Marker for liver cancer diagnosis as well as detection system and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a marker for liver cancer diagnosis and a detection system and application of the marker, the marker is UDG and / or APE1, and an ECEER system for detecting the marker is a regular sequence system composed of a substrate chain Qc, a fuel chain Fu with a dU site and a fuel chain Fc and is used for detecting a target chain miR-(z); according to different target chains miR-(z), matched substrate chains Qc, fuel chains Fu and fuel chains Fc are designed, specific malignant transformed cells can be specifically recognized and analyzed, and high-sensitivity analysis of UDG and APE1 for different malignant transformed cells can be realized by simply changing an EDR reaction chain sequence. According to the method, the distinguishing precision of hierarchical decoding is ensured by adjusting the positions and the number of dU bases of a fuel chain and the number of mismatched bases; and the ECEER system is simple and convenient in preparation process, provides a new model for researching the relationship between UDG and APE1 and the process of malignant transformed cells, has clinical application potential, and can guide the diagnosis and treatment of the malignant transformed cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to biomarkers for liver cancer, and more particularly to a biomarker for liver cancer diagnosis, its detection system, and its application. Background Technology

[0002] Uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic (AP) endonuclease (APE1), as rate-limiting enzymes in the BER pathway, play an important role in maintaining the stability of the human cell genome; however, whether UDG and APE1 can serve as novel serum tumor markers in liver cancer remains unknown.

[0003] Base excision repair (BER) is an important DNA damage repair mechanism, primarily responsible for repairing DNA damage caused by endogenous and exogenous mutagens. If the body cannot rapidly and accurately repair damaged DNA, it leads to genomic instability and the accumulation of mutations, potentially resulting in apoptosis, accelerated aging, and various diseases, including cancer. Of particular concern is that under pathological conditions, the burden of malignant transformed cells and drug treatments can further exacerbate genomic instability. Therefore, the detection and tracking of genomic instability caused by BER pathway abnormalities demonstrates significant clinical application potential and value in the early diagnosis of malignant transformed cells, evaluation of treatment efficacy, and monitoring of relapse.

[0004] Currently, the main methods and strategies for detecting genomic instability include real-time quantitative PCR (qPCR) and gene sequencing. However, these methods have limitations such as insufficient accuracy, complex probe design, cumbersome operation, and high cost, making them unsuitable for precise analysis of subcellular UDG and APE1. Recently, emerging biosensing technologies have brought new hope for high-resolution BER enzyme system detection, but most reported detection methods target a single enzyme at a single time point, resulting in a lack of cell specificity and making it very difficult to explore UDG and APE1 in different malignant transformed cells.

[0005] To reduce the impact of non-specific signal interference and achieve highly sensitive and accurate in situ analysis of UDG and APE1 in malignant transformed cells, the inventors developed a method for real-time tracking and quantitative analysis of cell-specific markers at the cellular and subcellular levels. Utilizing miRNA as a trigger element, this method initiates an entropy-driven chain displacement reaction by recognizing a hidden toehold structure, thereby achieving sequential identification and readable signal output for multi-target enzyme activities. In malignant transformed cells, this strategy can be used to accurately analyze the expression dynamics of UDG and APE1, revealing their roles in abnormal DNA repair processes. This dual-validation mechanism effectively distinguishes signal level fluctuations directly caused by changes in UDG and APE1 activity in specific malignant transformed cells from interference by other non-specific factors, significantly improving the specificity of the analysis. However, its application also reveals significant problems, particularly the tendency for multiple reactions in the multiple validation steps to interfere with each other, leading to signal intensity attenuation.

[0006] Therefore, this invention proposes a biomarker for liver cancer diagnosis, its detection system and application, and the ECEER system based on dynamic DNA self-assembly technology, which can be used for in situ cascade detection of UDG and APE1 in malignant transformed cells with high sensitivity, providing a new method for accurate analysis of intracellular biomarkers UDG and APE1 in malignant transformed cells under controllable time conditions. Summary of the Invention

[0007] To address the problems in existing technologies, this invention provides a biomarker for liver cancer diagnosis, its detection system, and its applications. First, the feasibility of UDG and APE1 as liver cancer biomarkers is verified. Second, a dynamic DNA self-assembly technology—Entropy-Driven Chain Exchange Reaction (EDR)—is integrated into a strategy that converts the sequential recognition of UDG and APE1 by specific miRNAs in malignant transformed cells into a readable signal output, reflecting the relative abundance and spatial distribution of UDG and APE1 in malignant transformed cells. A fuel chain (Fuel) containing deoxyuracil (dU bases) is introduced into the EDR, which can be sequentially recognized by UDG and APE1. Subsequently, the entropy-driven reaction mediated by the recognition of target miRNAs on the Qc sequence of the substrate chain is analyzed. The resolution accuracy of hierarchical decoding can be ensured by optimizing the position and number of dU bases and the number of mismatched bases in the fuel chain. Furthermore, by changing the sequence of the EDR reaction chain, a targeted miRNA can be activated.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] On the one hand, a biomarker for the diagnosis of liver cancer, wherein the biomarker is UDG and / or APE1.

[0010] On the one hand, the present invention also provides the application of the above-mentioned markers in the preparation of diagnostic products for liver cancer.

[0011] Preferably, the detection product detects at least one of serum, cells, and tissues, and the detection product is a diagnostic kit or an ECEER system.

[0012] On one hand, the present invention provides an ECEER system for detecting the above-mentioned markers or a method for constructing the above-mentioned ECEER system, comprising the following steps:

[0013] S11. Fuel chain Fu and substrate chain Qc are mixed at a concentration ratio of 1:1 and annealed to form a stable QcFu hybrid double strand. The damaged bases of fuel chain Fu in the QcFu hybrid double strand are successively recognized by UDG and APE1 enzymes.

[0014] S12. Add fuel chain Fc to the QcFu hybrid double-stranded solution, so that the concentration of fuel chain Fc is not lower than the concentration of QcFu hybrid double-stranded solution, i.e. Fc:QcFu≥1:1. Mix thoroughly to obtain QcFu+Fc mixture, which is the ECEER system.

[0015] The fuel chain Fu contains at least one dU base and is labeled with a fluorescent group and a quenching group. The substrate chain Qc has a Toehold structure. The fuel chain Fc has the same sequence as the fuel chain Fu. Through a chain substitution reaction, it hybridizes with Qc to form a QcFc complex, which enters a new cycle reaction.

[0016] Preferably, the annealing conditions are a 95°C water bath for 5-10 minutes followed by an ice bath for 1 hour; the number of dU bases in the fuel chain Fu is 1-3.

[0017] Preferably, the concentration of the UDG enzyme is 0.075-0.01 U / μL, the concentration of the APE1 enzyme is 0.075-0.01 U / μL, and the number of dU bases in the fuel chain Fu is 2.

[0018] On the other hand, the present invention also provides an ECEER system constructed by the above-described ECEER system construction method.

[0019] On the other hand, the present invention also provides a method for constructing an ECEER detection system, comprising the following steps:

[0020] S21. Add serum-free and antibiotic-free culture medium and Lipofectamine to the EP tube. TM 3000 reagents;

[0021] S22, Add serum-free and antibiotic-free culture medium and P3000 to another EP tube. TM The ECEER system constructed using the reagents and the above-described ECEER system construction method, or the above-described ECEER system;

[0022] S23. Mix the two EP tubes thoroughly and incubate at 5-45℃ for 20 minutes. Then, add the DNA-lipid complex into the malignant transformed cells for the detection of the target miR-(z).

[0023] Preferably, the amount added to the ECEER system is based on Lipofectamine. TM Add the reagent according to the dosage instructions in the 3000 reagent manual, adjusting the dosage based on the bottom area of ​​the well plate. For example, use the dosage per well of a 24-well plate. The instructions specify the dosage of serum-free, antibiotic-free culture medium and Lipofectamine. TM The volumes of reagent 3000 were 25 μL and 0.75 μL, respectively, and the serum-free and antibiotic-free culture medium and P3000 were used. tM The volumes of the reagent and the ECEER system were 25 μL, 1 μL, and 10 μL, respectively.

[0024] Preferably, the malignant transformed cells are cancer cells.

[0025] Preferably, the cancer cells are liver cancer cells.

[0026] Preferably, the liver cancer cells are Huh-7 cells.

[0027] Preferably, the target miR-(z) is one or more of miR-21, miR-375, and miR-185, wherein the sequence of the target miR-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3'; the sequence of the target miR-375 is 5'-UGCGCUCGGCUUGCUUGUUUU-3'; and the sequence of the target miR-185 is 5'-UGGAGAGAAAGGCAGUUCCUGA-3'.

[0028] Preferably, the incubation temperature is 25-37°C.

[0029] Preferably, the incubation temperature is 37°C.

[0030] Preferably, when the target miR-(z) is miR-21, the corresponding substrate chain Qc, fuel chain Fu, and fuel chain Fc are substrate chain Qc-21, fuel chain Fu-21, and fuel chain Fc-21, respectively.

[0031] The nucleotide sequence of the substrate chain Qc-21 is: 5'-TCAACATCAGTCTGATAAGCTAAGGGACCCGTAAGTTAGTTGGAGACGTAGG-3',

[0032] The nucleotide sequences of both fuel chain Fu-21 and fuel chain Fc-21 are: 5'-CCTACGTCTCCA ACTAA(FAM)CTTACGG(X)CCCTTAGC(BHQ1)TTATCAGACT-3';

[0033] When the target miR-(z) is miR-375, the corresponding substrate chain Qc, fuel chain Fu, and fuel chain Fc are substrate chain Qc-375, fuel chain Fu-375, and fuel chain Fc-375, respectively.

[0034] The nucleotide sequence of the substrate chain Qc-375 is: 5'-AAAACAAGCAAGCCGAGCGCAAGGGCCGTAAGTTAGTTGGAGACGTAGG-3',

[0035] The nucleotide sequences of the fuel chain Fu-375 and the fuel chain Fc-375 are both: 5'-CCTACGT CTCCAACTAA(FAM)CTTACGG(X)CCCT(BHQ1)TGCGCTCGGCTTG-3';

[0036] When the target miR-(z) is miR-185, the corresponding substrate chain Qc, fuel chain Fu, and fuel chain Fc are substrate chain Qc-185, fuel chain Fu-185, and fuel chain Fc-185, respectively.

[0037] The nucleotide sequence of the substrate chain Qc-185 is: 5'-TCAGGAACTGCCTTTCTCTCCAAGGGCCGTAAGTTAGTTGGAGACGTAGG-3',

[0038] The nucleotide sequences of the fuel chain Fu-185 and the fuel chain Fc-185 are both 5'-CCTACGTCT CCAACTAA(FAM)CTTACGG(X)CCCT(BHQ1)TGGAGAGAAAGGCA-3';

[0039] X represents a dU base, and there are two dU bases.

[0040] The ECEER system provided by this invention utilizes UDG to recognize damaged uracil bases in single / double-stranded DNA, initiating a base excision repair mechanism, and APE1 to excise AP sites in double-stranded DNA. These are integrated into a dynamic DNA self-assembly entropy-driven chain displacement reaction (EDR) to construct the ECEER. The single-stranded DNA fuel chain (Fu) containing dU bases is successively recognized by UDG and APE1, forming a stable QcFu hybrid double strand with the substrate chain (Qc) containing a Toehold structure, thus constituting the basic framework of the entropy-driven reaction. A chain displacement reaction occurs under miRNA initiation, fluorescence is restored, and fluorescence is continuously enhanced under the action of the fuel chain (Fc). This ECEER system combines the logical encoding of DNA molecules, the self-assembly framework, and the target molecule-driven signal linkage mechanism. As the EDR reaction chain sequence changes, the detected target miRNA changes, corresponding to the malignant transformed cell types rich in that target miRNA, indicating that the ECEER can be flexibly edited for UDG and APE1-specific analysis of malignant transformed cells.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] 1. This invention clarifies that both APE1 and UDG can serve as biomarkers for HCC diagnosis. Furthermore, it successfully constructs an enzyme-based entropy-driven chain exchange reaction (ECEER) for highly sensitive in-situ detection of UDG and APE1 in malignant transformed cells. Utilizing the specific biological properties of enzymes—UDG recognizing damaged uracil bases in single / double-stranded DNA to initiate a base excision repair mechanism, and APE1's endonuclease activity at the AP site of double-stranded DNA (AP-endo)—and integrating these into dynamic DNA self-assembly technology via DNA logical encoding, a high-precision and time-dependent cyclic ECEER can be developed, achieving signal amplification and enhancement of the entropy-driven chain exchange reaction.

[0043] 2. The ECEER system provided by this invention has three strands: a substrate strand Qc, a fuel strand Fu (with dU sites), and a fuel strand Fc (with dU sites) forming a regular sequence system that can be used to detect target strand miR-(z). This ECEER system does not refer to a specific sequence; rather, the substrate strand Qc, fuel strand Fu, and fuel strand Fc need to be designed according to different target strand miR-(z). Furthermore, this invention has demonstrated that the ECEER system can specifically identify and analyze specific malignant transformed cells. In the entire targeted detection process, the EDR reaction strand sequence can be simply modified to achieve specific activation by miRNAs from different types of malignant transformed cells, thereby achieving highly sensitive analysis of UDG and APE1 for different malignant transformed cells. In addition, the resolution accuracy of hierarchical decoding can be optimized by adjusting the position and number of dU bases and the number of mismatched bases in the fuel strand. Furthermore, the preparation process is simple and suitable for analyzing the expression levels of UDG and APE1 in cells at different times. This provides a new model for studying the relationship between UDG and APE1 and the process of malignant transformation cells, and has the potential for clinical application, which can guide the diagnosis and treatment of malignant transformation cells. Attached Figure Description

[0044] Figure 1 This is a quantitative analysis of UDG and APE1 activities in peripheral blood samples from healthy individuals and HCC patients (A is the quantitative analysis of UDG activity, B is the quantitative analysis of APE1 activity, C is the receiver operating characteristic (ROC) curve for HCC diagnosis based on UDG activity, and D is the ROC curve based on APE1 activity; where Ncs represents healthy individuals, HCC represents liver cancer patients, CNLCI represents stage I liver cancer patients, CNLCII represents stage II liver cancer patients, and CNLCIII represents stage III liver cancer patients).

[0045] Figure 2 This is a quantitative analysis of UDG and APE1 activities in hepatocellular carcinoma cells and normal hepatocytes (A: Immunofluorescence co-localization analysis of UDG expression in Huh7 and THLE-2 cells; B: Immunofluorescence co-localization analysis of APE1 expression in Huh7 and THLE-2 cells; C: ROC curves based on UDG and APE1 immunofluorescence co-localization analysis; D: Western blot analysis of UDG expression in Huh7 and THLE-2 cells; E: Western blot analysis of APE1 expression in Huh7 and THLE-2 cells; F: ROC curves based on UDG and APE1 Western blot analysis; where THLE-2 represents normal hepatocyte cell line and Huh7 represents hepatocellular carcinoma cell line).

[0046] Figure 3This is a quantitative analysis of UDG and APE1 activities in stromal and cancerous tissues (A is the expression analysis of UDG in HCC stromal and cancerous tissues; B is the expression analysis of APE1 in HCC stromal and cancerous tissues; C is the ROC curve based on immunohistochemical analysis of UDG and APE1; where Stroma represents stromal tissue and Tumor represents tumor tissue).

[0047] Figure 4 These are representative images characterizing the ECEER system (A is PAGE electrophoresis characterizing the ECEER system, Qc represents the substrate chain, Fu represents the fuel chain, R represents the display recovery gene, Fc represents the fuel chain, T represents the promoter, and QcFu represents the hybrid double strand of the substrate and fuel chain; B is fluorescence spectroscopy characterizing the ECEER system, Q'Fu2 represents the QcFu hybrid double strand, Q'Fu2+F' represents the ECEER system, and Q'Fu2+F'+T represents the simultaneous presence of the ECEER system and the promoter).

[0048] Figure 5 These are fluorescence spectra of different targets (A is the ECEER system used to detect target miR-21; B is the ECEER system used to detect target miR-375; C is the ECEER system used to detect target miR-185; where QFuB represents QcFu hybrid double strand, QFuB+F represents the ECEER system, and QFuB+F+T represents the simultaneous presence of the ECEER system and the promoter).

[0049] Figure 6 This is a diagram showing the optimization results of the number of dU bases and the binding state of Fu in the fuel chain;

[0050] Figure 7 This is a graph showing the temperature optimization results of the ECEER system;

[0051] Figure 8 The graph shows the effect of enzyme concentration on the ECEER system (A is the effect of UDG enzyme concentration on the ECEER system; B is the effect of APE1 enzyme concentration on the ECEER system).

[0052] Figure 9 These are laser confocal images of Huh-7 and THLE-2 cells at different time points after ECEER treatment;

[0053] Figure 10 The images show the flow cytometry results of Huh-7 and THLE-2 cells at different time points after ECEER treatment (A shows the flow cytometry results of Huh-7 and THLE-2 cells at different time points after ECEER treatment; B shows the flow cytometry results of Huh-7 and THLE-2 cells at different time points after ECEER treatment). Detailed Implementation

[0054] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to explain the present application and not to limit it. For those skilled in the art, the present application can be implemented without some of these specific details. The following description of the embodiments is merely to provide a better understanding of the present application by illustrating examples.

[0055] Example 1: Clinical value of UDG and APE1 activity in the diagnosis of HCC

[0056] 1. Research Subjects

[0057] Serum samples were collected from the Second Affiliated Hospital of Chongqing Medical University from January to June 2025. The average age of liver cancer patients was 58 years, with a male-to-female ratio of 9:1. Informed consent was obtained from all patients. The average age of healthy volunteers was 41 years, with a male-to-female ratio of 1:2. Liver cancer results were confirmed by magnetic resonance imaging. The study protocol was conducted in accordance with the ethical guidelines of the Declaration of Helsinki, 1975, with ethical approval number 2025(165). Tissue microarrays were provided by Hunan Aifang Biotechnology Co., Ltd., with ethical approval number HN20250401.

[0058] 2. Experimental Methods

[0059] (1) Serum enzyme-linked immunosorbent assay

[0060] Serum samples were collected from liver cancer patients (n=80) and healthy volunteers (n=50). Following the manufacturer's instructions, human depurinylpyrimidine endonuclease 1 (APE1) ELISA kit (ml037741, Mlbio) and human uracil DNA glycosidase (UNG) ELISA kit (ml106336, Mlbio) were used to group patients according to their tumor clinical stage, and enzyme activities were compared and analyzed.

[0061] (2) Cell line immunofluorescence experiment

[0062] Cells cultured on coverslips were fixed with 4% paraformaldehyde for 30 minutes, then permeabilized with 0.1% Triton X-100 for 15 minutes. Subsequently, samples were blocked with ready-to-use normal goat serum (AR009, Boster) and incubated overnight at 4°C with primary antibodies diluted in ready-to-use normal goat serum: anti-UDG (IF: 1:100) (MA5-25680, Thermo Fisher Scientific), anti-APE1 (IF: 1:250) (ab189474, Abcam). After primary antibody incubation, the cells were washed three times with PBST, and then incubated for 1 hour at room temperature in the dark with fluorescein-conjugated secondary antibody in PBST. 594 conjugated with goat anti-rabbit IgG (IF: 1:100) (AS039, ABclonal); 488 conjugated with goat anti-mouse IgG (IF: 1:100) (AS037, ABclonal). After washing three times with PBST, the sample was immersed in mounting medium containing DAPI (G1407-25ML, Seville) for imaging analysis.

[0063] (3) Cell line immunoblotting experiment

[0064] Cells were lysed in RIPA buffer (P0013, Beyotime). Proteins were separated by 10% SDS-PAGE gel chromatography and analyzed by Western blot. The following specific primary antibodies were used: anti-UDG (WB: 1:2500) (67490-1-Ig, Proteintech); anti-APE1 (WB: 1:1000) (ab189474, Abcam); and anti-β-tubulin (WB: 1:1000) (M05613-4, Boster Biological). Subsequently, corresponding horseradish peroxidase (HRP)-labeled anti-mouse or anti-rabbit IgG (Boster Biological, China) were applied and visualized and detected using a chemiluminescence (ECL) detection kit (G20140-500ML, Savill).

[0065] (4) Immunofluorescence assay of liver cancer tissue

[0066] Tissue sections were baked at 60°C for two hours for dewaxing and hydration. This process included dewaxing with fresh xylene for 10 minutes, repeated twice. Sections were then immersed in 100% ethanol for 5 minutes, followed by immersion in 95%, 90%, and 80% ethanol for 5 minutes each, and finally washed three times in PBS for 5 minutes each, followed by immersion in PBST for 30 seconds. Antigen retrieval was performed by microwave heating on medium heat for 8 minutes, followed by standing for 8 minutes. Then, the temperature was lowered and heated on medium-low heat for 7 minutes. After natural cooling, sections were placed in PBS at pH 7.4 and washed three times on a decolorizing shaker for 5 minutes each. Afterward, they were treated with 3% hydrogen peroxide for 15 minutes to block endogenous catalase, followed by immersion and washing three times in PBST for 1 minute each. Finally, 100 μL of non-immune normal goat serum was added, and the sections were incubated at room temperature for 30 minutes. Primary antibody against UDG (1:200, MA5-25680, Thermo Fisher Scientific) was incubated overnight at 4°C, followed by washing three times with PBST for 5 min each time. Secondary antibody was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 30 min, followed by washing five times with PBST for 5 min each time. The signal amplification stage was completed by adding TSA fluorescent dye (AFIHC025, Aifang Biotechnology) for 5 min, followed by washing three times with PBST for 5 min each time. The blocking step was repeated until the signal amplification step for additional staining (anti-APE1 (1:250, ab189474, Abcam), anti-AFP (1:100, AFRM81134, Aifang Biotechnology), anti-KI67 (1:300, AFRM9189, Aifang Biotechnology). Sections were stained with DAPI for 10 min, washed three times with PBST for 5 min each time, and then mounted with anti-quenching mounting medium. Finally, the results were observed and interpreted using a multispectral imaging system.

[0067] 3. Experimental Results

[0068] (1) Compared with the healthy group, UDG expression was increased in HCC and its different stages (p<0.0001), and ROC curve analysis showed that serum UDG had high diagnostic value for HCC; compared with the healthy group, APE1 expression was increased in HCC and its different stages, that is, APE1 activity in the same cohort showed a trend of gradually increasing with tumor progression (p<0.0001), and ROC curve analysis showed that serum APE1 had high diagnostic value for HCC, that is, APE1 has diagnostic potential in distinguishing HCC patients from healthy controls (see Figure 1 ).

[0069] (2) Immunofluorescence and Western blotting results showed that, compared with the normal hepatocyte cell line THLE-2, the expression of UDG in the hepatocellular carcinoma cell line Huh7 was increased, and ROC curve analysis showed that UDG at the cellular level had high diagnostic value for HCC, i.e., it has diagnostic potential in distinguishing hepatocellular carcinoma cells from healthy cells; compared with the normal hepatocyte cell line THLE-2, the expression of APE1 in the hepatocellular carcinoma cell line Huh7 was increased, and ROC curve analysis showed that APE1 at the cellular level had high diagnostic value for HCC (see Figure 2 ).

[0070] (3) Immunofluorescence assays of liver cancer tissues showed that UDG expression was increased in the tumor region compared to the interstitial region, and ROC curve analysis indicated that tissue UDG had a higher diagnostic value for HCC. Conversely, APE1 expression was increased in the tumor region compared to the interstitial region, and ROC curve analysis showed that tissue APE1 had a higher diagnostic value for HCC (see [link to study].) Figure 3 ).

[0071] In conclusion, both APE1 and UDG can serve as biomarkers for the diagnosis of HCC.

[0072] Example 2: Construction of an ECEER Detection System

[0073] 1. Materials and Methods

[0074] 1.1 Materials

[0075] DNA purified by HPLC was synthesized at Sangon Biotech (Shanghai) Co., Ltd. Tris, NaCl, and MgCl2 were purchased from Chuandong Chemical (Chongqing, China) Co., Ltd. Lipofectamine TM The 3000 reagent was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0076] 1.2 Characterization Instruments

[0077] TCSSP8 laser scanning confocal microscope (LEICA, Germany), flow cytometer (Beckman Coulter, USA), upright and inverted fluorescence microscope (Leica, Germany), fluorescence chemiluminescence agglutination imaging system (Bio-Rad, USA), protein and nucleic acid quantification analyzer (Beckman Coulter, USA), electrophoresis system (Bio-Rad, USA), and fully automated microplate reader (Bio-Rad, USA).

[0078] 1.3 Principles for Constructing the ECEER System

[0079] The fuel chain Fu contains at least one dU base and is labeled with a fluorescent group and a quenching group. The fuel chain Fu and the substrate chain Qc form a stable QcFu hybrid double strand through annealing. The damaged base (dU base) on the fuel chain Fu in the QcFu hybrid double strand is recognized and excised by UDG to form an AP site, and further forms a notched triple-stranded complex under the action of APE1.

[0080] 2. Sequence preparation

[0081] 2.1 Prepare Tris-NaCl-MgCl2 buffer solution

[0082] All glassware was soaked in chromic acid overnight, then rinsed with deionized water. The solution was prepared according to the formula (Tris 10mM, NaCl 480mM, MgCl2 5mM, pH 7.5) and diluted to volume in volumetric flasks. The prepared Tris-NaCl-MgCl2 solution was stored at 4°C for later use.

[0083] 2.2 Dissolution sequence

[0084] Pack the dry sequence powder (substrate chain Qc, fuel chain Fu, and fuel chain Fc) into 1.5 ml EP tubes and centrifuge at 12000 rpm for 5 min. According to the label on the EP tube, add an appropriate amount of Tris-NaCl-MgCl2 buffer to prepare a 10 μM solution. Vortex for 3 min, then cool on ice for 3 min; repeat three times. Aliquot and label the dissolved sequence, and store at -20°C.

[0085] 3. Construction of the ECEER System

[0086] The substrate chain Qc and the fuel chain Fu sequence were mixed and annealed at a concentration ratio of 1:1. That is, 50 μL of substrate chain Qc (10 μM) and 50 μL of fuel chain Fu (10 μM) were thoroughly mixed, placed in a 95°C water bath for 5 min, and then rapidly cooled in a container filled with crushed ice for 1 hour to obtain stable QcFu hybrid double strands.

[0087] To ensure a continuous supply of fuel chain Fu, add at least 50 μL of fuel chain Fc (10 μM) to a solution containing QcFu hybrid double strands, maintaining the concentration ratio of fuel chain Fc to QcFu hybrid double strands at Fc:QcFu ≥ 1:1, and mix thoroughly to obtain a mixture of QcFu + Fc, which is the ECEER system, and store at -20℃.

[0088] The above-mentioned fuel chain Fc has the same sequence as the fuel chain Fu. Through chain substitution reaction, it can hybridize with Qc to form QcFc complex, which enters a new cycle reaction.

[0089] 4. Construction of the ECEER Detection System

[0090] The required amount of ECEER varies depending on the bottom area of ​​the plate. Refer to the Thermo Fisher Scientific Lipo3000 transfection reagent instructions for specific dosage. For example, using one well of a 24-well plate, add 25 μL of serum-free and antibiotic-free culture medium and 0.75 μL of Lipofectamine to a 200 μL EP tube. TM 3000 reagent; take another 200μL EP tube and add 25μL serum-free and antibiotic-free culture medium, 1μL P3000. TM Prepare the reagents and 10 μL of QcFu + Fc mixture. Mix the two EP tubes thoroughly and incubate at room temperature for 20 minutes. Then, add the DNA-lipid complex into the cells for detection.

[0091] Example 3: Validation of the ECEER System

[0092] 1. Validation of the ECEER system

[0093] The ECEER system obtained in Example 2 was characterized by PAGE electrophoresis. Figure 4 As shown in Figure A, after the substrate chain Qc and the fuel chain Fu self-assembled via annealing, a distinct band was formed (see Figure A). Figure 4 Lane 7 in section A represents a successfully assembled QcFu hybrid double strand (QcFu). A distinct R band only appears when promoter T is present (see...). Figure 4 The results above demonstrate that the ECEER system designed in this invention can specifically identify targets.

[0094] The ECEER system obtained in Example 2 was characterized using fluorescence spectroscopy. The feasibility of a nick-based entropy-driven chain displacement reaction system was analyzed by labeling the dU bases of the fuel chain Fu with BHQ1 and FAM. Figure 4 Figure B shows that ECEER exhibits a significant fluorescence signal (FL) in the presence of promoter T (here, promoter T is miR-21), with a signal-to-noise ratio as high as 10.4 (see Figure B). Figure 4 (B) This is consistent with the electrophoresis results above. This demonstrates that the ECEER designed in this invention can specifically identify targets, and the substrate-self-supplying EDR has the advantage of a high signal-to-noise ratio, making it feasible for implementation.

[0095] 2. ECEER Logic Encoding Function Verification

[0096] To modify the reaction chain sequence of entropy-driven chain displacement reactions for various targets, an adapted ECEER system was constructed, and the universality of the ECEER system was analyzed by fluorescence spectroscopy.

[0097] When detecting the target miR-21 (5'-UAGCUUAUCAGACUGAUGUUGA-3'), the compatible ECEER system includes substrate chain Qc-21 with the sequence 5'-TCAACATCAGTCTGATAAGCTAAGGGACCCGTAA GTTAGTTGGAGACGTAGG-3', fuel chain Fu-21 with the sequence 5'-CCTACGTCTCCAACT AA(FAM)CTTACGG(X)CCCTTAGC(BHQ1)TTATCAGACT-3', and fuel chain Fc-21.

[0098] When detecting the target miR-375 (5'-UGCGCUCGGCUUGCUUGUUUU-3'), the compatible ECEER systems include substrate chain Qc-375 with the sequence 5'-AAAACAAGCAAGCCGAGCGCAAGGGCCGTAAGT TAGTTGGAGACGTAGG-3', fuel chain Fu-375 with the sequence 5'-CCTACGTCTCCAACT AA(FAM)CTTACGG(X)CCCT(BHQ1)TGCGCTCGGCTTG-3', and fuel chain Fc-375.

[0099] When detecting the target miR-185 (5'-UGGAGAGAAAGGCAGUUCCUGA-3'), the compatible ECEER systems include the substrate chain Qc-185 with the sequence 5'-TCAGGAACTGCCTTTCTCTCCAAGGGCCGTAAGT TAGTTGGAGACGTAGG-3', the fuel chain Fu-185 with the sequence 5'-CCTACGTCTCCAACT AA(FAM)CTTACGG(X)CCCT(BHQ1)TGGAGAGAAAGGCA-3', and the fuel chain Fc-185.

[0100] In the above, X represents a dU base, and there are two dU bases.

[0101] like Figure 5 The results show that the ECEER system can still produce a high signal-to-noise ratio when detecting different targets. When detecting the miR-21 target, the signal-to-noise ratio is 4.60 (see...). Figure 5 (A) When detecting the target miR-375, the signal-to-noise ratio is 5.32 (A). Figure 5 (B in the text); When detecting the target miR-185, the signal-to-noise ratio is 4.40 (in the text). Figure 5 (C) This demonstrates that the ECEER of the present invention is universal and provides strong guidance for the practical detection of various miRNAs.

[0102] Example 4: Optimization of the ECEER System and its Resolution

[0103] This embodiment further optimizes the number and binding state of dU bases in the fuel chain Fu, the reaction temperature, and the enzyme concentration during the construction of the ECEER system in Example 2, to ensure the resolution accuracy of hierarchical decoding.

[0104] To investigate the effect of the number of dU bases in the fuel chain Fu on the ECEER reaction, fuel chains Fu containing different numbers of dU bases (1, 2, and 3) were designed, along with two binding states: fully complementary pairing (no convex ring) and partially convex dU bases (with convex ring). Fluorescence spectroscopy was then used for detection. Figure 6 The results show that the optimal number of dU bases in the fuel chain Fu is 2, and the optimal binding state is a convex ring.

[0105] To optimize the reaction temperature for the entire experiment, at least five points (4℃, 16℃, 25℃, 37℃, and 45℃) were selected from low to high for the experiment. Figure 7 The results show that the optimal reaction temperature for the ECEER system is between 25-37℃, with the optimal temperature among the five points mentioned above being 37℃.

[0106] To verify the signal feedback mechanism of the ECEER system for the recognition of UDG and APE1, the concentrations of one enzyme in UDG and APE1 were kept constant, and the signal changes caused by changes in the concentration of the other enzyme were examined. Figure 8 As shown, at a constant APE1 concentration, FL increases with increasing UDG concentration (see...). Figure 8 (A in the middle), and vice versa (see A in the middle). Figure 8 (B in the text). This indicates that the logical coding of DNA molecules has a high resolution and signal amplification capability for UDG and APE1.

[0107] Example 5: ECEER System Detection Performance Analysis

[0108] This embodiment utilizes the EDR combined with the UDG base excision repair mechanism and APE1 endonuclease activity. UDG and APE1 work synergistically to excise damaged bases and create nucleotide gaps, thereby establishing a specific cellular in-situ signal transduction, amplification, and reporting mechanism for UDG and APE1. This approach aims to develop a novel in-situ detection technology for UDG and APE1 in malignant transformed cells, characterized by high spatiotemporal precision and high specificity, targeting the EDR reaction chain sequence encoded by the cell type.

[0109] To evaluate the detection performance of the ECEER system, hepatocellular carcinoma cell lines (Huh-7 cells) highly expressing UDG and APE1 and control cells (THLE-2 cells) were selected for cytological experiments. Cells were incubated with the ECEER system modified with a fluorescent dye (FAM) and a quencher (BHQ1) to complete the hierarchical encoding of target molecules. Intracellular FAM fluorescence signals from both cell lines were collected using laser scanning confocal microscopy and flow cytometry, respectively. Specifically, appropriate amounts of both cell lines at the same concentration were seeded in 24-well plates, and transfection was performed when the cell confluence reached 80%. For example, using one well of a 24-well plate, 200 μL of EP tube was added to 25 μL of serum-free and antibiotic-free culture medium and 0.75 μL of Lipofectamine. TM 3000 reagent; take another 200μL LEP tube and add 25μL serum-free and antibiotic-free culture medium and 1μL P3000. TM The reagents and 10 μL of QcFu + Fc mixture were added. The two EP tubes were mixed thoroughly and incubated at room temperature for 20 minutes. Then, the DNA-lipid complex was added to the cells, and the tubes were transferred to a CO2 incubator for further incubation. ECEER treatment times were 0 min, 30 min, 60 min, 90 min, 120 min, and 180 min, followed by observation and analysis using a laser confocal microscope and flow cytometry.

[0110] like Figure 9 As shown, under 488nm laser irradiation, the fluorescence of both cell lines generally increased with increasing reaction time. At the same time point, Huh-7 cells showed a more obvious fluorescence signal than THLE-2 cells, suggesting that ECEER was successively recognized by UDG, APE1, and miR-21, and initiated substrate self-supply EDR, which allowed FAM to continuously move away from BHQ1 and recover fluorescence, mainly distributed in the cytoplasm. This confirms the feasibility of ECEER-mediated encoding of UDG, APE1, and miR-21.

[0111] like Figure 10 As shown, flow cytometry results indicate that Huh-7 cells exhibit more pronounced fluorescence signals than THLE-2 cells, with the largest difference in fluorescence signals between Huh-7 and THLE-2 cells observed after 90 minutes of ECEER treatment. Consistent with the laser confocal results, this demonstrates that the ECEER system of this invention can specifically identify multiple targets, and that substrate-self-supplying EDR is highly feasible in improving the signal-to-noise ratio.

[0112] UDG and APE1, as rate-limiting enzymes in the base excision repair (BER) pathway, play a crucial role in maintaining the stability of the human cell genome. Precise assessment of their spatiotemporal variations holds promise for monitoring the development and progression of malignant transformed cells, evaluating treatment efficacy, and predicting relapse risk.

[0113] This invention develops a high-precision and time-dependent recyclable ECEER, which utilizes the biologically specific properties of enzymes—UDG recognizing damaged uracil bases in single / double-stranded DNA to initiate a base excision repair mechanism, and APE1's endonuclease activity excising AP sites in double-stranded DNA—to integrate into the dynamic DNA self-assembly technology EDR to construct the ECEER.

[0114] This invention utilizes the EDR combined with the UDG base excision repair mechanism and APE1 endonuclease activity. UDG and APE1 work synergistically to excise damaged bases and form nucleotide gaps, thereby establishing a specific cellular in-situ signal conversion, amplification, and reporting mechanism for UDG and APE1. This mechanism possesses strong signal discrimination and collection capabilities, and encodes EDR reaction chain sequences for malignant transformed cell types. This invention develops a new technology for in-situ detection of UDG and APE1 in malignant transformed cells with high spatiotemporal precision and high specificity.

[0115] The ECEER established in this invention has good universality and can be activated by miRNAs highly expressed in malignant transformed cells. ECEER probes with AND logical coding function were designed targeting the highly expressed UDG and APE1 in cells. The results show that miRNA-21, UDG, and APE1 can effectively activate the ECEER signal switch in an enzyme concentration-dependent manner. Furthermore, their fluorescence signals in Huh7 cells are significantly higher than in THLE-2 cells, demonstrating that this system can be used for the cascade detection and analysis of UDG and APE1 in malignant transformed cells. Therefore, by simply changing the reaction chain sequence of the entropy-driven chain displacement reaction, precise in-situ high-sensitivity analysis of UDG and APE1 in malignant transformed cells can be performed. This invention provides a novel tracking model for studying the correlation between intracellular UDG and APE1 and the process of malignant transformation, clarifies the clinical application of this new technology, and helps guide its application in the diagnosis and treatment of malignant transformation in clinical testing.

[0116] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A biomarker for the diagnosis of liver cancer, characterized in that, The markers are UDG and / or APE1.

2. The use of the biomarker according to claim 1 in the preparation of a diagnostic product for liver cancer.

3. The application according to claim 2, characterized in that, The detection product detects at least one of serum, cells, and tissues, and the detection product is a diagnostic kit or an ECEER system.

4. An ECEER system for detecting the markers described in claim 1 or a method for constructing the ECEER system described in claim 3, characterized in that, Includes the following steps: S11. Fuel chain Fu and substrate chain Qc are mixed at a concentration ratio of 1:1 and annealed to form a stable QcFu hybrid double strand. The damaged bases of fuel chain Fu in the QcFu hybrid double strand are successively recognized by UDG and APE1 enzymes. S12. Add fuel chain Fc to the QcFu hybrid double-stranded solution, so that the concentration of fuel chain Fc is not lower than the concentration of QcFu hybrid double-stranded solution, i.e. Fc:QcFu≥1:

1. Mix thoroughly to obtain QcFu+Fc mixture, which is the ECEER system. The fuel chain Fu contains at least one dU base and is labeled with a fluorescent group and a quenching group. The substrate chain Qc has a Toehold structure. The fuel chain Fc has the same sequence as the fuel chain Fu. Through a chain substitution reaction, it hybridizes with Qc to form a QcFc complex, which enters a new cycle reaction.

5. The method for constructing the ECEER system according to claim 4, characterized in that, The annealing conditions are: 95℃ water bath for 5-10 minutes, followed by ice bath for 1 hour; the number of dU bases in the fuel chain Fu is 1-3.

6. The method for constructing the ECEER system according to claim 4, characterized in that, The concentration of the UDG enzyme is 0.075-0.01 U / μL, the concentration of the APE1 enzyme is 0.075-0.01 U / μL, and the number of dU bases in the fuel chain Fu is 2.

7. An ECEER system constructed using the method for constructing an ECEER system as described in any one of claims 4-6.

8. A method for constructing an ECEER detection system, characterized in that, Includes the following steps: S21. Add serum-free and antibiotic-free culture medium and Lipofectamine to the EP tube. TM 3000 reagents; S22, Add serum-free and antibiotic-free culture medium and P3000 to another EP tube. TM The ECEER system constructed using the reagents and the method for constructing the ECEER system according to any one of claims 4-6, or the ECEER system according to claim 7; S23. Mix the two EP tubes thoroughly and incubate at 5-45℃ for 20 minutes. Then, add the DNA-lipid complex into the malignant transformed cells for the detection of the target miR-(z).

9. The method for constructing an ECEER detection system according to claim 5, characterized in that, The dosage of the ECEER system is based on Lipofectamine. TM Add the reagent according to the dosage instructions in the 3000 reagent manual.

10. A method for constructing an ECEER detection system according to claim 5, characterized in that, The malignant transformed cells are cancer cells, including liver cancer cells, which include Huh-7 cells.

11. A method for constructing an ECEER detection system according to claim 5, characterized in that, The target miR-(z) is one or more of miR-21, miR-375, or miR-185, wherein the sequence of the target miR-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3'; the sequence of the target miR-375 is 5'-UGCGCUCGGCUUGCUUGUUUU-3'; and the sequence of the target miR-185 is 5'-UGGAGAGAAAGGCAGUUCCUGA-3'.

12. The method for constructing an ECEER detection system according to claim 5, characterized in that, The incubation temperature is 25-37℃.

13. The method for constructing an ECEER detection system according to claim 10, characterized in that, When the target miR-(z) is miR-21, the corresponding substrate chain Qc, fuel chain Fu, and fuel chain Fc are substrate chain Qc-21, fuel chain Fu-21, and fuel chain Fc-21, respectively. The nucleotide sequence of the substrate chain Qc-21 is: 5'-TCAACATCAGTCTGATAAGCTAAGGGACCCGTAAGTTAGTTGGAGACGTAGG-3', The nucleotide sequences of both fuel chain Fu-21 and fuel chain Fc-21 are: 5'-CCTACGTCTCCA ACTAA(FAM)CTTACGG(X)CCCTTAGC(BHQ1)TTATCAGACT-3'; When the target miR-(z) is miR-375, the corresponding substrate chain Qc, fuel chain Fu, and fuel chain Fc are substrate chain Qc-375, fuel chain Fu-375, and fuel chain Fc-375, respectively. The nucleotide sequence of the substrate chain Qc-375 is: 5'-AAAACAAGCAAGCCGAGCGCAAGGGCCGTAAGTTAGTTGGAGACGTAGG-3', The nucleotide sequences of the fuel chain Fu-375 and the fuel chain Fc-375 are both: 5'-CCTACGT CTCCAACTAA(FAM)CTTACGG(X)CCCT(BHQ1)TGCGCTCGGCTTG-3'; When the target miR-(z) is miR-185, the corresponding substrate chain Qc, fuel chain Fu, and fuel chain Fc are substrate chain Qc-185, fuel chain Fu-185, and fuel chain Fc-185, respectively. The nucleotide sequence of the substrate chain Qc-185 is: 5'-TCAGGAACTGCCTTTCTCTCCAAGGGCCGTAAGTTAGTTGGAGACGTAGG-3', The nucleotide sequences of the fuel chain Fu-185 and the fuel chain Fc-185 are both 5'-CCTACGTCT CCAACTAA(FAM)CTTACGG(X)CCCT(BHQ1)TGGAGAGAAAGGCA-3'; X represents a dU base, and the number of dU bases is 2.

Images