Application of biomarkers based on DEPTOR function in diagnosis and treatment of liver cancer

Abstract

The application provides a modified form of DEPTOR, DEPTOR K106 lactylation, as a biomarker in the preparation of a test agent for the preliminary evaluation or prediction of liver cancer. The application also provides a kit for quantitatively or semi-quantitatively detecting a protein, which contains an antibody capable of specifically binding to DEPTOR K106 lactylation. The application can more effectively preliminarily evaluate and predict whether a subject has a tumor by detecting the expression level of DEPTOR K106 lactylation. The application will help improve the diagnostic accuracy of liver cancer and provide an important basis for early treatment of patients. The kit of the application can be used for the diagnosis of early, medium and late liver cancer.

Description

Technical Field

[0001] This invention relates to the application of biomarkers based on the function of DEPTOR in the diagnosis and treatment of liver cancer, and belongs to the field of oncology medicine. Background Technology

[0002] Given the intricate mechanisms of tumorigenesis and development, current research remains inadequate, significantly limiting our in-depth understanding of the nature of tumors and the development of new diagnostic and therapeutic methods. Metabolism is a key factor in tumorigenesis and evolution, during which tumor cells undergo a significant shift in metabolic patterns, known as "metabolic reprogramming." As a hallmark of tumors, metabolic reprogramming fulfills three crucial needs of tumor cells: ensuring the energy supply required for sustained growth, synthesizing biomolecules such as nucleic acids, proteins, and lipids to sustain growth, and maintaining redox homeostasis. For example, in the field of tumor energy / glucose metabolism, there is a significant phenomenon known as the "Warburg effect." This describes the strong dependence of tumor cells on aerobic glycolysis, which differs greatly from the mitochondrial oxidative phosphorylation pathway preferred by normal cells. Although this approach produces ATP (adenosine triphosphate) less efficiently, it is precisely this unusual metabolic strategy that endows tumor cells with specific survival advantages. These advantages are crucial for tumor cell growth, metastasis, and coping with therapeutic stress. Therefore, only when we more comprehensively and systematically reveal the metabolic mechanisms of tumors can we effectively identify and target tumor metabolic biomarkers, thereby breaking through the current bottlenecks in tumor diagnosis and treatment and bringing more effective treatment options to cancer patients.

[0003] Currently, tumor diagnosis methods are divided into five levels: ① Clinical diagnosis: Based on the patient's clinical symptoms, signs, and imaging results, combined with the general progression of the disease, this method provides a preliminary direction, but its accuracy is limited. ② Surgical diagnosis: This relies on the presence of a visible mass during surgery or endoscopy. However, this method lacks pathological support, thus its reliability is relatively low. ③ Physicochemical diagnosis: This method can be used when the patient's clinical presentation matches the characteristics of cancer and is supported by imaging examinations such as X-ray, ultrasound, CT, and MRI, or biochemical indicators such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). It provides a more objective diagnostic basis, but also has certain limitations. ④ Cytopathological diagnosis: This is based on the examination results of various exfoliated cells or biopsied cells. This method allows for more direct observation of abnormal cellular changes, thus providing more accurate diagnostic information. ⑤ Histopathological diagnosis: This is currently the most ideal diagnostic method. It involves obtaining tissue samples through core needle biopsy and preparing pathological sections for detailed pathological analysis. This method allows for direct observation of the microstructure of the diseased tissue, thus providing the most accurate diagnostic results. Although the reliability of these five diagnostic methods increases sequentially, they all face a common challenge: the difficulty in achieving early detection and diagnosis of tumors. In most cases, these methods only become effective when the tumor has progressed to a certain stage, or even when the patient presents with severe symptoms, which undoubtedly increases the difficulty of treatment and the patient's suffering. Furthermore, some methods with better diagnostic results, such as histopathological diagnosis, require puncture to obtain cells or tissue sections, which may cause some harm and negative impact on the patient's mental and physical well-being. Therefore, we need to continuously explore new diagnostic technologies and methods to achieve early detection and diagnosis of tumors, improve treatment success rates, and alleviate patient suffering.

[0004] In tumor cells, mutations in metabolic enzyme genes or changes in their expression levels lead to significant alterations in the levels of intracellular metabolites. This "classical" metabolic reprogramming plays a crucial role in tumor progression. However, increasing evidence in recent years suggests that some metabolic enzymes and small metabolic molecules can also participate in various important life processes of tumor cells through "non-classical" functions that are not dependent on metabolic pathways, thereby promoting tumor development and progression.

[0005] Given the above background, in order to more accurately grasp the proliferation of cancer cells, achieve early diagnosis of cancer, and select treatment plans more rationally, it is necessary to find key biomarkers involved in the key mechanisms of tumor cell proliferation and use them for early diagnosis and treatment of tumors. Summary of the Invention

[0006] The technical problem or primary objective of this invention is to address the issue of early preliminary assessment or prediction of liver cancer using novel tumor markers.

[0007] Another object of the present invention is to provide a product for the preliminary assessment or prediction of the early stage of liver cancer.

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

[0009] In one aspect, the present invention uses the modified form of DEPTOR, DEPTOR K106 lactation, as a biomarker to prepare a reagent for the preliminary assessment or prediction of early-stage liver cancer.

[0010] In the present invention, DEPTOR K106 lactation refers to lactation at position 106 of the amino acid sequence of DEPTOR.

[0011] In the scheme described in this invention, by using an immunoassay reagent targeting the lactation of DEPTOR K106 to detect the expression level of DEPTOR K106 lactation in samples from the subject, the subject's liver cancer status can be effectively preliminarily assessed and predicted. Furthermore, the sample from the subject can be any one of the subject's peripheral venous blood, adjacent normal tissue, or tumor tissue.

[0012] In a preferred embodiment of the present invention, the reagent for early preliminary assessment or prediction of liver cancer is an immunoassay reagent based on ELISA or Western Blot methods.

[0013] Secondly, the present invention also provides an antibody that is capable of specifically binding to the lactated DEPTORK106.

[0014] Thirdly, the present invention also provides a kit for quantitative or semi-quantitative detection of proteins, containing the antibody described in the second aspect of the present invention.

[0015] In a preferred embodiment of the present invention, the reagent kit for quantitative or semi-quantitative detection of proteins is a reagent kit for detecting proteins by enzyme-linked immunosorbent assay (ELISA) or Western blotting; the reagent kit contains a first antibody and a second antibody; the first antibody is the antibody DEPTOR1aK106 described in the second aspect of the present invention, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

[0016] Fourthly, this invention also provides the application of antibodies that specifically bind to lactated DEPTOR K106 in the preparation of liver cancer diagnostic kits.

[0017] In a further preferred embodiment of the present invention, the kit further comprises one or more substances selected from the group consisting of: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

[0018] The instruction manual describes how to use the kit for testing, and how to use the test results to assess the development of liver cancer and select a treatment plan.

[0019] This invention addresses the technical challenges in the early diagnosis of liver cancer by providing a novel specific biomarker, DEPTOR K106 lactation, for early assessment and prediction of liver cancer. By detecting the expression level of this biomarker, early diagnosis of liver cancer can be achieved. This biomarker not only provides a powerful tool for early diagnosis of liver cancer but also, by detecting its expression level, enables a more effective preliminary assessment and prediction of the tumor status in a subject. The application of this technology will help improve the accuracy of liver cancer diagnosis and provide important evidence for early treatment of patients. The detection kit prepared based on the aforementioned biomarker is applicable to the diagnosis of early, intermediate, and advanced stages of liver cancer.

[0020] The lactation of DEPTOR K106 described in this invention, as a specific post-translational modification of DEPTOR, is highly correlated with cancer cell growth and poor prognosis in hepatocellular carcinoma (HCC). Specifically, our experimental studies revealed that CD8+ T cells secrete interferon-γ (IFNγ), which can stabilize the protein level of transcriptional coactivator (TAZ) with a PDZ binding sequence in HCC cells. As the central transcriptional regulatory module of the Hippo pathway, TAZ has been shown in numerous studies to play a crucial role in the progression of various cancers, including liver cancer, lung cancer, breast cancer, skin cancer, pancreatic cancer, and brain cancer. Generally, high TAZ activity in the cell nucleus is closely related to tumor formation and is considered a key factor in tumor development. Under IFNγ stimulation, DEPTOR is lactated at the K106 site, which increases the stability of TAZ protein and promotes liver cancer growth. Furthermore, TAZ levels in human hepatocellular carcinoma (HCC) specimens were positively correlated with lactation levels of DEPTOR K106 and were associated with poor prognosis in HCC patients. These findings reveal a key mechanism by which tumor cells promote HCC proliferation through IFNγ activation and DEPTOR's enhanced TAZ stability, and highlight the potential of DEPTOR protein targeting for the diagnosis and treatment of HCC.

[0021] The above findings were obtained through the following research:

[0022] Experimental steps:

[0023] 1. Huh7 liver cancer cells were stimulated with IFNγ (2 ng / mL) for 0 h, 0.5 h, 1 h, 6 h and 12 h. Cells were then collected, proteins were lysed, and TAZ protein levels were detected by wb.

[0024] 2. Stimulate Huh7 hepatocellular carcinoma cells with or without IFNγ (2 ng / mL) for 1 hour, and then detect the binding of DEPTOR and TAZ using CO-IP technology.

[0025] 3. In Huh7 hepatocellular carcinoma cells, after transfection with HA-TAZ, FLAG-DEPTOR WT or its mutant plasmid was transfected. Stimulation with IFNγ (2 ng / mL) for 1 hour was performed with or without, and the binding level of DEPTOR to TAZ was detected using CO-IP technology.

[0026] 4. Huh7 hepatocellular carcinoma cells expressing FLAG-DEPTOR WT or its mutants were stimulated with or without IFNγ (2 ng / mL), and then the hepatocellular carcinoma cells were treated with CHX (100 μg / mL) for a specified time for immunoblotting analysis to detect their TAZ protein.

[0027] Experimental results:

[0028] Figure 1 The results suggest that TAZ protein gradually increases in Huh7 liver cancer cells after IFNγ stimulation.

[0029] Figure 2 The results suggest that IFNγ stimulation can increase the stability of TAZ protein; however, IFNγ cannot increase the stability of TAZ protein when DEPTOR K106 cannot be lactated.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] 1. Based on the metabolic mechanism of liver cancer, this invention has identified new biomarkers, providing strong support for the early diagnosis of tumors;

[0032] 2. The test can be completed by collecting peripheral venous blood, without the need for complicated surgery or invasive procedures, which greatly reduces the burden on the test subjects. Attached Figure Description

[0033] Figure 1 This study reflects the changes in TAZ protein levels in Huh7 hepatocellular carcinoma cells under IFNγ stimulation.

[0034] Figure 2 This reflects the TAZ protein levels in Huh7 hepatocellular carcinoma cells DEPTOR WT and K106R mutant cell lines treated with CHX (100 μg / mL) for a specified time and with or without IFNγ stimulation, as analyzed by immunoblotting. Detailed Implementation

[0035] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following tests are conventional methods; for tests where specific techniques or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials described are commercially available.

[0036] This invention provides the application of DEPTOR K106 lactation as a biomarker in the early assessment or prediction of liver cancer.

[0037] This invention also provides the application of DEPTOR K106 lactation in the preparation of reagents for early assessment or prediction of liver cancer.

[0038] The reagents include those for detecting the expression level of DEPTOR K106 lactation in samples. The samples are peripheral venous blood, adjacent normal tissue, and / or tumor tissue.

[0039] The reagent comprises a first antibody and a second antibody. The first antibody is a lactated antibody, DEPTOR laK106. The second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0040] This invention also provides a diagnostic kit for early liver cancer based on ELISA or Western Blot methods. The kit contains reagents for detecting DEPTOR K106 lactation. Each kit includes a first antibody and a second antibody. The first antibody is DEPTOR K106 lactation antibody (DEPTOR laK106), and the second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase. The kit may also include a container, instructions for use, a positive control, a negative control, a buffer, adjuvants, and a solvent. The instructions for use describe how to use the kit for detection and how to use the test results to assess tumor development and select treatment options. The kit components can be packaged in an aqueous medium.

[0041] The method for assessing or predicting early-stage liver cancer in patients using the kit described in this invention includes the following steps:

[0042] 1) Detect the expression levels of the biomarkers mentioned in the samples;

[0043] When using peripheral venous blood as a sample, the sample processing method includes: after collecting peripheral venous blood, it should be placed on ice for 15 minutes, and then centrifuged at 3500 rpm for 15 minutes to obtain serum or plasma, which can then be tested or stored at -80℃. Samples that cannot be tested immediately should be stored at -80℃ and thawed on ice before testing.

[0044] The processed samples were analyzed using methods such as ELISA or Western Blot to detect the lactation expression level of DEPTOR K106.

[0045] 2) Based on the lactation expression level of DEPTOR K106 in the samples obtained in 1), the subjects were used for early assessment or prediction of liver cancer.

[0046] The Western blotting method mentioned in the evaluation or prediction method described in this invention, also known as protein immunoblotting, is a technique commonly used in research for the isolation and identification of proteins. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample. The separated proteins are then transferred to a nitrocellulose or PVDF membrane, which is subsequently incubated with a specific antibody against the target protein. During membrane washing, unbound antibodies are washed away, leaving only antibodies bound to the target protein. Finally, the bound antibodies are detected by developing film or fluorescence scanning. Because the antibody binds only to the target protein, typically only a single, clear band is observed, with the band thickness corresponding to the protein content. By analyzing the location and intensity of specific reactions, information about the expression of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the high specificity and sensitivity of immunoblotting, Western blotting analysis can detect target proteins as low as 1 ng. This method is widely used in molecular biology fields such as molecular biology, biochemistry, and immunogenetics.

[0047] Based on the above description of specific embodiments, the present invention provides the following examples to further illustrate the technical solution and its technical effects.

[0048] Example 1.

[0049] An ELISA detection kit contains a pre-coated microplate, standards, enzyme-labeled secondary antibody, substrate solution, stop solution, washing buffer, sample diluent, control sample, instructions, and sealing film; the wells of the microplate are pre-immobilized with the capture antibody DEPTOR laK106; the enzyme-labeled secondary antibody is an antibody homologous to the capture antibody DEPTOR laK106 and labeled with horseradish peroxidase.

[0050] Example 2.

[0051] A Western blot (WB) test kit containing the following reagents and items:

[0052] Primary antibody DEPTOR laK106;

[0053] Secondary antibody (antibody homologous to DEPTOR laK106 and labeled with horseradish peroxidase);

[0054] Substrate solution;

[0055] Sealing solution: Milk powder sealing solution;

[0056] Washing buffer: TBS / T;

[0057] Transfer buffer;

[0058] PVDF membrane;

[0059] Electro-hydraulic transfer;

[0060] Membrane staining solution;

[0061] Sample buffer solution;

[0062] Standard protein or marker protein: used to estimate the molecular weight of the target protein;

[0063] Instruction manual.

[0064] Example 3.

[0065] A method for early assessment or prediction of liver cancer in subjects includes the following steps:

[0066] I. The expression level of DEPTOR K106 lactation in the peripheral serum of subjects was detected using the WB detection kit described in Example 2.

[0067] The specific testing steps are as follows:

[0068] 1. Prepare samples

[0069] 1) Sample collection: Collect peripheral blood from the subjects' veins, let it stand on ice for 15 minutes, centrifuge at 3500 rpm for 15 minutes at 4°C to obtain serum, and test it for later use.

[0070] 2) Make ice and prepare ice boxes;

[0071] 3) Preparation of cell lysis buffer: Cell lysis buffer containing protease inhibitors and phosphatase inhibitors;

[0072] Resuspend the cells in a pipette and lyse them on ice for 30 minutes, vortexing them every 10 minutes to ensure complete lysis.

[0073] 4) After lysis, centrifuge at 12,000 rpm for 20 minutes at 4°C, and collect the supernatant into an EP tube to obtain the protein sample after lysis.

[0074] 5) Determine the protein concentration of the sample according to the instructions of the BCA protein quantification kit. Based on the protein quantification results, add an appropriate amount of protein sample and 4' Loading to a new EP tube and make up the volume with cell lysis buffer. After mixing, denature in a 70°C metal bath for 10 minutes.

[0075] 2. Protein gel electrophoresis:

[0076] Remove the comb from the top of the precast gel horizontally, rinse with water, and remove the bottom sealing strip. Fix the precast gel vertically in the electrophoresis tank, and pour in enough electrophoresis buffer to completely submerge the gel. Add the protein sample evenly to the wells of the precast gel, filling any blank wells with 1' Loading. Perform electrophoresis at a constant voltage of 140V, stopping when the bromophenol blue reaches the bottom of the gel.

[0077] 3. Transfer:

[0078] Remove the pre-made gel from the electrophoresis tank, pry open the gel plate to remove the gel, and immerse the gel, NC membrane, filter paper, and sponge pads in pre-cooled transfer buffer. Then, begin constructing the "sandwich": from bottom to top, two layers of sponge pads, one layer of filter paper, NC membrane, the completed electrophoresis gel, another layer of filter paper, and two more layers of sponge pads, avoiding air bubbles during the construction process. Place the completed "sandwich" horizontally into the transfer chamber, then insert the transfer chamber horizontally into the transfer tank. Pour the pre-cooled transfer buffer into the inside of the transfer chamber, and add an ice-water mixture to the outside to reduce heat generated during the transfer process.

[0079] 4. Enclosed:

[0080] After the transfer was completed, the NC membrane was removed, stained with Ponceau S, and the transfer quality was observed. The membrane was then placed in TBST buffer and cut according to the protein marker and the molecular weight of the target protein. After washing away the Ponceau S, the membrane was blocked with 5% skim milk on a shaker at room temperature for 45 minutes.

[0081] 5. Antibody incubation:

[0082] After blocking, the NC membrane was washed twice with TBST. Primary antibody was prepared according to the instructions using antibody diluent. The cut bands were placed in the prepared DEPTOR K106 lactated primary antibody diluent and incubated overnight at 4°C on a horizontal shaker. After incubation, the primary antibody diluent was recovered, and the NC membrane was washed three times with TBST for 5 minutes each time. Then, a specific ratio of secondary antibody diluent corresponding to the species was added, and the membrane was incubated for 2 hours at room temperature on a shaker.

[0083] 6. Exposure:

[0084] Prepare solutions A and B in a 1:1 ratio according to the instructions for the ultra-high sensitivity enhanced chemiluminescence (ECL) developer. Drop the prepared developer onto the NC membrane after antibody incubation, react in the dark for a certain period of time, and then expose and develop it in an exposure machine.

[0085] II. Based on the protein expression results detected in step one, perform early assessment or prediction of liver cancer in patients.

[0086] Diagnostic analysis of test results:

[0087] If the following protein bands of the corresponding size appear on the NC membrane: DEPTOR K106 lactation, it suggests that the subject may have invasive cancer, be in the advanced stage of invasive cancer, or have a poor prognosis. If this band is not present, it suggests that the subject has a good prognosis.

[0088] We collected serum samples from 80 individuals at the Second Affiliated Hospital of Zhejiang University, including 46 liver cancer patients and 34 healthy individuals. Diagnostic criteria for liver cancer:

[0089] 1. It has two typical imaging features of liver cancer (US, enhanced CT, MRI or selective hepatic arteriography) and the lesion is >2cm.

[0090] 2. A typical imaging finding of liver cancer: lesion >2cm, AFP >400ng / ml.

[0091] 3. Positive liver biopsy.

[0092] Following the early assessment or prediction method of this embodiment, the serum DEPTORK106 lactation expression level was detected in 80 cases using the aforementioned WB detection kit, and assessment and prediction were performed based on the obtained protein bands. The results showed that 41 out of 46 liver cancer patients were assessed as positive, and 3 out of 34 healthy individuals were assessed as positive. This indicates that the diagnostic sensitivity of this invention for liver cancer is 89.1%, and the specificity is 91.2%.

[0093] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of reagents for detecting the modified form of DEPTOR, DEPTOR K106 lactated biomarker, in the preparation of reagents for early preliminary assessment or prediction of liver cancer.

2. The application as described in claim 1, characterized in that: The aforementioned preliminary assessment or prediction reagent for early liver cancer is used to detect the expression level of DEPTOR K106 lactation in samples from subjects.

3. The application as described in claim 2, characterized in that: The expression level of DEPTOR K106 lactation in samples from subjects was detected by ELISA or Western Blot.

4. The application as described in any one of claims 2 or 3, characterized in that: The detection of the expression level of DEPTOR K106 lactation in samples from the subject refers to the detection of the expression level of DEPTOR K106 lactation in peripheral venous blood, adjacent normal tissue and / or tumor tissue from the subject.

5. A kit for quantitative or semi-quantitative detection of proteins, containing an antibody that specifically binds to lactated DEPTOR K106.

6. The kit as described in claim 5, characterized in that: The aforementioned kit for quantitative or semi-quantitative protein detection is a kit that can detect proteins using enzyme-linked immunosorbent assay (ELISA) or Western blotting. The kit contains a first antibody and a second antibody. The first antibody is an antibody that specifically binds to lactated DEPTORK106, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

7. The kit according to any one of claims 5-6, characterized in that: The antibody that specifically binds to DEPTORK106 lactation is a monoclonal antibody against DEPTOR K106 lactation.

8. Application of DEPTOR K106 lactated antibody in the preparation of liver cancer diagnostic kit.

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