EccDNA marker for early lung cancer diagnosis and application of eccDNA marker

Abstract

The invention discloses an eccDNA marker for early lung cancer diagnosis and application of the eccDNA marker, and relates to the technical field of biological medicines, and a nucleotide sequence of the eccDNA marker comprises a GPATCH2L gene. The invention also discloses an application of the eccDNA marker in preparation of an early lung cancer diagnosis product. The eccDNA marker can be used for auxiliary diagnosis of early lung cancer, and can significantly improve the sensitivity and accuracy of early lung cancer diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an eccDNA marker for early lung cancer diagnosis and application thereof. Background Art

[0002] Lung cancer is the type of cancer with the highest morbidity and mortality in the world. According to the pathological type, lung cancer is mainly divided into two types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is the most common, accounting for about 85% of the total lung cancer cases. For early NSCLC, low-dose spiral CT scan (LDCT) is used for early screening, combined with surgical resection, and the 5-year survival rate can reach 60-90%. However, for patients in the middle or even late stages of progression, the situation is not ideal, with a 5-year survival rate of less than 15%, and the tumor is prone to recurrence or metastasis after surgery. Therefore, exploring early screening and diagnosis of lung cancer is of great significance to improving the survival rate of lung cancer.

[0003] Extrachromosomal circular DNA (eccDNA) is a type of circular DNA derived from chromosomes, which has attracted widespread attention due to its important role in tumorigenesis, heterogeneity and drug resistance. With the rapid development of detection technology, more and more studies have shown that eccDNA is not only widely present in human tissues and organs, but also has obvious tissue specificity and physiological and pathological specificity. eccDNA has been shown to be common in human tissues and plasma, but due to its low abundance in blood, further research on eccDNA is insufficient. With the advancement of medical technologies such as NGS and rolling circle amplification, sequencing has been improved at a lower cost, releasing the possibility of eccDNA for clinical diagnosis of cancer. Therefore, cell-free eccDNA is not only functionally important, but also has good prospects for use as a marker for disease risk assessment and early clinical detection of tumors. However, it is still unclear whether eccDNA can contribute to the early diagnosis and progression of lung cancer.

[0004] The gold standard for NSCLC diagnosis is tumor pathological tissue examination. In recent years, liquid biopsy has been favored by many researchers due to its non-invasiveness and convenience. The occurrence and development of tumors is a complex and gradual process, which is closely related to changes in genomics and epigenetics in the body. Circulating free DNA (cfDNA) released from tumor cells into plasma can detect relevant tumor information. Since the discovery of cell-free nucleic acids in human plasma in the 1940s, research on the possibility of nucleic acids in body fluids as disease biomarkers has been ongoing. Normal human plasma also contains a small amount of cfDNA, which is derived from apoptotic cells produced by metabolism in the body. The cfDNA of tumor patients is not only more in quantity, but also can detect many related mutant tumor genes through molecular biological methods, which is named ctDNA (Circulating tumor DNA). In epigenetics, by detecting ctDNA methylation, the death of specific cell tissue types in the body can be identified, so as to dynamically monitor the human body and diagnose pathological conditions. Different research data show that ctDNA is a widely applicable, sensitive and specific biomarker that can be effectively monitored in different types of tumor patients. However, due to its linear structure, ctDNA is unstable and easily digested by nucleases, and there is an urgent need to seek new molecular markers for non-invasive diagnosis and research of tumors.

[0005] Extrachromosomal circular DNA (eccDNA) is widely present in eukaryotes, independent of chromosomes, with a covalently closed circular structure, not easily digested by exonucleases, and more stable than linear DNA. eccDNA can promote high heterogeneity within tumors, accelerate tumor evolution, play a vital role in the occurrence and development of tumors, and is more suitable as a clinical diagnostic molecular marker. However, there is currently no report on CirD-GPATCH2L eccDNA as a diagnostic marker for early lung cancer. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide an eccDNA marker for the diagnosis of early lung cancer and its application, which can be used for auxiliary diagnosis of early lung cancer and can significantly improve the sensitivity and accuracy of early lung cancer diagnosis.

[0007] The technical solution of the present invention to solve the above technical problem is as follows: an eccDNA marker for early lung cancer diagnosis is provided, the eccDNA marker is CirD-GPATCH2L eccDNA, and the nucleotide sequence of CirD-GPATCH2L eccDNA includes the GPATCH2L gene.

[0008] The present invention also provides the use of the above-mentioned eccDNA marker in the preparation of early lung cancer diagnosis products.

[0009] Furthermore, it includes the use of reagents for detecting eccDNA markers in the preparation of early lung cancer diagnosis products.

[0010] Furthermore, reagents for detecting eccDNA markers include exonuclease III, Lambda exonuclease, rCutsmartbuffer, random primers, phi29 DNA polymerase reaction buffer, BSA, dNTPs, phi29 polymerase, T7 endonuclease I and ddH2O.

[0011] Furthermore, the above products include detection reagents and test kits.

[0012] Furthermore, the above product diagnoses lung cancer by detecting the level of eccDNA markers.

[0013] Furthermore, the samples tested for the above products are plasma.

[0014] Furthermore, early lung cancer was diagnosed when the proportion of eccDNA markers in the total eccDNA entries in the sample exceeded 0.0165%.

[0015] Furthermore, the eccDNA log2(fc)=16.5 from the GPATCH2L gene in the test sample was diagnosed as early lung cancer.

[0016] Furthermore, a cut-off value of eccDNA from the GPATCH2L gene in the test sample of ≥0.0165% was diagnosed as early lung cancer.

[0017] Furthermore, the above-mentioned early lung cancer includes stage I and stage II lung cancer.

[0018] The present invention has the following beneficial effects:

[0019] By comparing and analyzing the eccDNA in plasma samples of healthy people, early lung cancer patients and advanced lung cancer patients, the present invention found that the eccDNA from the GPATCH2L gene can be used as a marker for the diagnosis and screening of early lung cancer, and the diagnostic model established using CirD-GPATCH2L eccDNA showed strong discrimination performance and had great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the length distribution diagram of eccDNA in the detection sample;

[0021] Figure 2 This is the receiver operating characteristic curve. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0023] Example 1

[0024] (1) Sample collection

[0025] Blood samples were collected from 6 healthy people, 17 patients with early lung cancer, and 8 patients with advanced lung cancer by venous blood sampling. The blood samples were centrifuged at 3000g and 4°C for 10 minutes, and the supernatant was plasma, which can be stored at 80°C for a long time. Free DNA was extracted using a free DNA extraction kit. The linear DNA in the extracted DNA samples was digested with exonuclease III and Lambda exonuclease, and eccDNA was purified and enriched. The purification system is shown in Table 1.

[0026] Table 1eccDNA purification system

[0027] name Dosage DNA 10-100ng Exonuclease III 10U Lambda Exonuclease 5U 10×rCutsmartbuffer 2μL <![CDATA[ddH2O]]> Add to 20 μL

[0028] The above system was reacted at 37°C for 2 h, and then eccDNA was recovered using twice the volume of SPRI beads (Beckman Coulter).

[0029] (2) Rolling circle amplification of eccDNA

[0030] The eccDNA and 500 pmol of random primers (sequence: NNNNNNNN) were mixed, treated at 95°C for 5 min, and immediately placed on ice for 5-10 min. Then, the amplification system was prepared according to Table 2.

[0031] Table 2 Amplification system

[0032] name Dosage 10×phi29DNApolymerasereactionbuffer 5μL BSA (20 mg / mL) 0.3μL dNTP(10mM) 5μL phi29polymerase 5U <![CDATA[ddH2O]]> Add to 50 μL

[0033] The amplification system was reacted at 30°C for 10 h, then at 65°C for 10 min to inactivate the enzyme, and then recovered using 0.8 times the volume of SPRI beads (Beckman Coulter).

[0034] (3) T7 Endonuclease I was used to remove the branch structure of the amplified product. The reaction system is shown in Table 3.

[0035] Table 3 Reaction system

[0036] name Dosage 10×T7EndonucleaseIReactionBuffer 2μL Step (2) Recovering the product 2-5 μg T7EndonucleaseI 10U <![CDATA[ddH2O]]> Add to 20 μL

[0037] The reaction was carried out at 37°C for 15 min, and the beads were recovered using 0.8 volumes of SPRI beads (Beckman Coulter).

[0038] (4) Sequencing was performed using the long-read DNA sequencing process of the nanopore sequencing platform.

[0039] (5) Generating eccDNA: Using the eccDNA capture process (eccDNA_RCA_nanopore software) suitable for sequencing data using rolling circle amplification technology combined with long-read sequencing, eccDNA is generated from the sequencing data, and eccDNA generated from raw reads containing at least two tandem repeat fragments is screened for subsequent analysis.

[0040] (6) Biomarker analysis

[0041] The R package geom_density was used to plot the eccDNA length distribution. Figure 1 As shown, the results showed that compared with healthy people, the eccDNA length distribution diagram of patients with early lung cancer had eight characteristic peaks in the range of 0-1500bp.

[0042] Annotation of the genomic origin of eccDNA: Annotation was performed using the annotatePeaks.pl algorithm of the homer software.

[0043] Evaluate the diagnostic ability of the proportion of eccDNA from the GPATCH2L gene: Statistically calculate the proportion of eccDNA entries annotated to the intron and exon sequences of the GPATCH2L gene in all eccDNA annotation entries, and draw the receiver operating characteristic curve (ROC curve) to evaluate its predictive ability for early lung cancer. The Cut off value is calculated according to the Youden index (Youden index = sensitivity + specificity - 1), and the calculated Cut off value is used to identify early lung cancer. The results are as follows Figure 2 shown.

[0044] Depend on Figure 2 It can be seen that the cut off value of the eccDNA proportion from the GPATCH2L gene is 0.0165% (greater than or equal to 0.0165% is early stage), and the area under the ROC curve is 0.75 (95% confidence interval is 0.531-0.969), indicating that the diagnostic accuracy is relatively high.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An eccDNA marker for early lung cancer diagnosis, characterized in that: The eccDNA marker is CirD-GPATCH2L eccDNA, and the nucleotide sequence of the CirD-GPATCH2L eccDNA includes the GPATCH2L gene.

2. Use of the eccDNA marker according to claim 1 in the preparation of early lung cancer diagnosis products.

3. The use according to claim 2, characterized in that The invention also includes the use of a reagent for detecting the eccDNA marker in the preparation of an early lung cancer diagnosis product.

4. The use according to claim 2, characterized in that The products include detection reagents and test kits.

5. The use according to claim 2, characterized in that The product diagnoses lung cancer by detecting the level of the eccDNA marker.

6. The use according to claim 2, characterized in that The sample tested by the product is plasma.

7. The use according to claim 5, characterized in that When the proportion of the eccDNA marker to the total eccDNA entries in the sample exceeds 0.0165%, early lung cancer is diagnosed.

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