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

Abstract

The invention provides an application of DEPTOR K106 lactic acid in a modified form of DEPTOR as a biomarker in preparation of a reagent for early preliminary evaluation or prediction of liver cancer. The invention further provides a kit capable of quantitatively or semi-quantitatively detecting the protein, and the kit contains the lactic acid antibody capable of being specifically combined with DEPTOR K106. According to the application disclosed by the invention, by detecting the expression level of DEPTOR K106 lactylation, the tumor suffering condition of a subject can be more effectively and preliminarily evaluated and predicted. The application of the invention is helpful for improving the diagnosis accuracy of liver cancer, and provides an important basis for early treatment of patients. The kit disclosed by the invention can be suitable for diagnosis of early stage, middle stage and late stage of liver cancer.

Description

Technical Field

[0001] The present invention relates to the application of biomarkers based on DEPTOR function in the diagnosis and treatment of liver cancer, and belongs to the field of tumor medicine. Background Art

[0002] Given the intricate mechanisms of tumor development, current research still has many deficiencies, which greatly limits our in-depth understanding of the nature of tumors and the development of new diagnostic and therapeutic methods. Metabolism is a key factor in tumor occurrence and evolution. During this process, tumor cells undergo a significant change in metabolic pattern, namely "metabolic reprogramming". As a hallmark feature of tumors, metabolic reprogramming meets the three most important needs of tumor cells: ensuring the energy supply required for sustained growth, synthesizing biological macromolecules such as nucleic acids, proteins and lipids to maintain their growth needs, and maintaining redox homeostasis. For example, in the field of tumor energy / sugar metabolism, there is a significant phenomenon, the so-called "Warburg effect". This describes the strong dependence of tumor cells on aerobic glycolysis, which is very different from the mitochondrial oxidative phosphorylation pathway preferred by normal cells. Although this method is less efficient in producing ATP (adenosine triphosphate), it is this unusual metabolic strategy that gives tumor cells a specific survival advantage. These advantages are crucial for the growth, metastasis and response to treatment pressure of tumor cells. Therefore, only when we reveal the metabolic mechanism of tumors more comprehensively and systematically can we effectively identify and target the biomarkers of tumor metabolism, thereby breaking through the current bottleneck in tumor diagnosis and treatment and bringing more effective treatment options to cancer patients.

[0003] At present, the diagnosis methods of tumors are divided into five levels: ① Clinical diagnosis: based on the patient's clinical symptoms, signs and imaging examination results, combined with the general development law of the disease, it is speculated. Although this method can provide a preliminary direction, the accuracy of the diagnosis is limited. ② Surgical diagnosis: It relies on the tumor visible to the naked eye during surgery or endoscopic examination. However, this diagnostic method lacks pathological support, so its reliability is relatively low. ③ Physical and chemical diagnosis: This method can be used when the patient's clinical manifestations are consistent with the characteristics of cancer and are supported by imaging examinations such as X-ray, B-ultrasound, CT, MRI, or biochemical indicators such as carcinoembryonic antigen and alpha-fetoprotein. It provides a more objective basis for diagnosis, but it also has certain limitations. ④ Cytopathological diagnosis: It is based on the examination results of various exfoliated cells or puncture cells. This method can more directly observe abnormal changes in cells, thereby providing more accurate diagnostic information. ⑤ Histopathological diagnosis, which is currently the most ideal diagnostic method. It obtains tissue samples through coarse needle puncture and makes pathological sections for detailed pathological analysis. This method can directly observe the microstructure of the diseased tissue, thereby providing the most accurate diagnostic results. Although the reliability of these five diagnostic methods increases in turn, they all face a common challenge: it is difficult to achieve early detection and early diagnosis of tumors. In most cases, these methods can only work when the tumor develops to a certain extent, or even when the patient has serious symptoms, which undoubtedly increases the difficulty of treatment and the patient's pain. In addition, some methods with better diagnostic effects, such as histopathological diagnosis, require puncture to obtain cells or tissue sections, which may cause certain damage and negative effects on the patient's mind and body. Therefore, we need to continuously explore new diagnostic technologies and methods to achieve early detection and early diagnosis of tumors, improve the success rate of treatment, and alleviate the pain of patients.

[0004] In tumor cells, mutations in metabolic enzyme genes or changes in expression levels can lead to significant changes in the content of intracellular metabolites. This "classical" metabolic reprogramming plays a key role in tumor evolution. However, in recent years, more and more evidence has shown that some metabolic enzymes and metabolic small 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 the occurrence and development of tumors.

[0005] In view of the above background, in order to more accurately grasp the proliferation of cancer cells, achieve early diagnosis of cancer, and more reasonably select treatment plans, it is necessary to find key markers 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 purpose to be solved by the present invention is to solve the problem of early preliminary evaluation or prediction of liver cancer by using new tumor markers.

[0007] Another object of the present invention is to provide a product for the early preliminary evaluation or prediction of the above-mentioned liver cancer.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention uses the lactic acid (Lactylated) form of DEPTOR, DEPTOR K106, as a biomarker for preparing an early preliminary evaluation or prediction reagent for liver cancer.

[0010] In the solution of the present invention, DEPTOR K106 lactylation refers to lactylation at position 106 of the amino acid sequence of DEPTOR; the NCBI sequence number of DEPTOR is: NP_001269941.1.

[0011] In the scheme described in the present invention, by using an immunoassay reagent for DEPTOR K106 lactylation to detect the expression level of DEPTOR K106 lactylation in a sample from a subject, a preliminary assessment and prediction of liver cancer in the subject can be effectively performed. Furthermore, the sample from the subject is any one of the subject's venous peripheral blood, paracancerous tissue or tumor tissue.

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

[0013] In a second aspect, the present invention also provides an antibody, wherein the antibody is an antibody that can specifically bind to the lactylated DEPTORK106.

[0014] In a third aspect, the present invention further provides a kit for quantitative or semi-quantitative detection of proteins, comprising the antibody described in the second aspect of the present invention.

[0015] In a preferred embodiment of the present invention, the kit for quantitative or semi-quantitative detection of proteins is a kit for detecting proteins by enzyme-linked immunosorbent assay (Elisa) or Western Blot; the kit contains a first antibody and a second antibody; the first antibody is the antibody DEPTORlaK106 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, the present invention also provides the use of an antibody that specifically binds to the lactic acid version of DEPTOR K106 in the preparation of a liver cancer diagnosis kit.

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

[0018] Among them, the instruction manual records how to use the test kit for testing, and how to use the test results to judge the development of liver cancer and choose treatment plans.

[0019] The present invention is committed to solving the technical challenges in the field of early diagnosis of liver cancer, and provides a new specific biomarker DEPTOR K106 lactylation, and uses it in the early evaluation or 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 the early diagnosis of liver cancer, but also, by detecting its expression level, it can more effectively conduct preliminary evaluation and prediction of the subject's tumor condition. The application of this technology will help improve the diagnostic accuracy of liver cancer and provide an important basis for the early treatment of patients. The detection kit prepared based on the biomarker of the present invention can be used for the diagnosis of early, middle and late stages of liver cancer.

[0020] The lactation of DEPTOR K106 described in the present invention, as a specific post-translational modification form of DEPTOR, is highly correlated with cancer cell growth and poor prognosis of hepatocellular carcinoma (HCC). Specifically, we found through experimental studies that CD8+T cells secrete interferon-γ (IFNγ), which can stabilize the protein level of transcription coactivator (TAZ) with PDZ binding sequence in hepatocellular carcinoma (HCC) cells. As the central transcriptional regulatory module of the Hippo pathway, a large number of literatures have revealed that TAZ plays a pivotal role in the progression of various cancers such as human liver cancer, lung cancer, breast cancer, skin cancer, pancreatic cancer and brain cancer. Generally speaking, the high activity of TAZ in the cell nucleus is closely related to the formation of tumors and is regarded as a key factor in tumor development. Under IFNγ stimulation, DEPTOR is lactylated at the K106 site, and this lactylation increases the binding of DEPTOR to TAZ, which increases the stability of TAZ protein and promotes liver cancer growth. In addition, TAZ levels in human HCC specimens were positively correlated with lactylation 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-enhanced TAZ stability, and highlight the potential of DEPTOR proteins for HCC diagnosis and treatment.

[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) at 0 h, 0.5 h, 1 h, 6 h, and 12 h, and then the cells were collected, the proteins were lysed, and the TAZ protein level was subsequently detected by WB.

[0024] 2. Huh7 liver cancer cells were stimulated with or without IFNγ (2 ng / mL) for 1 hour, and the binding of DEPTOR and TAZ was detected using CO-IP technology.

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

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

[0027] Experimental results:

[0028] Figure 1 These results suggest that after IFNγ stimulation, the expression of TAZ protein in Huh7 liver cancer cells gradually increased.

[0029] Figure 2 This suggests that IFNγ stimulation promotes the interaction between DEPTOR and TAZ.

[0030] Figure 3 These results suggest that IFNγ causes DEPTOR to lactylate at the K106 site, thereby promoting its interaction with TAZ.

[0031] Figure 4 This suggests that IFNγ stimulation can increase the stability of TAZ protein. However, when DEPTOR K106 cannot be lactylated, IFNγ cannot increase the stability of TAZ protein.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. Based on the metabolic mechanism of liver cancer, the present invention has found new biomarkers, which provides strong support for the early diagnosis of tumors;

[0034] 2. The test can be completed by collecting venous peripheral blood, without the need for complicated surgery or invasive operations, which greatly reduces the burden on the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It reflects the changes in the protein level of TAZ in Huh7 liver cancer cells under IFNγ stimulation.

[0036] Figure 2 Immunoprecipitation analysis showed that in Huh7 hepatoma cells, IFNγ stimulation promoted the interaction between DEPTOR and TAZ.

[0037] Figure 3 Immunoprecipitation analysis showed that under IFNγ stimulation, DEPTOR K106 increased its binding to TAZ after lactylation.

[0038] Figure 4 Shown are immunoblot analyses of TAZ protein levels in Huh7 hepatoma cell lines DEPTOR WT and K106R mutant treated with CHX (100 μg / mL) for the indicated times with or without IFNγ stimulation. DETAILED DESCRIPTION

[0039] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings. The experimental methods described in the following experiments are all conventional methods unless otherwise specified; if no specific technology or conditions are specified in the experiment, the technology or conditions described in the literature in the field or the product instructions are used; the reagents and materials described can be obtained from commercial channels unless otherwise specified.

[0040] The present invention provides the use of DEPTOR K106 lactylation as a biomarker in the early evaluation or prediction of liver cancer.

[0041] The present invention also provides the use of DEPTOR K106 lactylation in preparing a reagent for early evaluation or prediction of liver cancer.

[0042] The reagent includes a reagent for detecting the lactic acid expression level of DEPTOR K106 in a sample. The sample is venous peripheral blood, paracancerous tissue and / or tumor tissue.

[0043] The reagents include a first antibody and a second antibody. The first antibody is the lactic acidified antibody DEPTOR K106, DEPTOR laK106. The second antibody is homologous to the first antibody and is labeled with horseradish peroxidase.

[0044] The present invention also provides a liver cancer early diagnosis kit based on Elisa or Western Blot method, wherein the kit contains a reagent for detecting DEPTOR K106 lactation, wherein the reagents in each kit include a first antibody and a second antibody, wherein the first antibody is a 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, an adjuvant and a solvent. The instructions for use record how to use the kit for detection, and how to use the detection results to judge tumor development and select a treatment plan. The components of the kit may be packaged in the form of an aqueous medium.

[0045] The method for using the kit of the present invention to evaluate or predict early stage liver cancer in patients comprises the following steps:

[0046] 1) detecting the expression level of the biomarker in the sample;

[0047] When venous peripheral blood is used as a sample, the sample processing method includes: after collecting venous peripheral blood, it needs to be placed on ice for 15 minutes, and then centrifuged at 3500rpm for 15 minutes to obtain serum or plasma, which is then tested or stored at -80°C. Samples that cannot be tested immediately need to be stored at -80°C and thawed on ice during testing.

[0048] Using Elisa or Western Blot to detect the lactic acid expression level of DEPTOR K106 in the treated samples;

[0049] 2) Performing early assessment or prediction of liver cancer in a subject based on the lactic acid expression level of DEPTOR K106 in the sample detected in 1).

[0050] The Western Blot method mentioned in the evaluation or prediction method of the present invention, i.e., protein immunoblotting experiment, is a technique commonly used to separate and identify proteins in research. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample, and then transfers the separated proteins to a nitrocellulose or PVDF membrane, and then incubates the membrane with a specific antibody to the target protein. During the membrane washing process, unbound antibodies are washed away, leaving only antibodies bound to the target protein, and finally the bound antibodies are detected by developing film or fluorescence scanning. Since the antibody is only bound to the target protein, generally only a clear band can be seen, and the thickness of the band corresponds to the amount of protein. By analyzing the position and intensity of a specific reaction, the expression information of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the strong specificity and high sensitivity of immunity, Western blot analysis can detect target proteins as low as 1ng. This method is widely used in molecular biology, biochemistry, immunogenetics and other molecular biology fields.

[0051] Based on the description of the above specific implementation methods, the present invention specifically lists the following embodiments to further illustrate the technical solution and technical effects of the present invention in detail.

[0052] Example 1.

[0053] An ELISA detection kit comprises a pre-coated microplate, a standard substance, an enzyme-labeled secondary antibody, a substrate solution, a stop solution, a washing buffer solution, a sample diluent, a control sample, an instruction manual and a sealing film; the wells of the microplate are pre-fixed with a capture antibody DEPTOR laK106; the enzyme-labeled secondary antibody is an antibody homologous to the capture antibody DEPTOR laK106 and labeled with horseradish peroxidase.

[0054] Example 2.

[0055] A WB detection kit containing the following reagents and items:

[0056] Primary antibody: DEPTOR laK106;

[0057] secondary antibody (antibody homologous to DEPTOR laK106 and labeled with horseradish peroxidase);

[0058] Substrate solution;

[0059] Blocking solution: milk powder blocking solution;

[0060] Wash buffer: TBS / T;

[0061] Transfer buffer;

[0062] PVDF membrane;

[0063] Electroporation fluid;

[0064] Membrane staining solution;

[0065] Sample buffer;

[0066] Standard protein or marker protein: used to estimate the molecular weight of the target protein;

[0067] Instructions for use.

[0068] Example 3.

[0069] A method for early assessment or prediction of liver cancer in a subject, comprising the following steps:

[0070] 1. The WB detection kit described in Example 2 was used to detect the lactic acid expression level of DEPTOR K106 in the venous peripheral serum of the subjects.

[0071] The specific detection steps are as follows:

[0072] 1. Sample Preparation

[0073] 1) Sample collection: Collect peripheral blood from the subject's vein, place it on ice for 15 min, and centrifuge it at 4°C, 3500 rpm for 15 min to obtain serum for testing.

[0074] 2) Make ice and prepare ice boxes;

[0075] 3) Prepare cell lysis solution: cell lysis solution containing protease inhibitors and phosphatase inhibitors;

[0076] Resuspend the cells with a pipette and lyse on ice for 30 minutes, vortexing every 10 minutes to ensure complete lysis.

[0077] 4) After lysis, centrifuge at 12000 rpm for 20 minutes at 4°C, and recover the supernatant into an EP tube as the protein sample after lysis.

[0078] 5) Determine the protein concentration of the sample according to the instructions of the BCA protein quantification kit. According to the protein quantification results, add appropriate amount of protein sample and 4′ Loading to a new EP tube and make up the volume with cell lysis buffer. Mix well and denature in a 70°C metal bath for 10 minutes.

[0079] 2. Protein gel electrophoresis:

[0080] Pull out the comb on the top of the precast gel horizontally, clean it with water and tear off the bottom seal. Fix the precast gel vertically in the electrophoresis tank, and pour an appropriate amount of electrophoresis solution into the tank to immerse the precast gel. Add the protein sample evenly into the wells of the precast gel, and make sure that the blank wells are filled with 1′ Loading. Perform electrophoresis at a constant voltage of 140V and end the electrophoresis when the bromophenol blue runs to the bottom of the gel.

[0081] 3. Transfer membrane:

[0082] Take out the precast gel from the electrophoresis tank, pry open the gel plate and take out the gel, soak the gel, NC membrane, filter paper and sponge pad in the pre-cooled transfer solution, and then start making a "sandwich", that is, from bottom to top, two layers of sponge pads, a layer of filter paper, NC membrane, gel after electrophoresis, a layer of filter paper, and two layers of sponge pads. Avoid bubbles during the production process. Put the finished "sandwich" horizontally into the transfer liner, and then insert the transfer liner horizontally into the transfer tank, and then pour the pre-cooled transfer solution into the inner side of the transfer liner, and add ice water mixture to the outside to reduce the heat generated during the transfer process.

[0083] 4. Closure:

[0084] After the transfer, the NC membrane was taken out and stained with Ponceau red to observe the transfer quality, and 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 red, the membrane was blocked with 5% skim milk on a shaker at room temperature for 45 minutes.

[0085] 5. Antibody incubation:

[0086] After blocking, wash the NC membrane twice with TBST, prepare the primary antibody with antibody diluent according to the instructions, put the cut bands into the prepared DEPTOR K106 lactic acid primary antibody diluent, and incubate overnight on a horizontal shaker at 4°C. After incubation, recover the primary antibody diluent and rinse the NC membrane three times with TBST, 5 minutes each time. Then add a certain proportion of the corresponding species of secondary antibody diluent and incubate on a shaker at room temperature for 2 hours.

[0087] 6. Exposure:

[0088] According to the instructions of the ultra-high sensitivity enhanced chemiluminescence (ECL) developer, solution A and solution B were prepared in a ratio of 1:1. The prepared developer was dropped onto the NC membrane incubated with the antibody, and after a certain period of light-proof reaction, it was exposed and developed in an exposure machine.

[0089] 2. Based on the protein expression results detected in step 1, early liver cancer assessment or prediction is performed on the patient.

[0090] Diagnostic analysis of test results:

[0091] If the following protein bands corresponding to the size appear on the NC membrane: DEPTOR K106 lactylation, it indicates that the subject may have invasive cancer, be in the advanced stage of invasive cancer, or have a poor prognosis. If the band is not present, it indicates that the subject has a good prognosis.

[0092] We collected 80 serum samples from Zhejiang University Second Hospital, including 46 patients with liver cancer and 34 healthy people; liver cancer diagnostic criteria:

[0093] 1. Two typical imaging manifestations of liver cancer (US, enhanced CT, MRI or selective hepatic artery angiography), with the lesion >2 cm.

[0094] 2. A typical imaging manifestation of liver cancer: lesion >2cm, AFP>400ng / ml.

[0095] 3. Liver biopsy is positive.

[0096] According to the early evaluation or prediction method of this embodiment, the lactic acid expression level of serum DEPTORK106 in 80 cases was detected with the above-mentioned WB detection kit, and then the evaluation and prediction were performed based on the obtained protein bands. The results showed that 41 of the 46 liver cancer patients were evaluated as positive, and 3 of the 34 healthy people were evaluated as positive. It was suggested that the diagnostic sensitivity of the present invention for liver cancer was 89.1% and the specificity was 91.2%.

[0097] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of DEPTOR K106 lactylation, a modified form of DEPTOR, as a biomarker in the preparation of early preliminary evaluation or prediction reagents for liver cancer.

2. The use according to claim 1, characterized in that: The early preliminary evaluation or prediction reagent for liver cancer is used to detect the expression level of DEPTOR K106 lactylation in a sample from a subject.

3. The use according to claim 2, characterized in that: Detection of the expression level of DEPTOR K106 lactylation in samples from subjects is achieved by Elisa or Western Blot method.

4. The use according to any one of claims 2 or 3, characterized in that: The detection of the expression level of DEPTOR K106 lactate in the sample from the subject is to detect the expression level of DEPTOR K106 lactate in the venous peripheral blood, paracancerous tissue and / or tumor tissue from the subject.

5. A kit for quantitative or semi-quantitative detection of proteins, comprising an antibody that can specifically bind to lactylated DEPTOR K106.

6. The kit according to claim 5, characterized in that: The kit for quantitative or semi-quantitative detection of proteins is a kit for detecting proteins by enzyme-linked immunosorbent assay (Elisa) or Western Blot; the kit contains a first antibody and a second antibody; the first antibody is an antibody that can specifically bind to DEPTORK106 lactate, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

7. The kit according to any one of claims 5 to 6, characterized in that: The antibody that can specifically bind to DEPTOR K106 lactate is a monoclonal antibody against DEPTOR K106 lactate.

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

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