Application of biomarker based on WDHD1 function in diagnosis and treatment of hepatocellular carcinoma

Abstract

The invention provides an application of phosphorylated WDHD1S141 in the modification form of WDHD1 as a biomarker in preparation of a reagent for early diagnosis or prediction of liver cancer. The invention also provides a kit capable of quantitatively or semi-quantitatively detecting the protein, and the kit contains an antibody capable of specifically amplifying the phosphorylated WDHD1S141. According to the application disclosed by the invention, by detecting the expression level of phosphorylation of the WDHD1S141, the condition of a subject suffering from liver cancer 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. The invention also provides an application of WDHD1S141 phosphorylation in preparation of a medicine for treating liver cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of a biomarker based on the function of WDHD1 in the diagnosis and treatment of hepatocellular carcinoma. Background Art

[0002] Liver cancer is one of the most common malignancies worldwide, with the fastest-growing mortality rate over the past few 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 with clinical features of insidious onset, high invasiveness, 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 approaches have been applied to HCC in recent years, such as surgery, chemotherapy, radiotherapy, immunotherapy, natural chemicals, and nanotechnology; however, the prognosis of HCC patients remains poor, with a 5-year overall survival rate of less than 20%. Therefore, it is crucial to reveal the molecular mechanisms of HCC progression and find new therapeutic targets to improve prognosis.

[0003] WDHD1 is a protein that fuses WD repeats and HMG-box DNA binding domains, and is characterized by multiple N-terminal WD40 domains and a C-terminal HMG box. WD40 domains are ubiquitous in many eukaryotic proteins and may serve as adapters or regulatory modules for 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, and plays a vital role in cell proliferation, embryonic development, and sister chromatid cohesion. In addition, WDHD1 dysregulation has been found to be associated with the development of multiple types of cancer.

[0004] In the prior art, studies have reported that WDHD1 has potential as a marker in the diagnosis of HCC and the prediction of patient prognosis, for example, patent documents CN103194532A, CN114854834A and scientific research papers Rong-Quan He et al, Prognosis prediction ability and prospective biological mechanisms of WDHD1in hepatocellμlar carcinoma tissues, Electronic Journal of Biotechnology, 55:78-90. However, the mechanism of the influence of unmodified WDHD1 protein on cancer development in such methods is not deep enough, so the sensitivity and specificity of these existing methods for the diagnosis of liver cancer still need to be improved.

[0005] In view of this, in order to better warn the occurrence of liver cancer, it is necessary to find the key markers of modified WDHD1 and use them for early diagnosis and treatment of HCC. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, one of the purposes of the present invention is to provide a product for diagnosing early liver cancer so that patients can be treated at an early stage, thereby improving the survival rate and quality of life.

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

[0008] The third object of the present invention is to provide a method for screening substances to be screened for preventing or treating liver cancer.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention uses the modified form of WDHD1, WDHD1 S141 phosphorylation, as a biomarker for preparing an early diagnosis or prediction reagent for liver cancer.

[0011] In the scheme of 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 a gene having a sequence number of NC_0000014.9 in the international public nucleic acid sequence database GeneBank.

[0012] In the scheme described in the present invention, by using an immunoassay reagent for WDHD1 S141 phosphorylation to detect the expression level of WDHD1 S141 phosphorylation in a sample from a subject, a preliminary assessment and prediction of liver cancer in the subject can be effectively performed.

[0013] In a preferred embodiment of the present invention, by using immunoassay reagents for WDHD1 and WDHD1 S141 phosphorylation to detect the expression level of WDHD1 and the expression level of WDHD1 S141 phosphorylation in samples from subjects, the subject's liver cancer condition can be more effectively preliminarily evaluated and predicted.

[0014] Furthermore, the sample from the subject is a tumor tissue of the subject. In a more preferred embodiment, in order to improve the accuracy of diagnosis, the sample from the subject also includes adjacent cancer tissue.

[0015] 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 the Elisa or Western Blot method; the most preferred is an immunoassay reagent based on the Western Blot method.

[0016] In a second aspect, the present invention further provides an antibody, wherein the antibody is an antibody capable of specifically amplifying the phosphorylation of WDHD1S141.

[0017] 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.

[0018] In a preferred embodiment of the present invention, the kit capable of quantitatively or semi-quantitatively detecting proteins is a kit capable of detecting proteins by Western Blot method; the 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] In a fourth aspect, the present invention also provides use of an antibody capable of specifically amplifying the phosphorylation of WDHD1 S141 in the preparation of a liver cancer diagnosis kit.

[0020] 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.

[0021] 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.

[0022] In a fifth aspect, the present invention also provides the use of WDHD1 S141 phosphorylation in the preparation of drugs for treating liver cancer.

[0023] In the application, the S at site 141 of the WDHD1 protein is mutated to A to obtain the WDHD1 S141A polypeptide, which can inhibit the phosphorylation modification of WDHD1 at site 141 and reduce the endogenous WDHD1 S141 phosphorylation level in the body. Therefore, it is used as a WDHD1 S141 phosphorylation inhibitor for the preparation of liver cancer treatment drugs.

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

[0025] In a further preferred application, a mixture of the first inhibitor and the second inhibitor is selected as the main active ingredient, and is prepared together with other drugs and pharmaceutically acceptable carriers and / or excipients to prepare a liver cancer treatment drug; the second inhibitor is a substance that can inhibit the expression of WDHD1 or a substance involved in the upstream or downstream pathway of WDHD1; it can be selected from Bazedoxifene acetate (BZA) or (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol. In this way, the combination of the WDHD1 S141 phosphorylation inhibitor WDHD1 S141A polypeptide and the WDHD1 inhibitor can be achieved, and a more ideal therapeutic effect can be obtained.

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

[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 a substance involved in an upstream or downstream pathway of WDHD1; and can 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 the present invention is not limited to quantifying the gene expression of any specific variant of the marker gene of the present invention. In a specific embodiment, as a non-limiting example, the marker gene WDHD1 has the sequence shown in the WDHD1 gene (NC_0000014.9) in the current international public nucleic acid sequence database GeneBank.

[0029] In the present invention, pharmaceutically acceptable carriers cover a variety of ingredients, including but not limited to diluents, excipients (such as lactose, sodium chloride, glucose, urea, starch, water), fillers (such as starch, sucrose), binders (such as simple syrup, glucose solution, starch solution, cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone), wetting agents (such as glycerol), disintegrants (such as dry starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate), absorption accelerators (such as quaternary ammonium compounds, sodium lauryl sulfate), surfactants (such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol), wetting agents (such as glycerol, starch), adsorption carriers (such as starch, lactose, bentonite, silica gel, kaolin and bentonite) and lubricants (such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder).

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

[0031] The pharmaceutical composition of the present invention provides a variety of routes of administration, including but not limited to oral administration, parenteral administration (such as subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracorporeal, intralesional and intracranial injection or infusion), inhalation spray administration, topical administration, rectal administration, nasal administration, buccal administration and vaginal administration. In addition, administration through an implanted drug storage device is also a feasible option. Among these modes of administration, oral administration and injection administration are highly favored because of their convenience and effectiveness. In order to enhance the stability and efficacy of the pharmaceutical composition, the pharmaceutical composition may contain various commonly used non-toxic, pharmaceutically acceptable carriers, excipients or excipients. In certain specific cases, medicinal acids, bases or buffers may be used to adjust the pH value of the preparation to ensure the stability of the compound or its administration form. Regarding parenteral 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 selection of these modes of administration depends on the characteristics of the target tissue and the needs of the drug. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be administered to the receptor by any suitable route.

[0032] The pharmaceutical composition of the present invention can be administered orally in a variety of oral dosage forms, such as capsules, tablets, emulsions, aqueous suspensions, dispersants and solutions. For oral tablets, commonly used carriers include lactose and corn starch, and lubricants such as magnesium stearate are usually added to improve the smoothness of the tablets. If you choose to administer in capsule form, lactose and anhydrous corn starch are ideal diluents. When an aqueous suspension or emulsion is selected as an oral administration method, the active ingredient can be suspended or dissolved in an oil phase and mixed with an emulsifier and / or a suspending agent to ensure its stability and uniformity. Sweeteners, flavoring agents and / or coloring agents can also be added as needed to improve mouthfeel and patient acceptance. When necessary, the dosage unit preparation for oral administration can be microencapsulated, for example, by coating or embedding in materials such as polymers, waxes, etc., to achieve the effect of extending or maintaining release.

[0033] The pharmaceutical composition of the present invention may also be a pharmaceutical composition for topical administration, and may be formulated into an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil.

[0034] The pharmaceutical composition of the present invention should be administered in a pharmaceutically effective amount, where "pharmaceutically effective amount" refers to a dose sufficient to treat or prevent a 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, administration time, route of administration and excretion rate, duration of treatment, and coordination or simultaneous use with other drugs.

[0035] The pharmaceutical composition of the present invention can be used alone as an independent therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously. In addition, the administration method can be flexibly selected as a single or multiple administration. It is important to fully consider the above factors when determining the dosage to ensure that the maximum therapeutic effect is achieved with the minimum amount of side effects.

[0036] The term "treatment" as used herein means that it does not seek a complete cure for the disease, but focuses on slowing down (reducing) the progression of a specific pathological condition or disorder, or preventing its recurrence. When a patient receives an effective therapeutic dose of a drug, if successfully "treated", it will show a reduction or disappearance of observable and / or quantifiable signs and symptoms of a specific disease. For example, this includes a significant reduction in the number of cancer cells or the disappearance of cancer cells, a reduction in tumor size; inhibition of tumor metastasis (i.e., slowing down to a certain extent, preferably completely stopping); a significant slowing of tumor growth; a prolonged relief of one or more symptoms associated with a specific cancer; a reduction in disease morbidity and mortality, and an improvement in quality of life. The alleviation of these signs or symptoms of the disease can be perceived by the patient. The effect of the 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 a reduction of more than 50%, and more ideally more than 75%. In addition, even if the patient feels that the disease state remains stable, it is considered to be effective.

[0037] In the present invention, the term "sample" includes (but is not limited to) cells, tissues, organs, body fluids (blood, lymph, etc.), digestive fluid, sputum, alveolar bronchial washings, urine, feces, etc. Preferably, the sample is liver tissue and / or paracancerous tissue.

[0038] In a seventh aspect, the present invention also provides a method for screening substances to be screened for preventing or treating liver cancer using the phosphorylation level of WDHD1 S141 as a standard.

[0039] Furthermore, the step of screening the substance to be screened for preventing or treating liver cancer comprises: 1) using the substance to be screened to treat a system expressing or containing the WDHD1 gene; 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 by more than 20%, preferably by more than 50%, and more preferably by more than 80%), it indicates that the substance to be screened can prevent or treat liver cancer.

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

[0041] The present invention provides a new specific biomarker WDHD1 S141 phosphorylation, and uses it in the early evaluation or prediction and treatment of liver cancer. The present invention also provides an early diagnosis kit for liver cancer prepared using WDHD1 S141 phosphorylation, and a liver cancer treatment drug with an inhibitor of WDHD1 S141 phosphorylation as the main active ingredient, so that patients can be diagnosed and treated at an early stage, thereby improving survival rate and life treatment. The liver cancer treatment drug described in the present invention can achieve precise molecular treatment of liver cancer. In addition, the present invention also provides a method for screening substances to be screened for preventing or treating liver cancer, which plays an important role in the diagnosis, prognosis and prediction of liver cancer.

[0042] The WDHD1 S141 phosphorylation described in the present invention is a specific post-translational modification form of WDHD1, which is highly correlated with cancer cell growth and poor prognosis of hepatocellular carcinoma (HCC). Specifically, through comparative experimental studies on post-translational modified proteins at many sites of WDHD1, we found that the phosphorylation of the WDHD1 S141 site is significantly more correlated with the occurrence and development of liver cancer than the proteins modified at other sites. In samples of patients with liver cancer, not only the expression level of WDHD1 increases, but more importantly, the phosphorylation level of the WDHD1 S141 site is upregulated, suggesting that intervening in the phosphorylation of the WDHD1 gene may become a new approach for the treatment of liver cancer. For further verification, by reducing the levels of modification of multiple sites of WDHD1, including the phosphorylation of site 141, it was found that the reduction of the phosphorylation water of the WDHD1 S141 site can significantly reduce the proliferation, migration and invasion of liver cancer cells compared with the reduction of the modification levels of other sites. It shows that the phosphorylation level of WDHD1 S141 can be used as an important basis for early diagnosis and prognosis evaluation of liver cancer. This discovery provides new strategies and options for the treatment of diseases such as liver cancer.

[0043] The above-mentioned discovery of the present invention is specifically obtained through the following research:

[0044] 1. Western blot analysis revealed that the phosphorylation level of WDHD1 S141 increased in liver cancer cells

[0045] 1. Cell Culture

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

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

[0048] 2.1. Preparation of cell samples

[0049] 2.1.1 Cell sample collection: Remove the culture medium, add 1× PBS to wash three times, and transfer to a 1.5 ml 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 the samples according to the experimental requirements: add 1×PBS to the samples in the EP tube and wash them 3 times (to remove impurities in the samples). Add 50μl of lysis buffer containing protease / phosphatase inhibitors to each well of the cell sample (six-well plate). Place on ice for 20 minutes, shake and mix, and then place on ice for 10 minutes.

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

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

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

[0055] 2.1.7 Load the sample directly onto the gel or aliquot and store at -80℃ for long term storage.

[0056] 2.2 SDS-PAGE polyacrylamide gel electrophoresis

[0057] 2.2.1 Prepare separation gel (6 ml / gel).

[0058] 2.2.2 Inject the separation gel, taking care not to generate bubbles, leaving about 2 cm of space for the concentrated gel, cover the top layer with deionized water, and let it stand for about 30 minutes.

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

[0060] 2.2.4 Carefully and slowly inject the concentrated gel into the upper end of the separation gel, taking care to avoid generating bubbles.

[0061] 2.2.5 Insert the comb immediately, wait for the concentrated gel to solidify (there should be a clear dividing line between the gel and the comb, and the time for adding the separation gel should be no less than 2 hours), clean the wells with double distilled water to remove the gel fragments, and then use filter paper to absorb the excess water on 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 (repeated use should not exceed 3 times).

[0063] 2.2.7 Loading: Take 5μl prestained marker from 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 15-25μl. First, boil it in a heating block at 95℃ for 5-10 minutes, oscillate once during the period, and then quickly centrifuge it to load the sample and run the gel; finally, add an equal volume of 1× loading buffer to the well without load sample. 2.2.8 Electrophoresis: Start with a constant voltage of 60-80V. After running through the concentrated gel, increase the current to 100-120V. The time of electrophoresis is determined according to the size of the target protein and the position of the marker. Generally, the electrophoresis is stopped when the target protein runs to two-thirds of the separation gel.

[0064] 2.3. Membrane transfer

[0065] 2.3.1 Cut the gel according to the instructions of the marker and the position of the target band (make sure to mark the cutting angle of the gel). First, immerse the eluted gel in the transfer buffer for 15 minutes.

[0066] 2.3.2 After marking, PVDF membrane was completely immersed in methanol for at least 1 minute, and then immersed in transfer buffer together with 4 pieces of 3 mm filter paper and sponge 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 are no bubbles.

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

[0071] 2.4. Membrane blocking and antibody incubation

[0072] 2.4.1 After 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 5 ml of 5% milk powder blocking solution at room temperature for 2 hours or shake gently at 4°C overnight. Since milk powder is difficult to dissolve, it should be prepared at least 1 hour in advance.

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

[0075] 2.4.4 Add 5 ml of primary antibody dilution buffer (antibodies are diluted according to the instructions), gently shake at room temperature for 2 hours or at 4°C overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml primary antibody solution, and store at 4°C (uncommon antibodies can be stored at -20°C for a long time), which can be reused.

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

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

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

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

[0080] 2.5.1 Color development steps: First lay out plastic wrap, then lay out plastic wrap on absorbent paper; pour water, developer (cannot be used if the color becomes darker) and fixer into their respective EP tubes. Mix 2.5ml ECL-A and 2.5ml ECL-B, and protect from light. Take the ECL mixed solution, membrane, etc. into the darkroom. Close the door, turn off the light, and pull back the cloth. Pour the ECL mixed solution into a small box, dry the membrane slightly with absorbent paper, put it into the ECL mixed solution and shake it at room temperature for 5 minutes (so that the ECL evenly immerses the membrane). After drying with absorbent paper, place it on plastic wrap, wrap the membrane face down and place it on a clip, cut the X-film (note that you can only hold the edge of the X-film with your hands) and place it on the membrane. The side with the cut corner is the marker (note: when taking the film, adjust the light to the minimum and do not face the light, but face away from the light). Adjust the exposure time according to the brightness of the strip. Generally, expose for 30 seconds first, observe the depth of the strip, and then determine the best exposure time.

[0081] 2.5.2 Development and fixation: Take out the X-film and place it in the developer for a certain period of time (depending on the intensity of the target band and the background), wash it with water once and place it in the fixer for no less than 5 minutes. Finally, the band of the target protein on the PVDF membrane is obtained. By comparing it with the protein marker, the size and position of the target protein can be determined (the size of WDHD1 pS141 protein is 141KD), and the expression result of the target protein can be obtained.

[0082] 3. Results

[0083] Figure 1 The results shown show that the phosphorylation level of WDHD1 S141 in liver cancer cells is significantly increased compared with normal liver cells. The above results indicate that the phosphorylation level of WDHD1 S141 will increase in liver cancer cells.

[0084] 2. CCK8 detection revealed that increased phosphorylation of WDHD1 S141 can promote the growth of liver cancer cells

[0085] 1. Cell Culture

[0086] Human hepatoma cell line Huh-7 was cultured in DMEM 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 digested and passaged using 0.25% trypsin containing EDTA.

[0087] 2. CCK8 detection of cell proliferation

[0088] 1) Huh7 cells in the logarithmic proliferation phase were seeded in 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 replicate wells in each group;

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

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

[0091] 3. Statistical methods

[0092] The experiments were repeated three times and statistically analyzed using GraphPad Prism software. The differences between the two were tested using t-test and were considered statistically significant when P < 0.05.

[0093] 4. Results

[0094] Figure 2 The results shown in the figure showed that the cell growth rate of the WDHD1 S141A group was significantly lower than that of the WDHD1 WT group, WDHD1S72A group, and WDHD1 S113A group, and the difference was statistically significant (P<0.05). The above results indicate that increased phosphorylation level of WDHD1 S141 can promote the growth of liver cancer cells.

[0095] 3. Cell scratch assay revealed that increased phosphorylation level of WDHD1 S141 can promote liver cancer cell migration

[0096] 1. Cell Culture

[0097] Human hepatoma cell line Huh-7 was cultured in DMEM 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 digested and passaged using 0.25% trypsin containing EDTA.

[0098] 2. Scratch assay to detect cell migration

[0099] 2.1 Huh7 cells in the logarithmic growth phase were seeded in 6-well plates, with 1×10 6 cells; the experiment was divided into WDHD1WT group, WDHD1 S72A group, WDHD1 S113A group, and WDHD1 S141A group, with 3 replicate wells in each group;

[0100] 2.2 After Huh7 cells have grown all over the 6-well plate, use a 10-μl pipette tip to make scratches of uniform thickness, replace the culture medium with serum-free DMEM, and photograph the width of the scratches under a microscope.

[0101] 2.3 After 24 hours, take another picture of the scratch width under a microscope.

[0102] 3. Statistical methods

[0103] The experiments were repeated three times. ImageJ was used to analyze the width of the scratches, and GraphPadPrism software was used for statistical analysis. The difference between the two was analyzed using the t-test, and it was considered statistically significant when P < 0.05.

[0104] 4. Results

[0105] Figure 3 The results showed that the width of cell scratches in the WDHD1 WT group was significantly narrower than that in the WDHD1 S141A group, and the scratches in the WDHD1 S141A group were significantly wider than those in the WDHD1 S72A group and the WDHD1 S113A group. The width differences were statistically significant (P<0.05). Figure 4 The statistical results also show that phosphorylation of WDHD1 S141 site has the greatest effect on cell migration among different modification sites. The above results indicate that increased phosphorylation level of WDHD1 S141 can promote the migration of liver cancer cells.

[0106] 4. Transwell assay revealed that increased phosphorylation of WDHD1 S141 can promote the invasion of liver cancer cells

[0107] 1. Cell Culture

[0108] Human hepatoma cell line Huh-7 was cultured in DMEM 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 digested and passaged using 0.25% trypsin containing EDTA.

[0109] 2. Transwell assay to detect cell invasion

[0110] 2.1 Take an appropriate amount of cells and inoculate them in a 6-well plate. When the cells are in the logarithmic growth phase, perform cell transfection. 24 hours after transfection, collect the cells routinely, resuspend 10,000 cells in 100 μL serum-free culture medium, and inoculate them in the upper chamber of the Transwell;

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

[0112] 3. Statistical methods

[0113] The experiments were repeated three times. ImageJ was used to analyze the number of cells passing through the Transwell membrane, and GraphPad Prism software was used for statistical analysis. The difference between the two was tested using the t test, and it was considered statistically significant when P < 0.05.

[0114] 4. Results

[0115] Figure 5 , 6 The results shown in the figure showed that the number of cell crossings in the WDHD1 WT group was significantly greater than that in the WDHD1 S141A group, and the number of cell crossings in the WDHD1 S141A group was significantly less than that in the WDHD1 S72A group and the WDHD1 S113A group, and the difference was statistically significant (P<0.05). The above results indicate that increased phosphorylation levels of WDHD1 S141 can promote the invasion of liver cancer cells.

[0116] Compared with the prior art, the present invention has the following beneficial effects: based on the mechanism of hepatocellular carcinoma metabolism, the present invention has found a new biomarker for hepatocellular carcinoma, and early diagnosis and treatment of liver cancer can be achieved by detecting the expression level of the biomarker. The drug of the present invention is particularly suitable for reducing the endogenous WDHD1 S141 phosphorylation level, and by reducing the endogenous WDHD1 S141 phosphorylation level, liver cancer and other related diseases caused by increased endogenous WDHD1 S141 phosphorylation level can be treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 This is a diagram of the expression of WDHD1 pS141 in normal hepatocytes and various liver cancer cells detected by WB.

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

[0119] Figure 3 This figure shows the effect of different WDHD1 phosphorylation sites on the migration of Huh-7 liver cancer cells detected 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 detected by scratch assay.

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

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

[0123] Below, we will combine the accompanying drawings and examples to explain the present invention in more detail. Please note that the following examples are only intended to demonstrate the application of the present invention, rather than to limit its scope of protection. For the specific conditions of the experimental methods not explicitly mentioned in the examples, generally follow the standard operating procedures recognized in the industry, such as "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1989) compiled by Sambrook et al., or operate according to the recommended conditions provided by the reagent manufacturer.

[0124] Example 1

[0125] A WB detection kit containing the following reagents and items:

[0126] The primary antibody was WDHD1 S141 phosphorylation antibody;

[0127] secondary antibody (an antibody homologous to the primary antibody and labeled with horseradish peroxidase);

[0128] Substrate solution;

[0129] Blocking solution: milk powder blocking solution;

[0130] Wash buffer: TBS / T;

[0131] Transfer buffer;

[0132] PVDF membrane;

[0133] Electroporation fluid;

[0134] Membrane staining solution;

[0135] Sample buffer;

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

[0137] Instructions for use.

[0138] Example 2

[0139] The detection of WDHD1 S141 phosphorylation level using the kit described in Example 1 is used to diagnose liver cancer or determine the prognosis of liver cancer patients.

[0140] The following is the detailed operation process:

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

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

[0143] Western Blot, 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 the sample, and then transfers the separated proteins to nitrocellulose or PVDF membranes. Next, the membrane is incubated with specific antibodies against the target protein to bind the antibodies to the target protein. In the membrane washing step, unbound antibodies are removed, leaving only antibodies that are firmly bound to the target protein. Finally, these bound antibodies are detected by developing film or fluorescent scanning technology to form specific bands on the membrane. Due to the high specificity of antibodies, usually only one clear band can be observed, and its thickness reflects the content of the target protein. By analyzing the position and intensity of these bands, researchers can obtain the expression of the target protein in specific cells or tissue homogenates. Western Blot, with its high-resolution gel electrophoresis technology, strong specificity, and high-sensitivity immunoassay, can detect target proteins as low as 1ng, making it widely used in molecular biology, biochemistry, and immunogenetics. The specific detection steps are as follows:

[0144] 1. Collect samples:

[0145] The tumor tissues of the subjects were collected, placed on ice for 15 min, and centrifuged at 4°C, 3500 rpm for 15 min to obtain the tumor tissue precipitate for testing.

[0146] 2. Preparation of Tumor Tissue Samples

[0147] 2.1 Tumor tissue sample collection: Remove the culture medium, add 1× PBS to wash three times, and transfer to a 1.5 ml centrifuge tube.

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

[0149] 2.3 Prepare the samples according to the experimental requirements: add 1×PBS to the EP tube and wash the samples 3 times (to remove impurities in the samples), add 50μl of lysis buffer containing protease / phosphatase inhibitors to each well of the cell sample (six-well plate), place on ice for 20 minutes, shake and mix, and then place on ice for 10 minutes.

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

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

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

[0153] 2.7 Load the sample directly onto the gel or aliquot and store at -80℃ for long term storage.

[0154] 3. SDS-PAGE polyacrylamide gel electrophoresis

[0155] 3.1 Prepare separation gel (6 ml / gel).

[0156] 3.2 Inject the separation gel, be careful not to generate bubbles, leave about 2 cm of space for the concentrated gel, cover the top layer with deionized water, and let it stand for about 30 minutes.

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

[0158] 3.4 Carefully and slowly inject the concentrated gel into the upper end of the separation gel, taking care to avoid generating bubbles.

[0159] 3.5 Insert the comb immediately, wait for the concentrated gel to solidify (there is a clear dividing line between the gel and the comb, and the time for adding the separation gel should be no less than 2 hours), wash the holes with double distilled water to remove the gel fragments, and then use filter paper to absorb the excess water on 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 (repeated use should not exceed 3 times).

[0161] 3.7 Loading: Take 5μl prestained marker from 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 15-25μl. First, cook it in a heating block at 95℃ for 5-10 minutes, shake it once during the period, and then quickly centrifuge it to load the sample and run the gel; finally, add an equal volume of 1× loading buffer to the well without loading sample.

[0162] 3.8 Electrophoresis: Start with a constant voltage of 60-80V. After running through the concentrated 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, the electrophoresis is stopped when the target protein runs to two-thirds of the separation gel.

[0163] 4. Membrane Transfer

[0164] 4.1 Cut the gel according to the instructions of the marker and the position of the target band (make sure to mark the cutting angle of the gel). First, immerse the eluted gel in the transfer buffer for 15 minutes.

[0165] 4.2 After marking, PVDF membrane was completely immersed in methanol for at least 1 minute, and then immersed in transfer buffer together with 4 pieces of 3 mm filter paper and sponge 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 are no bubbles.

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

[0170] 5. Membrane Blocking and Antibody Incubation

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

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

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

[0174] 5.4 Add 5 ml of primary antibody dilution buffer (antibodies are diluted according to the instructions), gently shake at room temperature for 2 hours or at 4°C overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml primary antibody solution, and store at 4°C (uncommon antibodies can be stored at -20°C for a long time), which can be reused.

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

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

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

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

[0179] 6.1 Color development steps: First lay out plastic wrap, then lay out plastic wrap on absorbent paper; pour water, developer (cannot be used if the color becomes darker) and fixer into their respective EP tubes. Mix 2.5ml ECL-A and 2.5ml ECL-B, and protect from light. Take the ECL mixed solution, membrane, etc. into the darkroom. Close the door, turn off the light, and pull back the cloth. Pour the ECL mixed solution into a small box, dry the membrane slightly with absorbent paper, put it into the ECL mixed solution and shake it at room temperature for 5 minutes (so that the ECL evenly immerses the membrane). After drying with absorbent paper, place it on plastic wrap, wrap the membrane face down and place it on a clip, cut the X-film (note that you can only hold the edge of the X-film with your hands) and place it on the membrane. The side with the cut corner is the marker (note: when taking the film, adjust the light to the minimum and do not face the light, but face away from the light). Adjust the exposure time according to the brightness of the strip. Generally, expose for 30 seconds first, observe the depth of the strip, and then determine the best exposure time.

[0180] 5.2 Development and fixation: Take out the X-film and place it in the developer for a certain period of time (depending on the intensity of the target band and the background), wash it with water once and place it in the fixer for no less than 5 minutes. Finally, the band of the target protein on the PVDF membrane is obtained. By comparing it with the protein marker, the size and position of the target protein can be determined (the size of WDHD1 pS141 protein is 141KD), and the expression result of the target protein can be obtained.

[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 indicates that the subject may have invasive cancer, is in the progressive stage of invasive cancer, or has 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 excipient.

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

[0186] The carrier and auxiliary materials include lactose, corn starch, magnesium stearate and sweetener.

[0187] Example 4. Combination drug for treating liver cancer

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

[0189] The WDHD1 S141 phosphorylation inhibitor is a WDHD1S141A polypeptide obtained by mutating the S at site 141 of the WDHD1 protein to A, and is a substance capable of reducing the endogenous WDHD1 S141 phosphorylation level 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 can be selected from Bazedoxifene acetate (BZA) or (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol.

[0191] The carrier and auxiliary materials include gelatin, lactose, anhydrous corn starch and sweetener.

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

2. The use according to claim 1, characterized in that: The reagent for early diagnosis or prediction of liver cancer is a reagent for detecting the expression level of WDHD1 S141 phosphorylation in a sample from a subject.

3. The use according to claim 2, characterized in that: The expression level of WDHD1 S141 phosphorylation in samples from subjects was detected based on the Western Blot method.

4. A kit for quantitative or semi-quantitative detection of proteins, comprising an antibody that can specifically amplify the phosphorylation of WDHD1 S141.

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

6. Application of WDHD1 S141 phosphorylation in the preparation of drugs for treating liver cancer, characterized in that: A first inhibitor is selected as the main active ingredient and is prepared together with other drugs and pharmaceutically acceptable carriers and / or excipients to prepare a liver cancer therapeutic drug; the first inhibitor is a WDHD1 S141A polypeptide obtained by mutating the S at site 141 of the WDHD1 protein to A.

7. The use according to claim 6, characterized in that: The main active ingredient also includes a second inhibitor; the second inhibitor is selected from Bazedoxifene acetate (BZA) or (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol or a mixture of two thereof; preferably BZA.

8. A drug for treating liver cancer, comprising a WDHD1 S141 phosphorylation inhibitor and a pharmaceutically acceptable carrier and / or excipient; the WDHD1 S141 phosphorylation inhibitor is a WDHD1S141A polypeptide obtained by mutating the S at site 141 of the WDHD1 protein to A.

9. The drug according to claim 8, characterized in that: Further comprising a WDHD1 inhibitor; the WDHD1 inhibitor is Bazedoxifene acetate (BZA) or (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol.

10. A method for screening substances for preventing or treating liver cancer using WDHD1 S141 phosphorylation level as a standard, comprising: 1) Treating a system expressing or containing the WDHD1 gene with a substance to be screened; and 2) detecting the expression of 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 by more than 20%, preferably by more than 50%, and more preferably by more than 80%), it indicates that the substance to be screened can prevent or treat liver cancer; the system includes: a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system.

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