leader-ValAAC-1-2 in plasma exosomes in blood; compared with patients with benign lung nodules, the expression level of 5’Leader-ValAAC-1-2 in plasma exosomes in the malignant lung nodule group is significantly increased. ROC curve analysis shows that the AUC is 0.914, the sensitivity is 80.3%, and the specificity is 89.8%. It has the characteristics of high sensitivity and high accuracy, can be used for the preliminary diagnosis and early warning of early-stage lung cancer, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a diagram showing the expression levels of 5’Leader-ValAAC-1-2 in plasma exosomes of patients with malignant lung nodules and patients with benign lung nodules; Figure 2 It is the ROC curve of 5’Leader-ValAAC-1-2 for diagnosing patients with malignant lung nodules; Figure 3 It is a diagram showing the expression levels of 5’Leader-ValAAC-1-2 in patients with early-stage lung cancer at different stages. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0014] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be obtained through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in the conventional manner in the art or according to the product instructions.

[0015] In the specific embodiment of the present invention, the hematological marker for the early diagnosis of lung cancer in plasma exosomes is plasma exosome 5’Leader-ValAAC-1-2, and the sequence of 5’Leader-ValAAC-1-2 is shown as SEQ ID NO.1 in Table 1.

[0016] In the specific embodiments of the present invention, for the detection of plasma exosome 5’Leader-ValAAC-1-2, the fluorescence PCR reaction system includes a forward primer for detecting 5’Leader-ValAAC-1-2, a reverse primer for detecting 5’Leader-ValAAC-1-2, SYBR Green mixture and water. The SYBR Green mixture includes PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ ; the forward primer for detecting 5’Leader-ValAAC-1-2 is shown as SEQ ID NO.2 in Table 1, and the reverse primer for detecting 5’Leader-ValAAC-1-2 is shown as SEQ ID NO.3 in Table 1.

[0017] In the specific embodiments of the present application, the early lung cancer diagnosis kit based on plasma exosomes further includes a reverse transcription reaction system and an internal reference system; the internal reference system includes a forward primer for internal reference U6 and a reverse primer for internal reference U6. The sequence of the forward primer for internal reference U6 is shown as SEQ ID NO.4 in Table 1, and the sequence of the reverse primer for internal reference U6 is shown as SEQ ID NO.5 in Table 1; the reverse transcription reaction system includes polyadenylate polymerase, reverse transcriptase mixture, reverse transcription buffer and water.

[0018] Table 1 Related sequence list of the early lung cancer diagnosis kit based on plasma exosomes 。

[0019] The present invention will be further described in conjunction with the accompanying drawings of the specification and specific embodiments.

[0020] Example 1 (1) Experimental design Collect plasma samples from patients with newly diagnosed malignant pulmonary nodules and patients with benign pulmonary nodules in Shandong First Medical University Affiliated Tumor Hospital, and extract exosomal RNA. Detect the expression of 5’Leader-ValAAC-1-2 by qPCR, statistically analyze the expression differences between patients with malignant pulmonary nodules and patients with benign pulmonary nodules, and analyze its clinical diagnostic efficacy.

[0021] (2) Patient samples involved in the experiment 178 patients with malignant pulmonary nodules and 88 patients with benign pulmonary nodules admitted to the Affiliated Tumor Hospital of Shandong First Medical University from June 2022 to January 2023. The enrolled patients were classified as benign or malignant and TNM by pathological methods. Clinical data mainly included age, gender, tumor stage, smoking history, drinking history, and nodule size, etc. All patients with malignant pulmonary nodules were diagnosed by a combined method of clinical, pathological, and radiological diagnosis, and the tumor stage was determined according to the eighth edition of the lung cancer TNM staging standard formulated by the International Association for the Study of Lung Cancer (IASLC). Approximately 3 ml of peripheral blood samples were collected from the patients before surgery, chemotherapy, and radiotherapy and placed in a vacuum blood collection tube containing a coagulant promoter.

[0022] (3) Determination of the level of 5’Leader-ValAAC-1-2 in plasma exosomes The qPCR method was used to analyze the expression level of 5’Leader-ValAAC-1-2 in plasma exosomes of patients with early lung cancer and the benign pulmonary nodule group. The specific steps were as follows: 1) Extraction of plasma exosomes: The collected peripheral blood of the patients was centrifuged at 3000 g for 10 minutes at room temperature. The separated plasma was centrifuged at 12,000 g for 30 minutes at 4°C. The supernatant was taken and then centrifuged at 100,000 g for 2 hours at 4°C. The supernatant was discarded. 500 μL of Trizol reagent was added to the exosome precipitate, and after lysing at 4°C for 30 minutes, it was transferred to a clean enzyme-free EP tube and stored at -80°C for later use.

[0023] 2) Extraction of total RNA from plasma exosomes The operation was carried out in a biosafety cabinet; 100 μL of chloroform was added to 500 μL of plasma exosomes, and it was shaken vigorously for 30 seconds. After standing for 3 minutes, it was centrifuged at 12000 g for 15 minutes at 4°C (the mixture was separated into a lower pink phenol-chloroform liquid phase, a middle white flocculent phase, and an upper colorless aqueous phase, and RNA was distributed in the upper aqueous phase); 250 μL of the upper aqueous phase was aspirated into a clean enzyme-free EP tube, an equal volume of isopropanol was added, and it 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 it was gently inverted up and down to mix evenly, and then centrifuged at 7500 g for 5 minutes at 4°C; as much supernatant as possible was discarded, 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 - 15 minutes. 20 μL of RNase-free water was added, and it was mixed thoroughly with a pipette to completely dissolve the RNA; the dissolved RNA sample could be stored at -80°C for later use; the concentration and purity of the RNA were detected with a Nanodrop ultra-micro spectrophotometer.

[0024] 3) Reverse transcription reaction Reverse transcription reaction system: 3.75 μL of Total RNA (dissolved in Step 2), 1.25 μL of RT Enzyme Mix, 5 μL of RT Reaction Solution; Reverse transcription reaction conditions: 37 °C for 60 minutes, 85 °C for 15 minutes, store at 4 °C to obtain cDNA.

[0025] 4) Preparation before real-time fluorescence PCR amplification Prepare primer solution: The primer reagent is in dry powder form. First, centrifuge at 3000 rpm for 60 seconds, then slowly open the lid, add an appropriate amount of nuclease-free double-distilled water to make its final concentration 10 μM, close the lid and shake well to dissolve and mix evenly; 5) Fluorescence PCR amplification reaction The fluorescence PCR reaction system is shown in Table 2 below: Table 2 Fluorescence PCR reaction system Add each component in the fluorescence PCR reaction system to the corresponding amplification reaction plate in sequence, and add the sample cDNA. To avoid reagent contamination, immediately attach a transparent sealing film after loading the samples (Note: To avoid exposure contamination during the operation, the system preparation and sample loading should be carried out in a biosafety cabinet). Centrifuge at 800 g for 3 minutes at room temperature to mix evenly and then load onto the machine. Use the LightCycler 480 RT-PCR instrument to set according to the fluorescence PCR reaction conditions in Table 3 below. One replicate is set for each sample in this reaction, and the mean value is taken to reduce errors. The experiment is repeated three times.

[0026] Table 3 Fluorescence PCR reaction conditions 。

[0027] 6) Amplification reaction of the internal reference system Use the U6 gene as the internal reference gene. Internal reference system: 10 μL of SYBR Green mixture, 0.8 μL of the forward primer of internal reference U6, 0.8 μL of the reverse primer of internal reference U6, 6.4 μL of enzyme-free water, 2 μL of cDNA; Internal reference system reaction conditions: Pre-denaturation at 95 °C for 30 seconds, denaturation at 95 °C for 5 seconds, annealing at 60 °C for 20 seconds, extension at 65 °C for 15 seconds, for a total of 40 cycles.

[0028] (4)Data statistical analysis Use Prism 9.0 to statistically analyze the qPCR data. For two groups of measurement data that meet the normal distribution, use tThe tests were expressed as mean ± standard deviation. When not satisfied, the Mann-Whitney test was used and expressed as median ± interquartile range. SPSS 26.0 was used to calculate the ROC curve to evaluate the clinical diagnostic efficacy of 5’Leader-ValAAC-1-2. P A difference with < 0.05 was considered statistically significant; all tests were two-tailed.

[0029] (5)Summary of the clinicopathological features of patients in the benign pulmonary nodule group and the malignant pulmonary nodule group and the relationship with the expression level of 5’Leader-ValAAC-1-2 Table 4 shows the clinical characteristics of the above-mentioned benign and malignant pulmonary nodule patients, including: age, gender, smoking history, drinking history, etc. The expression level of 5’Leader-ValAAC-1-2 is related to the size, age and smoking factors of pulmonary nodules, and is not related to gender and drinking.

[0030] Table 4 Summary of the clinicopathological features of patients with benign and malignant pulmonary nodules and the relationship with the expression level of 5’Leader-ValAAC-1-2 .

[0031] (6)Difference in the expression level of plasma exosomal 5’Leader-ValAAC-1-2 between patients with malignant pulmonary nodules and patients with benign pulmonary nodules The qPCR method was used to verify the samples of the plasma exosomal 5’Leader-ValAAC-1-2 levels in patients with malignant pulmonary nodules and patients with benign pulmonary nodules; the expression levels of plasma exosomal 5’Leader-ValAAC-1-2 in patients with malignant pulmonary nodules and patients with benign pulmonary nodules were as Figure 1 shown. Compared with patients with benign pulmonary nodules, the expression level of 5’Leader-ValAAC-1-2 was significantly increased in patients with malignant pulmonary nodules, and the difference was statistically significant ( P < 0.0001).

[0032] (7)Analysis of the diagnostic efficacy of plasma exosomal 5’Leader-ValAAC-1-2 for malignant pulmonary nodules By constructing an ROC curve, the clinical diagnostic efficacy of 5’Leader-ValAAC-1-2 for malignant pulmonary nodules was evaluated. The ROC curve of 5’Leader-ValAAC-1-2 for diagnosing patients with malignant pulmonary nodules was as Figure 2 shown. The results showed that the AUC was 0.914, the sensitivity was 80.3%, and the specificity was 89.8%.

[0033] (8)Difference in the level of plasma exosomal 5’Leader-ValAAC-1-2 between patients with early lung cancer and patients with benign pulmonary nodules As Figure 3 , the qPCR test results showed that, compared with patients with benign lung nodules, the expression levels of plasma exosome 5’Leader-ValAAC-1-2 in stage 0-II lung cancer patients were significantly increased, and the differences were statistically significant (all stages were P < 0.0001).

Claims

1. A hematological marker for early diagnosis of lung cancer based on plasma exosomes, characterized in that, The hematological marker is plasma exosome 5’Leader-ValAAC-1-2; The gene sequence of the plasma exosome 5’Leader-ValAAC-1-2 is shown in SEQ ID NO.

1.

2. A kit for early diagnosis of lung cancer based on plasma exosomes, characterized in that, For detecting the plasma exosome 5’Leader-ValAAC-1-2 described in claim 1; The early lung cancer diagnosis kit based on plasma exosomes includes a fluorescence PCR reaction system, and the fluorescence PCR reaction system includes a forward primer for detecting plasma exosome 5’Leader-ValAAC-1-2 and a reverse primer for 5’Leader-ValAAC-1-2; The sequence of the forward primer for detecting plasma exosome 5’Leader-ValAAC-1-2 is shown in SEQ ID NO.2; The sequence of the reverse primer for detecting plasma exosome 5’Leader-ValAAC-1-2 is shown in SEQ ID NO.

3.

3. The early lung cancer diagnosis kit based on plasma exosomes according to claim 2, characterized in that, The fluorescence PCR reaction system also includes a SYBR Green mixture and water.

4. The early lung cancer diagnosis kit based on plasma exosomes according to claim 2, wherein The early lung cancer diagnosis kit based on plasma exosomes also includes a reverse transcription reaction system and an internal reference system; The internal reference system includes a forward primer for internal reference U6 and a reverse primer for internal reference U6; The sequence of the forward primer for internal reference U6 is shown in SEQ ID NO.4; The sequence of the reverse primer for internal reference U6 is shown in SEQ ID NO.

5.

5. The early lung cancer diagnosis kit based on plasma exosomes according to claim 2, characterized in that, The reverse transcription reaction system includes polyadenylate polymerase, reverse transcriptase mixture, reverse transcription buffer and water.

6. The early lung cancer diagnosis kit based on plasma exosomes according to claim 2, characterized in that, The amplification conditions for the fluorescence PCR reaction in the fluorescence PCR reaction system are 95°C for 30 s, 95°C for 5 s, 60°C for 20 s, 65°C for 15 s, for 40 cycles.

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