Application of biomarkers based on wdhd1 function in diagnosis and treatment of hepatocellular carcinoma

Abstract

The application provides application of a modified form of WDHD1, WDHD1 S141 phosphorylation, as a biomarker in preparation of a liver cancer early diagnosis or prognosis reagent. The application also provides a kit for quantitatively or semi-quantitatively detecting a protein, which contains an antibody capable of specifically amplifying the WDHD1 S141 phosphorylation. The application can more effectively preliminarily evaluate and predict the condition of a subject suffering from liver cancer by detecting the expression level of the WDHD1 S141 phosphorylation. The application will help improve the diagnosis accuracy of liver cancer and provide an important basis for early treatment of patients. The kit can be applied to the diagnosis of early, middle and late liver cancer. The application also provides application of the WDHD1 S141 phosphorylation in preparation of a liver cancer treatment drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of WDHD1-based biomarkers in the diagnosis and treatment of hepatocellular carcinoma. Background Technology

[0002] Liver cancer is one of the most common malignant tumors worldwide, and its mortality rate has been rising rapidly for decades. Hepatocellular carcinoma (HCC) is the most common pathological type of liver cancer, accounting for approximately 90% of cases. HCC is a highly heterogeneous disease, characterized by insidious onset, high invasiveness, and high recurrence and metastasis rates. Most patients are diagnosed at an advanced stage, when liver resection and transplantation are not feasible. Although many new and effective methods have been applied to HCC in recent years, such as surgery, chemotherapy, radiotherapy, immunotherapy, natural chemicals, and nanotechnology, the prognosis for HCC patients remains poor, with a 5-year overall survival rate of less than 20%. Therefore, elucidating the molecular mechanisms of HCC progression and identifying new therapeutic targets to improve prognosis is crucial.

[0003] WDHD1 is a protein that integrates a WD repeat sequence and an HMG-box DNA-binding domain, characterized by multiple N-terminal WD40 domains and a C-terminal HMG box. The WD40 domain is ubiquitous in many eukaryotic proteins and may serve as an adapter or regulatory module in signal transduction, pre-mRNA processing, and cytoskeleton assembly. Studies have shown that WDHD1 is considered a key contributor to DNA replication and DNA damage repair, playing a crucial role in cell proliferation, embryonic development, and sister chromatid adhesion. Furthermore, dysregulation of WDHD1 has been found to be associated with the development of multiple types of cancer.

[0004] In existing technologies, studies have reported the potential of WDHD1 as a biomarker in the diagnosis and prognosis prediction of HCC, for example, patent documents CN103194532A and CN114854834A and research paper Rong-Quan He et al, Prognosis prediction ability and prospective biological mechanisms of WDHD1 in hepatocell microcarcinoma tissues, Electronic Journal of Biotechnology, 55:78-90. However, the mechanism by which unmodified WDHD1 protein affects cancer development is not yet fully understood in these methods, therefore the sensitivity and specificity of these existing methods for diagnosing liver cancer still need to be improved.

[0005] Therefore, in order to better predict the occurrence of liver cancer, it is necessary to find key biomarkers of modified WDHD1 and use them for early diagnosis and treatment of HCC. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, one of the objectives of this invention is to provide a product for diagnosing early-stage liver cancer, enabling patients to receive treatment at an early stage, thereby improving survival rates and quality of life.

[0007] The second objective of this invention is to provide a treatment method and pharmaceutical composition to achieve precise molecular treatment of liver cancer.

[0008] A third objective of this invention is to provide a method for screening substances for the prevention or treatment of liver cancer.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In one aspect, the present invention uses the modified form of WDHD1, WDHD1 S141 phosphorylation, as a biomarker for the preparation of reagents for early diagnosis or prediction of liver cancer.

[0011] In the present invention, the modified form of WDHD1, WDHD1 S141 phosphorylation, refers to phosphorylation at position 141 of the amino acid sequence of WDHD1; WDHD1 refers to the gene with the sequence number NC_0000014.9 in the international public nucleic acid sequence database GeneBank.

[0012] In the scheme described in this invention, by using an immunoassay reagent targeting WDHD1 S141 phosphorylation to detect the expression level of WDHD1 S141 phosphorylation in samples from subjects, the condition of subjects with liver cancer can be effectively preliminarily assessed and predicted.

[0013] In a preferred embodiment of the present invention, by using an immunoassay reagent targeting WDHD1 and WDHD1 S141 phosphorylation to detect the expression levels of WDHD1 and WDHD1 S141 phosphorylation in samples from subjects, the condition of subjects with liver cancer can be preliminarily assessed and predicted more effectively.

[0014] Furthermore, the sample from the subject is the subject's tumor tissue. In a more preferred embodiment, to improve diagnostic accuracy, the subject's sample also includes adjacent normal tissue.

[0015] 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; most preferably, it is an immunoassay reagent based on Western Blot methods.

[0016] Secondly, the present invention also provides an antibody that is capable of specifically amplifying the phosphorylation of WDHD1S141.

[0017] 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.

[0018] 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 Western blotting; the reagent kit contains a first antibody and a second antibody; the first antibody is the antibody described in the second aspect of the present invention, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

[0019] Fourthly, the present invention also provides the use of an antibody capable of specifically amplifying phosphorylated WDHD1 S141 in the preparation of a liver cancer diagnostic kit.

[0020] 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.

[0021] 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.

[0022] Fifthly, the present invention also provides the application of WDHD1 S141 phosphorylation in the preparation of hepatocellular carcinoma therapeutic drugs.

[0023] In the aforementioned application, the S-mutation at site 141 of the WDHD1 protein is changed to A to obtain the WDHD1 S141A polypeptide. This polypeptide can inhibit the phosphorylation modification of WDHD1 at site 141, thereby reducing the endogenous phosphorylation level of WDHD1 S141 in the body. Therefore, it is used as a WDHD1 S141 phosphorylation inhibitor in the preparation of drugs for the treatment of liver cancer.

[0024] In a preferred application, a first inhibitor is selected as the main active ingredient and co-prepared with other drugs and pharmaceutically acceptable carriers and / or excipients to prepare a liver cancer treatment drug; the first inhibitor is the WDHD1 S141A polypeptide.

[0025] In a further preferred application, a mixture of the first and second inhibitors is selected as the main active ingredient, and co-prepared with other drugs and pharmaceutically acceptable carriers and / or excipients to prepare a liver cancer treatment drug; the second inhibitor is a substance capable of inhibiting the expression of WDHD1 or substances involved in the upstream or downstream pathways of WDHD1; it can be selected from Bazedoxifene acetate (BZA) or (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol. This allows for the combined use of the WDHD1 S141 phosphorylation inhibitor WDHD1 S141A peptide and a WDHD1 inhibitor, achieving a more ideal therapeutic effect.

[0026] In a sixth aspect, the present invention also provides a medicament for treating liver cancer, comprising a WDHD1 S141 phosphorylation inhibitor and a pharmaceutically acceptable carrier and / or excipient; wherein the WDHD1 S141 phosphorylation inhibitor is a substance capable of reducing the phosphorylation level of endogenous WDHD1 S141 in the body, preferably a WDHD1S141A polypeptide obtained by mutating S to A at site 141 of the WDHD1 protein.

[0027] The preferred drug for treating liver cancer of the present invention further comprises a WDHD1 inhibitor; the WDHD1 inhibitor is a substance capable of inhibiting the expression of WDHD1 or substances involved in the upstream or downstream pathway of WDHD1; and may be selected from Bazedoxifene acetate (BZA) or (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol.

[0028] Those skilled in the art will recognize that the utility of this invention is not limited to quantifying the gene expression of any particular variant of the biomarker gene of this invention. In a specific embodiment, as a non-limiting example, the biomarker gene WDHD1 has the sequence shown in the WDHD1 gene (NC_0000014.9) currently available in the international public nucleic acid sequence database GeneBank.

[0029] In this invention, pharmaceutically acceptable carriers encompass a variety of components, including but not limited to diluents, excipients (such as lactose, sodium chloride, glucose, urea, starch, and water), fillers (such as starch and sucrose), binders (such as simple syrups, glucose solutions, starch solutions, cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone), humectants (such as glycerin), disintegrants (such as dry starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, and sodium bicarbonate), absorption enhancers (such as quaternary ammonium compounds and sodium lauryl sulfate), surfactants (such as polyethylene glycol sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol), humectants (such as glycerin and starch), adsorbent carriers (such as starch, lactose, bentonite, silica gel, kaolin, and soap clay), and lubricants (such as talc, calcium and magnesium stearate, polyethylene glycol, and boric acid powder).

[0030] In this invention, the pharmaceutical composition can be prepared using different additives, such as buffers, stabilizers, antibacterial agents, isotonic agents, chelating agents, pH control agents, and surfactants.

[0031] The pharmaceutical compositions of the present invention provide for a variety of routes of administration, including but not limited to oral administration, non-gastrointestinal administration (such as subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intramuscular, intra-injury site, and intracranial injection or infusion), inhalation spray administration, topical administration, rectal administration, nasal administration, buccal administration, and vaginal administration. Furthermore, administration via an implanted drug reservoir is also a viable option. Among these routes of administration, oral and injectable administration are favored due to their convenience and effectiveness. To enhance the stability and efficacy of the pharmaceutical compositions, these compositions may contain a variety of commonly used, non-toxic, pharmaceutically acceptable carriers, excipients, or formulations. In certain specific cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to ensure the stability of the formulated compound or its administered form. Regarding non-gastrointestinal administration, the injection or infusion techniques covered by the present invention are very extensive, including but not limited to the various in vivo injection methods mentioned above. The choice of these routes of administration depends on the characteristics of the target tissue and the requirements of the drug. As long as the target tissue can be reached, the pharmaceutical compositions of the present invention can be delivered to the receptor via any suitable route.

[0032] The pharmaceutical compositions of the present invention can be administered orally in various dosage forms, such as capsules, tablets, emulsions, aqueous suspensions, dispersants, and solutions. For oral tablets, common carriers include lactose and corn starch, and lubricants such as magnesium stearate are typically added to improve tablet dispensing. If capsule administration is chosen, lactose and anhydrous corn starch are ideal diluents. When aqueous suspensions or emulsions are chosen as the oral administration route, the active ingredient can be suspended or dissolved in the oil phase and mixed with emulsifiers and / or suspending agents to ensure its stability and homogeneity. Sweeteners, flavoring agents, and / or coloring agents may also be added as needed to improve palatability and patient acceptability. When necessary, dosage units for oral administration can be microencapsulated, for example, by coating or embedding in polymers, waxes, or other substances, to achieve a prolonged or sustained release effect.

[0033] The pharmaceutical compositions of the present invention can also be topical pharmaceutical compositions, which can be formulated into ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0034] The pharmaceutical compositions of the present invention shall be administered in a pharmaceutically effective amount, which means a dose sufficient to treat or prevent disease at a reasonable benefit-risk ratio, based on factors including but not limited to the severity of the disease, the activity of the drug, the patient's age, weight, health status, gender, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, and combination or simultaneous use with other drugs.

[0035] The pharmaceutical compositions of the present invention can be used alone as a standalone therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously. Furthermore, the administration method can be flexibly chosen as a single or multiple dose. Importantly, when determining the dosage, all the above factors must be fully considered to ensure that the maximum therapeutic effect is achieved with minimal side effects.

[0036] The term "treatment" here refers not to a complete cure of the disease, but rather to slowing (reducing) the progression of a specific pathological condition or symptom, or preventing its recurrence. When a patient receives an effective therapeutic dose of medication, a successful "treatment" is characterized by an observable and / or quantifiable reduction or disappearance of specific disease signs and symptoms. This includes, for example, a significant reduction or disappearance of cancer cells, a reduction in tumor size; suppression of tumor metastasis (i.e., slowing it to some extent, ideally stopping it completely); a significant slowing of tumor growth; a prolonged period of relief for one or more symptoms associated with a specific cancer; a reduction in disease morbidity and mortality; and an improvement in quality of life. These reductions in disease signs or symptoms can be perceptible to the patient. The effectiveness of treatment can be manifested as a complete response—that is, the complete disappearance of all signs of cancer—or a partial response—a significant reduction in tumor size, preferably more than 50%, and more ideally more than 75%. Furthermore, treatment is considered effective even if the patient perceives their disease state as stable.

[0037] In this invention, the term "sample" includes (but is not limited to) cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, sputum, bronchoalveolar lavage fluid, urine, feces, etc. Preferably, the sample is liver tissue and / or adjacent cancer tissue.

[0038] In a seventh aspect, the present invention also provides a method for screening substances for the prevention or treatment of liver cancer based on the phosphorylation level of WDHD1 S141.

[0039] Furthermore, the step of screening for substances to prevent or treat liver cancer includes: 1) treating a system expressing or containing the WDHD1 gene with the substance to be screened; and 2) detecting the expression of the WDHD1 gene in the system; wherein, if the substance to be screened can reduce the endogenous WDHD1 S141 phosphorylation level (preferably significantly reduced, such as more than 20%, more preferably more than 50%, and even more preferably more than 80%), it indicates that the substance to be screened is a substance that can prevent or treat liver cancer.

[0040] Furthermore, the systems described above include (but are not limited to): cellular systems, subcellular systems, solution systems, tissue systems, organ systems, or animal systems.

[0041] This invention provides a novel specific biomarker, WDHD1 S141 phosphorylation, and its application in the early assessment, prediction, and treatment of liver cancer. This invention also provides an early diagnostic kit for liver cancer prepared using WDHD1 S141 phosphorylation, and a liver cancer therapeutic drug with an inhibitor of WDHD1 S141 phosphorylation as the main active ingredient, enabling patients to be diagnosed and treated at an early stage, thereby improving survival rates and quality of life. The liver cancer therapeutic drug described in this invention enables precise molecular therapy for liver cancer. Furthermore, this invention provides a method for screening substances for the prevention or treatment of liver cancer, which plays an important role in the diagnosis, prognosis, and prediction of liver cancer.

[0042] Phosphorylation of WDHD1 S141, as a specific post-translational modification of WDHD1, is highly correlated with cancer cell growth and poor prognosis in hepatocellular carcinoma (HCC). Specifically, through comparative experiments targeting post-translational modifications of proteins at various sites on WDHD1, we found that phosphorylation at WDHD1 S141 was significantly more correlated with the occurrence and development of HCC than proteins modified at other sites. In samples from HCC patients, not only was the expression level of WDHD1 increased, but more importantly, the phosphorylation level of WDHD1 S141 was upregulated, suggesting that intervening in WDHD1 gene phosphorylation may be a new approach to HCC treatment. To further verify this, by reducing the levels of multiple site modifications of WDHD1, including phosphorylation at site 141, we found that compared with the reduction of other site modification levels, the reduction of phosphorylation at WDHD1 S141 significantly reduced the proliferation, migration, and invasion capabilities of HCC cells. This indicates that the phosphorylation level of WDHD1 S141 can serve as an important basis for early diagnosis and prognostic assessment of HCC. This finding provides new strategies and options for the treatment of diseases such as HCC.

[0043] The above-mentioned findings of this invention were specifically obtained through the following research:

[0044] I. Western blot analysis revealed increased phosphorylation levels of WDHD1 S141 in liver cancer cells.

[0045] 1. Cell Culture

[0046] Human hepatocellular carcinoma cell lines Huh-7, Bel-7405, LM3, JHH-7, and Hep-3B were cultured in DMEM medium containing 10% fetal bovine serum and 1% PBS at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2-3 days, and the cells were passaged using routine digestion with 0.25% EDTA-containing trypsin. Normal hepatocyte cell line THLE-2 was grown in BEGM (Biological Industries) basal medium containing 5 ng / ml EGF and 70 ng / ml phosphoethanolamine. The cells were then cultured in a humidified incubator at 37°C with 5% CO2, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The medium was changed every 2-3 days, and the cells were passaged using routine digestion with 0.25% EDTA-containing trypsin.

[0047] 2. Western blot was used to detect the expression of phosphorylated WDHD1 S141 in various cell lines.

[0048] 2.1. Preparing cell samples

[0049] 2.1.1 Cell sample collection: Remove the culture medium, wash 3 times with 1×PBS, and transfer to a 1.5ml centrifuge tube.

[0050] 2.1.2 Prepare an ice box and add ice; prepare cell lysis buffer: determine the required lysis buffer based on the sample volume: 50 μl / well (6-well plate) of cell sample; lysis buffer formula: 100 μl cell lysis buffer + 0.1 μl phosphatase inhibitor mixture + 1 μl protease inhibitor mixture.

[0051] 2.1.3 Prepare samples according to experimental requirements: Wash samples three times with 1×PBS in EP tubes (to remove impurities). Add 50 μl of lysis buffer containing protease / phosphatase inhibitors to each well of the cell sample (six-well plate). Incubate on ice for 20 min, vortex to mix, and then incubate on ice for another 10 min.

[0052] 2.1.4 After lysis, centrifuge at 12000g for 15 min at 4℃, and collect the supernatant into another 1.5ml tube.

[0053] 2.1.5 Take 2.5 μl of sample and dilute it with 22.5 μl of triple-distilled water for use in the BCA method to measure protein concentration.

[0054] 2.1.6 Add 5× Loading Buffer (2.5ml Buffer / 10ml protein) to the remaining samples and boil at 95℃ for 10 minutes, shaking once during the process.

[0055] 2.1.7 Directly load samples for gel electrophoresis or dispense into containers and store at -80℃ for long-term storage.

[0056] 2.2 SDS-PAGE polyacrylamide gel electrophoresis

[0057] 2.2.1 Preparation of separating gel (6 ml / gel).

[0058] 2.2.2 Pour in the separating gel, being careful not to generate air bubbles. Leave about 2 cm of space for the stacking gel. Cover the top with deionized water and let it stand for about 30 minutes.

[0059] 2.2.3 Prepare the stacking gel (2 ml / gel).

[0060] 2.2.4 Carefully and slowly pour the stacking gel into the top of the separating gel, taking care to avoid generating air bubbles.

[0061] 2.2.5 Immediately insert the comb and wait for the concentrated gel to solidify (there should be a clear boundary between the gel and the comb, and the time for separating the gel should be no less than 2 hours). Clean the pores with double-distilled water to remove gel fragments, and then use filter paper to absorb excess water from the gel.

[0062] 2.2.6 Place the prepared gel into the electrophoresis tank, and add 1× electrophoresis buffer to both the upper and lower tanks (the gel should not be reused more than 3 times).

[0063] 2.2.7 Sample Loading: Add 5 μl of prestained marker to the marker wells and an appropriate amount of 1× loading buffer to make the total volume consistent with the sample wells. The sample loading volume is generally 15-25 μl. First, heat the sample in a heating block at 95℃ for 5-10 minutes, shaking once during the process. Then, quickly centrifuge and load the sample onto the gel. Finally, add an equal volume of 1× loading buffer to the wells without sample loading. 2.2.8 Electrophoresis: Start with a constant voltage of 60-80V. After running the stacking gel, increase the current to 100-120V. The electrophoresis time depends on the size of the target protein and the position of the marker. Generally, stop electrophoresis when the target protein reaches two-thirds of the height of the separating gel.

[0064] 2.3. Membrane Transfer

[0065] 2.3.1 Cut the gel. Cut the gel according to the marker and the position of the target band (note to mark the cut corners of the gel). First, immerse the eluted gel in the transfer buffer for 15 minutes.

[0066] 2.3.2 After marking the PVDF membrane, immerse it completely in methanol for at least 1 minute, and then immerse it together with 4 sheets of 3mm filter paper and a sponge in transfer buffer for 15 minutes.

[0067] 2.3.3 Preparation of "Sandwich Cookies":

[0068] In the following order: fiber mat - filter paper - PVDF membrane - gel - filter paper - fiber mat;

[0069] Note: Each item needs to be aligned and there should be no air bubbles.

[0070] 2.3.4 Transfer: The transfer time is 300 mA for 70 minutes. One side of the PVDF membrane is connected to the positive terminal of the power supply (red), and the other side of the gel is connected to the negative terminal of the power supply (black).

[0071] 2.4. Membrane blocking and antibody incubation

[0072] 2.4.1 After the membrane transfer, wash the membrane three times with 10 ml of 1×TBS at room temperature for 10 minutes each time.

[0073] 2.4.2 Incubate 5ml of 5% milk powder sealing solution at room temperature for 2 hours or at 4°C with gentle shaking overnight. Since milk powder is relatively difficult to dissolve, it should be prepared at least 1 hour in advance.

[0074] 2.4.3 Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0075] 2.4.4 Add 5 ml of primary antibody dilution buffer (divide the antibody according to the instructions), incubate at room temperature for 2 h or at 4 °C with gentle shaking overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml of primary antibody solution, and store at 4 °C (less frequently used antibodies can be stored at -20 °C for a long time). It can be reused.

[0076] 2.4.5 Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0077] 2.4.6 Add the secondary antibody (usually diluted 1:2000) and gently shake at room temperature for 1 hour.

[0078] 2.4.7 Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0079] 2.5. Development and fixing (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibody)

[0080] 2.5.1 Developing Procedure: First, lay down plastic wrap, then place another layer of plastic wrap on top of absorbent paper. Pour water, developer (if the color darkens, it cannot be used), and fixer into their respective EP tubes. Mix 2.5ml of ECL-A and 2.5ml of ECL-B, and store away from light. Take the ECL mixture, membrane, etc., into the darkroom. Close the door, turn off the lights, and pull the cloth back. Pour the ECL mixture into a small box. Slightly pat the membrane dry with absorbent paper and place it in the ECL mixture at room temperature, shaking for 5 minutes (to ensure the ECL evenly submerges the membrane). After patting dry with absorbent paper, place it on plastic wrap, wrapping it with the membrane side down and placing it on a clip. Cut the X-film (note that you can only hold the edge of the X-film) and place it on the membrane. The cut corner is the marker. (Note: When handling the film, turn the light to the minimum and do not face the light; keep it away from the light.) Adjust the exposure time according to the brightness of the strip. Generally, you can expose for 30 seconds first, observe the depth of the strip, and then determine the optimal exposure time.

[0081] 2.5.2 Development and Fixing: After placing the X-film in the developing solution for a certain period of time (depending on the intensity of the target band and the background), wash it once with water and then fix it in the fixing solution for at least 5 minutes. Finally, the target protein band is obtained on the PVDF membrane. By comparing it with the protein marker, the size and location of the target protein can be determined (WDHD1 pS141 protein size 141KD), thus obtaining the result of the target protein expression.

[0082] 3. Results

[0083] Figure 1 The results show that phosphorylated WDHD1 S141 is significantly increased in liver cancer cells compared to normal liver cells, indicating that the phosphorylation level of WDHD1 S141 is increased in liver cancer cells.

[0084] II. CCK8 assay revealed that increased phosphorylation of WDHD1 S141 can promote the growth of liver cancer cells.

[0085] 1. Cell Culture

[0086] The human hepatocellular carcinoma cell line Huh-7 was cultured in DMEM medium containing 10% fetal bovine serum and 1% PBS in an incubator at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2-3 days, and the cells were routinely passaged using 0.25% trypsin containing EDTA.

[0087] 2. CCK8 assay for cell proliferation

[0088] 1) Log-proliferating Huh7 cells were seeded into 96-well plates, with 2 × 10³ cells per well; the experiment was divided into WDHD1WT group, WDHD1 S72A group, WDHD1 S113A group, and WDHD1 S141A group, with 3 replicates in each group.

[0089] 2) Add 10 μl / well of CCK8 reagent at 0 h, 24 h, 48 h, and 72 h after transfection, respectively;

[0090] 3) After 2 hours, use an ELISA reader to detect the absorbance of A450.

[0091] 3. Statistical methods

[0092] All experiments were performed in triplicate. Statistical analysis was conducted using GraphPad Prism software. Differences between the two experiments were analyzed using a t-test, and p < 0.05 was considered statistically significant.

[0093] 4. Results

[0094] Figure 2 The results showed that the cell growth rate of the WDHD1 S141A group was significantly lower than that of the WDHD1 WT group, WDHD1 S72A group, and WDHD1 S113A group, and the difference was statistically significant (P<0.05). The above results indicate that the increased phosphorylation level of WDHD1 S141 can promote the growth of liver cancer cells.

[0095] III. Cell scratch assays revealed that increased phosphorylation of WDHD1 S141 promoted the migration of liver cancer cells.

[0096] 1. Cell Culture

[0097] The human hepatocellular carcinoma cell line Huh-7 was cultured in DMEM medium containing 10% fetal bovine serum and 1% PBS in an incubator at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2-3 days, and the cells were routinely passaged using 0.25% trypsin containing EDTA.

[0098] 2. Scratch assay for cell migration

[0099] 2.1 Log-proliferating Huh7 cells were seeded into 6-well plates at a density of 1 × 10⁶ cells per well. 6 The experiment was divided into four groups: WDHD1WT, WDHD1 S72A, WDHD1 S113A, and WDHD1 S141A, with three replicates in each group.

[0100] 2.2 Once the Huh7 cells have filled the 6-well plate, use a 10 μL pipette tip to make a uniform scratch, and replace the culture medium with serum-free DMEM. Then, photograph the width of the scratch under a microscope.

[0101] 2.3 The width of the scratch was photographed again under a microscope 24 hours later.

[0102] 3. Statistical methods

[0103] All experiments were performed in triplicate. ImageJ was used to analyze the width of the scratches, and GraphPadPrism software was used for statistical analysis. The differences between the two were analyzed using a t-test, and P < 0.05 was considered statistically significant.

[0104] 4. Results

[0105] Figure 3 The results showed that the cell scratch width in the WDHD1 WT group was significantly narrower than that in the WDHD1 S141A group, and the scratch width in the WDHD1 S141A group was significantly wider than that in the WDHD1 S72A and WDHD1 S113A groups, with statistically significant differences (P<0.05). Furthermore, from... Figure 4 The statistical results also show that phosphorylation at the WDHD1 S141 site has the greatest impact on cell migration at different modification sites. These results indicate that increased phosphorylation levels of WDHD1 S141 can promote the migration of liver cancer cells.

[0106] IV. Transwell assays revealed that increased phosphorylation of WDHD1 S141 promoted the invasion of liver cancer cells.

[0107] 1. Cell Culture

[0108] The human hepatocellular carcinoma cell line Huh-7 was cultured in DMEM medium containing 10% fetal bovine serum and 1% PBS in an incubator at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2-3 days, and the cells were routinely passaged using 0.25% trypsin containing EDTA.

[0109] 2. Transwell assay for cell invasion

[0110] 2.1 Seed an appropriate amount of cells into a 6-well plate and transfect the cells when they are in the logarithmic growth phase. 24 h after transfection, collect the cells routinely, resuspend 10,000 cells in 100 μL of serum-free culture medium, and seed them into the upper chamber of a Transwell.

[0111] 2.2 Add 600 μL of cell culture medium containing 10% fetal bovine serum to the lower chamber. After 36 h, wipe the cells off the surface of the Transwell membrane with a cotton swab, fix with 4% paraformaldehyde for 10 min, stain with 0.1% crystal violet for 20 min, rinse with tap water, and take a picture.

[0112] 3. Statistical methods

[0113] All experiments were performed in triplicate. The number of cells that crossed the Transwell membrane was analyzed using ImageJ, and statistical analysis was performed using GraphPad Prism software. Differences between the two were analyzed using t-tests, and P < 0.05 was considered statistically significant.

[0114] 4. Results

[0115] Figure 5 , 6 The results showed that the number of cells crossing the WDHD1 WT group was significantly greater than that of the WDHD1 S141A group, and the number of cells crossing the WDHD1 S141A group was significantly less than that of the WDHD1 S72A and WDHD1 S113A groups, with statistically significant differences (P<0.05). These results indicate that increased phosphorylation of WDHD1 S141 can promote the invasion of liver cancer cells.

[0116] Compared with existing technologies, the beneficial effects of this invention are as follows: Based on the metabolic mechanism of hepatocellular carcinoma, this invention has identified novel biomarkers for hepatocellular carcinoma. Early diagnosis and treatment of liver cancer can be achieved by detecting the expression level of these biomarkers. The drug of this invention is particularly suitable for reducing endogenous WDHD1 S141 phosphorylation levels. By reducing endogenous WDHD1 S141 phosphorylation levels, it can treat liver cancer and related diseases caused by elevated endogenous WDHD1 S141 phosphorylation levels. Attached Figure Description

[0117] Figure 1 This image shows the expression of WDHD1 pS141 in normal hepatocytes and various liver cancer cells detected by Western blotting.

[0118] Figure 2 This is a graph showing the effect of different WDHD1 phosphorylation sites on the proliferation of Huh-7 liver cancer cells using CCK8 assay.

[0119] Figure 3 This is a diagram showing the effect of different WDHD1 phosphorylation sites on the migration of Huh-7 liver cancer cells using a scratch assay.

[0120] Figure 4This is a statistical graph showing the effect of different WDHD1 phosphorylation sites on the migration of Huh-7 liver cancer cells using a scratch assay.

[0121] Figure 5 This figure shows the effect of different WDHD1 phosphorylation sites on the invasion of Huh-7 liver cancer cells using a transwell assay.

[0122] Figure 6 This is a statistical graph showing the effect of different WDHD1 phosphorylation sites on the invasion of Huh-7 liver cancer cells using a transwell assay. Detailed Implementation

[0123] Below, we will explain the present invention in more detail with reference to the accompanying drawings and embodiments. Please note that the following embodiments are intended only to illustrate the application of the present invention and not to limit its scope of protection. For specific conditions of experimental methods not explicitly mentioned in the embodiments, generally accepted standard operating procedures in the industry should be followed, such as referring to *Molecular Cloning: A Laboratory Manual* edited by Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989), or operating according to the recommended conditions provided by reagent manufacturers.

[0124] Example 1

[0125] A Western blot (WB) test kit containing the following reagents and items:

[0126] The primary antibody is a phosphorylated antibody against WDHD1 S141;

[0127] Secondary antibody (antibody homologous to primary antibody and labeled with horseradish peroxidase);

[0128] Substrate solution;

[0129] Sealing solution: Milk powder sealing solution;

[0130] Washing buffer: TBS / T;

[0131] Transfer buffer;

[0132] PVDF membrane;

[0133] Electro-hydraulic transfer;

[0134] Membrane staining solution;

[0135] Sample buffer solution;

[0136] Standard protein or marker protein: used to estimate the molecular weight of the target protein;

[0137] Instruction manual.

[0138] Example 2

[0139] The kit described in Example 1 was used to detect the phosphorylation level of WDHD1 S141 for the diagnosis of liver cancer or to determine the prognosis of liver cancer patients.

[0140] The following are the detailed operating procedures:

[0141] The detection method of the kit is Western blotting.

[0142] Western blotting was used to detect the phosphorylation level of S141 in the tumor tissue of the subjects.

[0143] Western blotting, or protein immunoblotting, is a widely used protein separation and identification technique in research. This technique first uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to efficiently separate proteins in a sample, then transfers the separated proteins onto a nitrocellulose or PVDF membrane. Next, the membrane is incubated with a specific antibody against the target protein, allowing the antibody to bind to the target protein. During the washing step, unbound antibodies are removed, leaving only those firmly bound to the target protein. Finally, these bound antibodies are detected using developing film or fluorescence scanning techniques, resulting in specific bands on the membrane. Due to the high specificity of the antibodies, usually only one clear band is observed, its thickness reflecting the concentration of the target protein. By analyzing the position and intensity of these bands, researchers can obtain information on the expression of the target protein in specific cell or tissue homogenates. Western blotting, with its high-resolution gel electrophoresis and highly specific and sensitive immunoassay, can detect target proteins as low as 1 ng, making it widely used in molecular biology, biochemistry, and immunogenetics. The specific detection steps are as follows:

[0144] 1. Sample collection:

[0145] Tumor tissue was collected from the subjects, placed on ice for 15 minutes, and centrifuged at 3500 rpm for 15 minutes at 4°C to obtain the precipitate of tumor tissue for testing.

[0146] 2. Prepare tumor tissue samples

[0147] 2.1 Collection of tumor tissue samples: Remove the culture medium, wash 3 times with 1×PBS, and transfer to a 1.5ml centrifuge tube.

[0148] 2.2 Prepare an ice box and add ice; prepare cell lysis buffer: determine the required lysis buffer based on the sample volume: 50 μl / well (6-well plate) of cell sample; lysis buffer formula: 100 μl cell lysis buffer + 0.1 μl phosphatase inhibitor mixture + 1 μl protease inhibitor mixture.

[0149] 2.3 Prepare samples according to experimental requirements: Wash samples three times with 1×PBS in EP tubes (to remove impurities). Add 50 μl of lysis buffer containing protease / phosphatase inhibitors to each well of the cell sample (six-well plate). Incubate on ice for 20 min for lysis, vortex to mix, and then incubate on ice for another 10 min.

[0150] 2.4 After lysis, centrifuge at 12000g for 15 min at 4℃, and collect the supernatant into another 1.5ml tube.

[0151] 2.5 Take 2.5 μl of sample and dilute it with 22.5 μl of triple-distilled water for use in BCA method to measure protein concentration.

[0152] 2.6 Add 5× Loading Buffer (2.5ml Buffer / 10ml protein) to the remaining samples and boil at 95℃ for 10 minutes, shaking once during the process.

[0153] 2.7 Direct sample loading and gel running or aliquoting and long-term storage at -80℃.

[0154] 3. SDS-PAGE polyacrylamide gel electrophoresis

[0155] 3.1 Preparation of separating gel (6 ml / gel).

[0156] 3.2 Pour in the separating gel, being careful not to generate air bubbles. Leave about 2 cm of space for the stacking gel. Cover the top with deionized water and let it stand for about 30 minutes.

[0157] 3.3 Prepare the stacking gel (2 ml / gel).

[0158] 3.4 Carefully and slowly pour the stacking gel onto the top of the separating gel, taking care to avoid generating air bubbles.

[0159] 3.5 Immediately insert the comb and wait for the concentrated gel to solidify (there should be a clear boundary between the gel and the comb, and the time for separating the gel should be no less than 2 hours). Clean the holes with double-distilled water to remove gel fragments, and then use filter paper to absorb excess water from the gel.

[0160] 3.6 Place the prepared gel into the electrophoresis tank, and add 1× electrophoresis buffer to both the upper and lower tanks (the gel should not be reused more than 3 times).

[0161] 3.7 Sample loading: Take 5 μl of prestained marker into the marker well and add an appropriate amount of 1× loading buffer to make the total volume consistent with the sample well. The sample loading volume is generally 15-25 μl. First, heat it in a heating block at 95℃ for 5-10 minutes, shaking once during the process, and then quickly centrifuge to load the sample and run the gel. Finally, add an equal volume of 1× loading buffer to the wells without sample loading.

[0162] 3.8 Electrophoresis: Start with a constant voltage of 60-80V. After running through the stacking gel, increase the current to 100-120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, stop electrophoresis when the target protein has run to two-thirds of the separating gel.

[0163] 4. Membrane transfer

[0164] 4.1 Cut the gel according to the marker's instructions and the position of the target band (note to mark the cut corners of the gel). First, immerse the eluted gel in the transfer buffer for 15 minutes.

[0165] 4.2 After marking the PVDF membrane, immerse it completely in methanol for at least 1 minute, and then immerse it together with 4 sheets of 3mm filter paper and a sponge in transfer buffer for 15 minutes.

[0166] 4.3 Preparation of "Sandwich Cookies":

[0167] In the following order: fiber mat - filter paper - PVDF membrane - gel - filter paper - fiber mat;

[0168] Note: Each item needs to be aligned and there should be no air bubbles.

[0169] 4.4 Transfer: The transfer time is 300 mA for 70 minutes. One side of the PVDF membrane is connected to the positive terminal of the power supply (red), and the other side of the gel is connected to the negative terminal of the power supply (black).

[0170] 5. Membrane blocking and antibody incubation

[0171] 5.1 After the membrane transfer, wash the membrane three times with 10 ml of 1×TBS at room temperature for 10 minutes each time.

[0172] 5.2 Incubate 5ml of 5% milk powder blocking solution at room temperature for 2 hours or at 4°C with gentle shaking overnight. Since milk powder is relatively difficult to dissolve, it should be prepared at least 1 hour in advance.

[0173] 5.3 Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0174] 5.4 Add 5 ml of primary antibody dilution buffer (divide the antibody according to the instructions), incubate at room temperature for 2 h or at 4 °C with gentle shaking overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml of primary antibody solution, and store at 4 °C (less frequently used antibodies can be stored at -20 °C for a long time). It can be reused.

[0175] 5.5 Wash the membrane with 10ml of TBS / T solution 3 times, 5 minutes each time.

[0176] 5.6 Add the secondary antibody (usually diluted 1:2000) and gently shake at room temperature for 1 hour.

[0177] 5.7 Wash the membrane with 10ml of TBS / T solution 3 times, 5 minutes each time.

[0178] 6. Development and fixing (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibody)

[0179] 6.1 Developing Procedure: First, lay down plastic wrap, then place another layer of plastic wrap on top of absorbent paper. Pour water, developer (if the color darkens, do not use), and fixer into their respective EP tubes. Mix 2.5ml of ECL-A and 2.5ml of ECL-B, and store away from light. Take the ECL mixture, membrane, etc., into the darkroom. Close the door, turn off the lights, and pull the cloth back. Pour the ECL mixture into a small box. Slightly pat the membrane dry with absorbent paper and place it in the ECL mixture at room temperature, shaking for 5 minutes (to ensure the ECL evenly submerges the membrane). After patting dry with absorbent paper, place it on plastic wrap, wrapping it with the membrane side down and placing it on a clip. Cut the X-film (note that you can only hold the edge of the X-film) and place it on the membrane. The cut corner is the marker. (Note: When handling the film, turn the light to the minimum and do not face the light; keep it away from the light.) Adjust the exposure time according to the brightness of the strip. Generally, you can expose for 30 seconds first, observe the depth of the strip, and then determine the optimal exposure time.

[0180] 5.2 Development and Fixing: After placing the X-film in the developing solution for a certain period of time (depending on the intensity of the target band and the background), wash it once with water and then fix it in the fixing solution for at least 5 minutes. Finally, the target protein band is obtained on the PVDF membrane. By comparing it with the protein marker, the size and location of the target protein can be determined (WDHD1 pS141 protein size 141KD), thus obtaining the result of the target protein expression.

[0181] 7. Results

[0182] Diagnostic analysis of test results: If the concentration of the protein band corresponding to the size of WDHD1 pS141 appears on the PVDF membrane, it suggests that the subject may have invasive cancer, be in the advanced stage of invasive cancer, or have a poor prognosis.

[0183] Example 3. Drugs for treating liver cancer

[0184] An oral tablet for treating liver cancer, comprising a WDHD1 S141 phosphorylation inhibitor and a pharmaceutically acceptable carrier and / or excipients.

[0185] The WDHD1 S141 phosphorylation inhibitor is a WDHD1S141A polypeptide obtained by mutating the S at site 141 of the WDHD1 protein to A, which can reduce the phosphorylation level of endogenous WDHD1 S141 in the body.

[0186] The carrier and excipients include lactose, corn starch, magnesium stearate, and sweeteners.

[0187] Example 4. Combination therapy for liver cancer

[0188] An oral capsule for treating liver cancer, comprising a WDHD1 S141 phosphorylation inhibitor, a WDHD1 inhibitor, and a pharmaceutically acceptable carrier and / or excipients.

[0189] The WDHD1 S141 phosphorylation inhibitor is a WDHD1S141A polypeptide obtained by mutating the S at site 141 of the WDHD1 protein to A, which can reduce the phosphorylation level of endogenous WDHD1 S141 in the body.

[0190] The WDHD1 inhibitor is a substance capable of inhibiting the expression of WDHD1 or substances involved in the upstream or downstream pathways of WDHD1; it may be selected from Bazedoxifene acetate (BZA) or (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol.

[0191] The carrier and excipients include gelatin, lactose, anhydrous corn starch, and sweeteners.

[0192] 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 WDHD1, WDHD1 S141 phosphorylation biomarker, in the preparation of reagents for early diagnosis or prediction of liver cancer.

2. The application as described in claim 1, characterized in that: The aforementioned early diagnosis or prediction reagent for liver cancer is used to detect the expression level of WDHD1 S141 phosphorylation in samples from subjects.

3. The application as described in claim 2, characterized in that: The expression level of WDHD1 S141 phosphorylation in samples from subjects was detected using a Western blotting method.

4. A kit for quantitative or semi-quantitative detection of a protein, comprising an antibody that specifically amplifies phosphorylated WDHD1 S141.

5. The kit according to claim 4, characterized in that: The kit described herein is a kit for detecting proteins using the Western blotting method; the kit contains a first antibody and a second antibody; the first antibody is an antibody that specifically amplifies the phosphorylation of WDHD1 S141, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

6. A method for screening substances for the prevention or treatment of liver cancer based on WDHD1 S141 phosphorylation levels, comprising the following steps: 1) Treat systems expressing or containing the WDHD1 gene with the substance to be screened; 2) Detect the expression of the WDHD1 gene in the system; wherein, if the substance to be screened can reduce the endogenous WDHD1 S141 phosphorylation level by more than 20%, it indicates that the substance to be screened can prevent or treat liver cancer; the system includes: cell system, subcellular system, solution system, tissue system, organ system or animal system.

Images