Application of eccDNA-PIAS1 in early diagnosis of lung adenocarcinoma

Abstract

The invention belongs to the technical field of medical biological detection, and particularly relates to application of eccDNA-PIAS1 in early diagnosis of lung adenocarcinoma. The specific high expression of the eccDNA-PIAS1 in the lung adenocarcinoma cells is realized. The invention specifically provides application of eccDNA-PIAS1 as a marker in preparation of a reagent or a kit for early diagnosis of lung adenocarcinoma. The invention discloses the application of the eccDNA-PIAS1 in the early diagnosis of the lung adenocarcinoma for the first time. Through analysis of the relationship between the eccDNA-PIAS1 and the disease progression period, it can be known that the eccDNA-PIAS1 can be used as a marker for the early diagnosis of the lung adenocarcinoma. The diagnosis efficiency result shows that the AUC of the eccDNA-PIAS1 is 0.8440, which prompts that the eccDNA-PIAS1 can be used as a novel diagnosis target of lung adenocarcinoma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and relates to application of eccDNA-PIAS1 as a lung adenocarcinoma early diagnosis biomarker, in particular to application of eccDNA-PIAS1 in preparation of a lung adenocarcinoma early diagnosis reagent or kit. BACKGROUND

[0002] Extrachromosomal circular DNA (eccDNA) is a circular DNA formed due to shedding, recombination or abnormal repair of chromosomal DNA, and can carry genes (such as oncogenes, drug resistance genes) and regulatory elements (enhancers, promoters). In recent years, it has been found that eccDNA is highly prevalent in various cancers (such as glioma, breast cancer), and its dynamic amplification characteristics are closely related to tumor progression and drug resistance. eccDNA widely exists in human tissues and organs and has strong tissue specificity, and can be released from tissues into the peripheral circulation. Due to the special circular structure, eccDNA is resistant to degradation by nucleases and is more stable in body fluids. Therefore, eccDNA is a very potential tumor biomarker.

[0003] Lung adenocarcinoma (LUAD) is the most common type of non-small cell lung cancer, accounting for 40% of lung cancer. The diagnosis of lung adenocarcinoma needs to be based on clinical manifestations, imaging examination, pathological examination and molecular detection, and finally confirmed by histopathology. However, early diagnosis of lung adenocarcinoma is difficult, tissue biopsy has limitations, liquid biopsy has low sensitivity, and the specificity of commonly used tumor markers is insufficient. Therefore, it is urgent to find a new type of tumor biomarker for early diagnosis of lung adenocarcinoma.

[0004] At present, nucleic acid molecules have made breakthrough progress in the auxiliary diagnosis, individualized treatment and prognosis prediction of NSCLC. As a new type of nucleic acid molecule, eccDNA has been reported to be closely related to the malignant progression and early diagnosis of various tumors (lung cancer, pancreatic cancer, glioma, etc.). Therefore, eccDNA as a new tumor-specific biomarker for lung adenocarcinoma is extremely potential. By exploring the diagnostic value of eccDNA in lung adenocarcinoma, breakthroughs in existing technical bottlenecks are provided, and a high-sensitivity, non-invasive and dynamic monitoring solution is expected to become a standard tool for the next generation of lung cancer precision diagnosis. SUMMARY

[0005] The present application is based on the above research, and aims to provide a biomarker for early diagnosis of lung adenocarcinoma, and to provide a new use of eccDNA-PIAS1, i.e. application in preparation of a lung adenocarcinoma early diagnosis kit.

[0006] The biomarker eccDNA-PIAS1 is derived from chromosome 15 68073059-68073441, and the nucleotide sequence of the corresponding DNA is:

[0007] AGGCTGGAGTGCAGTGGCGCAATCTCGGCCCACTGCAACCTCCGCCTCCTGGGTTCATGCCATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGTACCTGCCACCACGCCCGGCTCATTTTTTTGTATTTTTTAGTAGAGACGGGGTTTCACTGTATTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCTACCCATCTCGGCCTCCCGAAGTGCTGGGATTACAGGTGTGAGCCACCGTGCCCGGCGAGGTTATATGTGTAATTTCTAATATATTTGCATTACCAATATGGTTTGTAATAATTGACAGGTGCCAAACCGTTGTTACTCATATTTATTAATGATGAACAAGACAGTCTCTATAGACAGAGCAT (SEQ ID NO.1).

[0008] The present application first selects tumor tissue and normal tissue of lung adenocarcinoma patients for eccDNA sequencing, and finds that eccDNA-PIAS1 (chr15: 68073059-68073441) derived from PIAS1 gene only exists in tumor tissue and has the highest content through differential analysis screening, and agarose gel electrophoresis and Sanger sequencing are used for verification; further in vitro synthesis of eccDNA and transfection of lung adenocarcinoma cells to detect cell phenotype, which shows that eccDNA-PIAS1 significantly increases the malignant proliferation ability of lung adenocarcinoma cells; then detect eccDNA in lung adenocarcinoma patient tissue and paracancerous tissue, and analyze the content difference and diagnostic efficiency, which shows that the content of eccDNA-PIAS1 in tumor tissue is much higher than that in normal tissue, and the results of nest PCR combined with CRISPR / Cas12a detection are consistent with the results of eccDNA sequencing, through the analysis of diagnostic efficiency of eccDNA-PIAS1 by receiver operating characteristic curve, the result shows that the AUC is 0.8440, which indicates that eccDNA-PIAS1 can be used as a new diagnostic target for lung adenocarcinoma.

[0009] Therefore, the eccDNA-PIAS1 involved in the present application can significantly promote the proliferation of lung adenocarcinoma cells, and can be used as a tumor marker for early diagnosis of lung adenocarcinoma, significantly improving the sensitivity and specificity of early diagnosis of lung adenocarcinoma.

[0010] Specifically, the present invention provides the following technical solution:

[0011] In a first aspect, the invention provides the application of eccDNA-PIAS1 as a diagnostic biomarker. Specifically, it provides the application of a reagent for detecting eccDNA-PIAS1 expression levels in the preparation of a diagnostic kit for early lung adenocarcinoma.

[0012] Preferably, the reagent for detecting the expression level of eccDNA-PIAS1 is a reagent for detecting the expression level of eccDNA-PIAS1 in biological samples at the gene level; the kit contains a reagent for detecting the expression level of eccDNA-PIAS1 in biological samples.

[0013] Further preferably, the reagent for detecting eccDNA-PIAS1 in biological samples is selected from one or more of the following detection techniques or methods: PCR reaction combined with high-throughput sequencing or Sanger sequencing, nested PCR combined with CRISPR / Cas12a method.

[0014] The primers used in the PCR reaction can be random primers (NEB, E1603S) or primers with sequences as shown in SEQ ID NO.2~5:

[0015] DNA-1F: AGGCTGGAGTGCAGTGGCGCAATCTC (SEQ ID NO.2);

[0016] DNA-1R:ATGCTCTGTCTATAGAGACTGTCTTG (SEQ ID NO.3);

[0017] DNA-2F:ATCTCCTGACCTCGTGATCTACCC (SEQ ID NO.4);

[0018] DNA-2R: CGAGACCATCCTGGCTAATACAG (SEQ ID NO. 5).

[0019] Further preferred, nested PCR primer sequences with detection specificity for the eccDNA-PIAS1 gene are shown in SEQ ID NO. 6~9, the CrRNA recognition sequence in the CRISPR / Cas12a method is shown in SEQ ID NO. 10, and the CrRNA sequence is shown in SEQ ID NO. 11.

[0020]

[0021] Preferably, the biological sample is selected from lung adenocarcinoma tumor tissue or peripheral blood. Since eccDNA can be released from tissue into the peripheral circulation, peripheral blood can also be used as a detection sample.

[0022] In a second aspect, the present invention provides a diagnostic kit for early lung adenocarcinoma, the kit comprising reagents for detecting the content of eccDNA-PIAS1 in biological samples.

[0023] Preferably, the kit comprises a primer system, an amplification system, and a CrRNA system, wherein the primer system includes PCR primers as shown in SEQ ID NO. 6 to 9 above, and the CrRNA system contains sequences as shown in SEQ ID NO. 10 and 11 above.

[0024] Preferably, the kit further includes a linear DNA digestion system containing Plasmid-safe ATP-dependent DNase.

[0025] In a third aspect, this invention provides the application of eccDNA-PIAS1 as a target in the preparation of therapeutic drugs for lung adenocarcinoma. This invention has demonstrated that eccDNA-PIAS1 exists only in tumor tissues and is highly expressed, while significantly promoting the proliferation of lung adenocarcinoma cells. Therefore, it can be used as a target for small molecule drug screening or for the design of shRNA or siRNA.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention discloses the application of a novel eccDNA—eccDNA-PIAS1—in the diagnosis and treatment of lung adenocarcinoma. The DNA nucleotide sequence corresponding to this biomarker is shown in SEQ ID NO.1, and it exists only in lung adenocarcinoma and is highly expressed. This invention is the first to disclose the application of eccDNA-PIAS1 in the early diagnosis of lung adenocarcinoma. Analysis of the relationship between eccDNA-PIAS1 and disease progression shows that eccDNA-PIAS1 can serve as a biomarker for the early diagnosis of lung adenocarcinoma. Diagnostic efficacy results show that its AUC is 0.8440, suggesting that eccDNA-PIAS1 can serve as a novel diagnostic target for lung adenocarcinoma.

[0028] In terms of detection technology, the detection of eccDNA-PIAS1 is essentially a nested PCR detection based on the gene expression status of blood cells or tumor tissues. It has the characteristics of simple operation, high sensitivity, good specificity and high repeatability. It is now increasingly used in clinical testing technology. This technology has high sensitivity and accuracy, is widely used in clinical practice, and the test technology is very mature.

[0029] In terms of efficacy, the indicator eccDNA-PIAS1 involved in this invention is specifically highly expressed in the tumor tissue of patients with lung adenocarcinoma, and the difference is statistically significant (P<0.05). It can be used as an early diagnostic marker for lung adenocarcinoma, and its clinical reference value and reliability are high, which has important clinical significance and social benefits. Attached Figure Description

[0030] Figure 1 The results of tissue eccDNA sequencing are shown: A shows the detection process; B shows a comparison of detection results between tumor tissue and adjacent normal tissue; C shows the volcano plot; and D shows the agarose gel electrophoresis and Sanger sequencing results of tumor tissue and adjacent normal tissue.

[0031] Figure 2 The synthesis and identification of eccDNA-PIAS1 are shown: A is the schematic diagram of the synthesis; B is the result of agarose gel electrophoresis; C is the result of Sanger sequencing; and D is the result of atomic force microscopy.

[0032] Figure 3 The tumor-promoting effect of eccDNA-PIAS1 was demonstrated: A represents the results of CCK8 assay; B and C represent cell colony formation assays; and D represents the edu assay.

[0033] Figure 4 The results of nested PCR combined with CRISPR / Cas12a method for detecting eccDNA-PIAS1 are shown: A is the detection result of tumor tissue; B is the detection result of normal tissue; C is a comparison between the detection results of nested PCR combined with CRISPR / Cas12a method and the eccDNA sequencing results.

[0034] Figure 5 The diagnostic efficacy of eccDNA-PIAS1 was demonstrated: A, eccDNA-PIAS1 was not correlated with the patient's TNM stage, but it was correlated with the clinical stage; B, ROC curve analysis. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0036] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer.

[0037] I. Experimental Methods

[0038] (1) Sample collection and testing

[0039] Tumor tissue and normal lung tissue were collected from patients clinically diagnosed with lung adenocarcinoma at Tongji Hospital in Shanghai, and the samples were frozen at -80°C. The testing procedure is described below. Figure 1 A.

[0040] Tissue DNA was extracted using the MagAttract HMW 855 DNA Kit (Qiagen, 67563). Linear DNA was digested with Plasmid-safe ATP-dependent DNase (Epicenter, E3110K), and eccDNA was amplified by rolling circles using phi29 DNA polymerase and random primers (NEB, E1603S). High-throughput sequencing was performed using an Illumina NovaSeq sequencer in 150bp paired-end mode. Finally, the eccDNA sequencing data were analyzed.

[0041] The digestion system consisted of 500 ng total DNA, 1 μl (10 U) DNase (10 U), 2 μl (25 mM) ATP, 5 μl 10× reaction buffer, and ddH2O to a final volume of 50 μl. The digestion system was placed in a 37°C water bath for 24 h, followed by DNase inactivation at 70°C for 30 min.

[0042] The rolling circle amplification system consisted of: 500 ng DNA, 5 μl 10×Phi29 buffer, 5 μl dNTP (10 mM), 5 U Phi29 polymerase, and ddH2O to a final volume of 50 μl. The reaction was carried out at 30°C for 10 h, followed by inactivation at 65°C for 10 min.

[0043] The sequence of eccDNA-PIAS1 is as follows: AGGCTGGAGTGCAGTGGCGCAATCTCGGCCCACTGCAACCTCCGCCTCCTGGGTTCATGCCATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGTACCTGCCACCACGCCCGGCTCATTTTTTTGTATTTTTTAGTAGAGACGGGGTTTCACTGTATTAGCCAGGATGGTCTCGATC TCCTGACCTCGTGATCTACCCATCTCGGCCTCCCGAAGTGCTGGGATTACAGGTGTGAGCCACCGTGCCCGGCGAGGTTATATGTGTAATTTCTAATATATTTGCATTACCAATATGGTTTGTAATAATTGACAGGTGCCAAACCGTTGTTACTCATATTTATTAATGATGAACAAGACAGTCTCTATAGACAGAGCAT (SEQ ID NO.1).

[0044] (2) Difference analysis

[0045] This invention uses differential analysis to screen for high-abundance eccDNA that exists only in tumor tissues, and verifies the target eccDNA through PCR reaction, product agarose gel electrophoresis, and Sanger sequencing.

[0046] The PCR reaction system consisted of 10 μl of purified product, 0.5 μl (10 μM) of upstream primer, 1 μl (10 μM) of downstream primer, 10 μl of 2×PCR premix (Sangon Biotech, B532073), and 6 μl of sterile deionized water. The PCR reaction program was as follows: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. After 40 cycles, the product was briefly stored at 4°C.

[0047] The products were detected using a gel with 2% agarose content and subjected to Sanger sequencing (Sangon Biotech).

[0048] (3) In vitro synthesis and transfection

[0049] The target eccDNA was synthesized in vitro using LAMA technology, and the consistency of the eccDNA sequence and structure was verified by Sanger sequencing and atomic force microscopy.

[0050] For the LAMA technology system, please refer to Figure 2A: 500 ng linear fragment A, 500 ng linear fragment B, 1 μl Taq DNA ligase (Beyotime, D7023S), 10 μl Taq DNA ligase buffer, and sterile deionized water to a final volume of 100 μL. The reaction mixture was incubated at 95°C for 20 s, 4°C for 1 min, and 65°C for 20 min for a total of 10 cycles. Subsequently, the linear DNA was treated with Exonuclease V (NEB, M0345S). The primer sequences used in the synthesis are shown in Table 1 below.

[0051] Table 1. Two primer pairs used for eccDNA-PIAS1 synthesis.

[0052]

[0053] The digestion procedure was as follows: 1 μg of the reaction product was added to 2 μl of Exonuclease V (10 U / μl) and 10 μl of Exonuclease V reaction buffer. Water was added to bring the total volume to 100 μL. After mixing, the mixture was incubated at 37°C overnight (approximately 16 hours). The next day, the digestion product was purified using magnetic beads (Novizan, N411-02) to obtain purified eccDNA, which was then quantitatively detected.

[0054] Subsequently, 1 μg of control DNA and target eccDNA were transfected into lung adenocarcinoma cells using the transfection reagent lipo3000 (ThermoFisher Scientific, L3000015). Transfection efficiency was detected by PCR, and cell proliferation phenotype was then examined.

[0055] (4) Nested PCR combined with CRISPR / Cas12a detection

[0056] Nested PCR combined with CRISPR / Cas12a was used to detect and analyze the target eccDNA, and the diagnostic value of the target eccDNA was further analyzed. Nested PCR primers and crRNA were designed for the target eccDNA using nested PCR combined with CRISPR / Cas12a.

[0057] Nested PCR reaction system: 2 μl purified product, 1 μl (10 μM) upstream primer, 1 μl (10 μM) downstream primer, 10 μl 2×PCR premix, 6 μl sterile deionized water. Pre-denaturation at 95℃ for 10 min, followed by denaturation at 95℃ for 15 s, annealing at 45℃ for 30 s, and extension at 72℃ for 30 s. The first-round PCR product was used as a template for the second-round PCR reaction: 8 μl template, 1 μl (10 μM) upstream primer, 1 μl (10 μM) downstream primer, 10 μl 2×PCR premix. The PCR reaction program was: pre-denaturation at 95℃ for 10 min, followed by denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s, for 40 cycles.

[0058] The primer sequences and crRNA sequences used in the reaction process are shown in Table 2 below:

[0059] Table 2 Nested PCR primers and crRNA sequences

[0060]

[0061] The target eccDNA was then detected using CRISPR / Cas12a (TOLOBIO, 32108). The 20 μl reaction mixture consisted of: 2 μl PCR product, 0.5 μl (10 μM) lbCas12a, 0.5 μl (10 μM) crRNA, 0.5 μl (10 μM) ssDNA reporter, 10× buffer, and deionized water to a final volume of 20 μl. Fluorescence signals were detected using a quantitative real-time PCR instrument. The reaction program was 37℃ for 20 min, with fluorescence signal values ​​measured every 30 seconds.

[0062] (5) Performance analysis

[0063] The target eccDNA in peripheral plasma was quantitatively detected by analyzing changes in fluorescence signal, and the difference in levels between lung adenocarcinoma patients and healthy controls was analyzed. The diagnostic efficacy of the target eccDNA was evaluated using receiver operating characteristic (ROC) curves.

[0064] II. Results Analysis

[0065] (1) Sample collection and eccDNA detection

[0066] eccDNA sequencing was performed on tumor tissue (Tumor) and normal tissue (Normal) from patients with lung adenocarcinoma. Tissue DNA was extracted from 200 mg of tissue samples, and linear DNA was digested using Plasmid-safe ATP-dependent DNase, followed by rolling circle amplification using phi29 enzyme, and finally library construction and sequencing.

[0067] Differential analysis of sequencing results showed that eccDNA-PIAS1 (chr15:68073059-68073441) derived from the PIAS1 gene was present only in tumor tissues and had the highest content. Figure 1 B, 1C). eccDNA-PIAS1 was detected using PCR, and the PCR products were analyzed by agarose gel electrophoresis and Sanger sequencing. The results showed that the electrophoresis and sequencing results were completely consistent with the eccDNA sequencing results. Figure 1 D).

[0068] (2) eccDNA-PIAS1 synthesis and transfection

[0069] Two pairs of primers were designed according to Table 1 for the synthesis of eccDNA-PIAS1. Linear DNA fragment A was amplified from cellular genomic DNA. After linear digestion and rolling circle amplification of the genomic DNA, linear DNA fragment B was amplified using primers. Subsequently, the two complementary linear DNA fragments were ligated to form circular DNA using T4 DNA ligase. The unligated linear DNA was digested with exonuclease and purified using magnetic beads. The eccDNA was then purified by restriction endonuclease reactions (…). Figure 2 B), Sanger sequencing ( Figure 2 C) and atomic force microscope ( Figure 2 D) Verify that the eccDNA sequence and structure are completely identical to eccDNA-PIAS1.

[0070] In lung adenocarcinoma cells, 500 ng of control DNA and eccDNA-PIAS1 were transfected using lipo3000. Twenty-four hours after transfection, PCR detection confirmed successful transfection of eccDNA-PIAS1 into the cells. The results were confirmed using a CCK8 assay. Figure 3 A) Cell clone formation experiment ( Figure 3 B, 3C) and edu experiments ( Figure 3 D) and others have demonstrated that eccDNA-PIAS1 significantly increases the malignant proliferation ability of lung adenocarcinoma cells.

[0071] (3) Detection of eccDNA-PIAS1 by nested PCR combined with CRISPR / Cas12a method

[0072] Nested PCR primers and crRNA were designed specifically for the junction site of eccDNA-PIAS1. gDNA was extracted from tumor and normal tissues using a kit. Linear DNA was digested with Plasmid-safe ATP-dependent DNase and then purified. Nested PCR was used to specifically amplify eccDNA-PIAS1, followed by detection of its content using CRISPR / Cas12a. The final results are presented as changes in fluorescence signal. The results showed that the content of eccDNA-PIAS1 in tumor tissues was significantly higher than that in normal tissues, and the results of nested PCR combined with CRISPR / Cas12a detection were consistent with the eccDNA sequencing results. Figure 4 ).

[0073] (4) Diagnostic efficacy of eccDNA-PIAS1

[0074] After detecting the level of eccDNA-PIAS1, the correlation between eccDNA-PIAS1 and TNM stage and pathological analysis of lung adenocarcinoma patients was further analyzed. The results showed that eccDNA-PIAS1 was not correlated with TNM stage, but it was correlated with clinical stage. Figure 5 A). Analyzing the diagnostic efficacy of eccDNA-PIAS1 using receiver operating characteristic (ROC) curves showed an AUC of 0.8440 (…). Figure 5 (B) This suggests that eccDNA-PIAS1 could serve as a novel diagnostic target for lung adenocarcinoma.

[0075] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. The application of a reagent for detecting eccDNA-PIAS1 expression levels in the preparation of a diagnostic kit for early lung adenocarcinoma, characterized in that, The nucleic acid sequence of eccDNA-PIAS1 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The reagent for detecting the expression level of eccDNA-PIAS1 is a reagent for detecting the expression level of eccDNA-PIAS1 in biological samples at the gene level; the kit contains reagents for detecting the expression level of eccDNA-PIAS1 in biological samples.

3. The application according to claim 2, characterized in that, The reagents used to detect eccDNA-PIAS1 in biological samples are selected from one or more of the following detection techniques or methods: PCR reaction combined with high-throughput sequencing or Sanger sequencing, nested PCR combined with CRISPR / Cas12a method.

4. The application according to claim 3, characterized in that, The primer sequences used in the PCR reaction are shown in SEQ ID NO.2~5.

5. The application according to claim 3, characterized in that, Nested PCR primer sequences with detection specificity for the eccDNA-PIAS1 gene are shown in SEQ ID NO. 6-9, the CrRNA recognition sequence in the CRISPR / Cas12a method is shown in SEQ ID NO. 10, and the CrRNA sequence is shown in SEQ ID NO.

11.

6. The application according to claim 2, characterized in that, The biological samples were selected from lung adenocarcinoma tumor tissue or peripheral blood.

7. A diagnostic kit for early lung adenocarcinoma, characterized in that, This kit contains reagents for detecting the content of eccDNA-PIAS1 in biological samples.

8. The reagent kit according to claim 7, characterized in that, The kit comprises a primer system, an amplification system, and a CrRNA system. The primer system includes PCR primers as shown in SEQ ID NO. 6–9, and the CrRNA system contains sequences as shown in SEQ ID NO. 10 and 11.

9. The reagent kit according to claim 7, characterized in that, It also includes a linear DNA digestion system containing Plasmid-safe ATP-dependent DNase.

10. Application of eccDNA-PIAS1 as a target in the preparation of drugs for the treatment of lung adenocarcinoma.

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