Use of eppin protein in the preparation of a medicament for treating liver cancer

Abstract

The application discloses application of Eppin protein in preparation of a liver cancer treatment drug and belongs to the technical field of biological medicines.The amino acid sequence of the Eppin protein is shown as SEQ ID No.1, and the Eppin protein is a Kunitz type serine protease inhibitor which is identified from pearl powder for the first time, has excellent anti-liver cancer potential, can produce significant inhibitory effect on the proliferation, cycle process, migration and invasion of liver cancer cells and the like and promote apoptosis of the liver cancer cells, and can be used for preparing a liver cancer treatment drug and promoting development of the medical industry.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of Eppin protein in the preparation of drugs for the treatment of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high incidence and mortality rates. Its development typically involves a long process of persistent inflammatory damage, necrosis, and fibrotic deposition, and the exploration of treatment strategies remains a major challenge in the field of tumor research. Current treatment methods for HCC include surgical resection, liver transplantation, local ablation, transarterial chemoembolization, targeted therapy, and immunotherapy. In recent decades, despite continuous progress in these treatment methods, problems such as low early detection rates, high recurrence rates, difficult prognosis, and a lack of therapeutic targets still exist. On the one hand, radical treatments such as surgical resection and liver transplantation are only suitable for early-stage patients, and the postoperative recurrence rate is high; on the other hand, advanced-stage patients are generally insensitive to traditional radiotherapy and chemotherapy, and although systemic therapies represented by molecularly targeted drugs and immune checkpoint inhibitors have improved the treatment outcomes of advanced liver cancer, their overall response rates are still not ideal, and adverse reactions and drug resistance issues exist.

[0003] Discovering highly effective and low-toxicity lead compounds or novel treatment strategies for liver cancer from natural products has become a current research hotspot. Pearl powder, a member of the traditional Chinese medicine treasure trove, has long been used to treat palpitations, insomnia, infantile convulsions, epilepsy, conjunctivitis, slow-healing sores, and skin pigmentation, but its pharmacological effects have not been fully elucidated. Discovering drugs from pearl powder for liver cancer treatment, filling the current gap in the development of natural product-based anti-liver cancer drugs, would have significant theoretical and clinical value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Eppin protein in the preparation of drugs for treating liver cancer.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The application of an Eppin protein in the preparation of a liver cancer treatment drug, wherein the amino acid sequence of the Eppin protein is shown in SEQ ID No. 1.

[0007] Furthermore, the cDNA sequence of the Eppin protein is shown in SEQ ID No. 2.

[0008] Furthermore, the Eppin protein is a Kunitz-type serine protease inhibitor extracted from pearl powder.

[0009] Furthermore, the liver cancer treatment drug is a drug that inhibits the proliferation of liver cancer cells.

[0010] Furthermore, the liver cancer treatment drug is a drug that inhibits the migration of liver cancer cells.

[0011] Furthermore, the liver cancer treatment drug is a drug that inhibits the invasion of liver cancer cells.

[0012] Furthermore, the liver cancer treatment drug is a drug that induces apoptosis in liver cancer cells.

[0013] Furthermore, the liver cancer treatment drug is a drug that inhibits the cell cycle of liver cancer cells.

[0014] In summary, the present invention has the following beneficial effects:

[0015] This invention, for the first time, focuses on Eppin (a Kunitz-type serine protease inhibitor), a functional protein identified from pearl powder. Through in vitro experiments, it systematically demonstrates the multi-target anti-tumor activity of Eppin protein against Huh-7 liver cancer cells (including inhibition of proliferation, induction of apoptosis, cell cycle arrest, and inhibition of migration and invasion). In vivo animal experiments further confirm the significant inhibitory effect of Eppin protein on liver cancer cells in a living environment (it can induce extensive coagulative necrosis and significant apoptosis in tumor tissue, triggering large-scale disintegration of tumor tissue). This not only provides crucial modern scientific evidence for the traditional efficacy of pearl in "calming the liver and suppressing yang," promoting the modernization of traditional Chinese medicine, but also lays a key preclinical experimental foundation for developing Eppin into a novel anti-liver cancer treatment drug derived from a natural product. Attached Figure Description

[0016] Figure 1 Bioinformatics analysis diagram of Eppin (In the diagram: (A) is the cDNA sequence and encoded amino acid sequence of Eppin: the boxed parts are the start codon and stop codon, the double underlined parts are polyadenylation signals, and the single underlined parts are the N-terminal signal peptides (amino acid positions 1-24); (B) is a schematic diagram of the domains of Eppin).

[0017] Figure 2 Figure 1 shows the results of Huh-7 cell viability testing after treatment with different Eppin protein concentrations. (p<0.01);

[0018] Figure 3 Morphological images of Huh-7 cells after treatment with different Eppin protein concentrations;

[0019] Figure 4 Figure 1 shows the apoptosis rate of Huh-7 cells treated with different concentrations of Eppin protein.

[0020] Figure 5 Apoptosis of Huh-7 cells treated with different concentrations of Eppin protein;

[0021] Figure 6 The figure shows the test results of the effect of different concentrations of Eppin protein on the cell cycle of human liver cancer cells Huh-7.

[0022] Figure 7 Figure 1: ROS level detection results of Huh-7 cells treated with different concentrations of Eppin protein ((A): Flow cytometry histogram; (B): Quantitative statistical graph of mean fluorescence intensity).

[0023] Figure 8 The figure shows the test results of the effect of different concentrations of Eppin protein on Huh-7 cell clones;

[0024] Figure 9 The figure shows the test results of the effect of different concentrations of Eppin protein on the invasion of Huh-7 cells;

[0025] Figure 10 The figure shows the test results of the effect of different concentrations of Eppin protein on the migration of Huh-7 cells;

[0026] Figure 11 This is a graph showing differential gene analysis from transcriptome sequencing.

[0027] Figure 12 The expression levels of different genes in Huh-7 cells are shown in the diagrams (A): BBC3 gene expression level; (B): TP53 gene expression level; (C): CDK1 gene expression level; (D): VEGFD gene expression level in cells.

[0028] Figure 13 Images showing the appearance of xenografts in different groups of mice;

[0029] Figure 14 Histopathological images of xenograft tumor tissues in different groups of mice;

[0030] Figure 15 The results of the analysis on the effect of Eppin protein on gene expression levels in hepatocellular carcinoma xenografts in mice are shown in the following figures: (A) Relative expression level of BBC3 gene; (B) Relative expression level of TP53 gene; (C) Relative expression level of Caspase-9 gene; (D) Relative expression level of Caspase-3 gene; (E) Relative expression level of VEGFD gene.

[0031] Figure 16 Immunohistochemical image to detect the effect of Eppin protein on p53 protein expression level. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Experimental materials:

[0034] (1) The human hepatocellular carcinoma Huh-7 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. The depository institution was the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-526.

[0035] (2) Eppin protein and its cDNA were prepared according to the paper (Analysis of Differential Proteins in Pearls and Shells and Functional Study of Eppin in Immunotherapy and Whitening. Jiang Rui. Shaoxing University, 2025, No. 6). The bioinformatics analysis results of the Eppin protein are as follows: Figure 1 As shown.

[0036] The amino acid sequence of Eppin protein is: MQWKPLFIVFIFLLALFINNYVEGGRKDKKKDKPGKCPTPTGASACVELCSSDRDCTGKMKCCVNSCGGHICTLPVDTCQLPKVVGPCLASKKRYYFNKETGKCRAFYYGGCFGNENNFRSRRSCRKRCEKKKDD (SEQ ID No. 1); where MQWKPLFIVFIFLLALFINNYVEG is the signal peptide.

[0037] The 992bp full-length cDNA sequence of Eppin protein is shown in SEQ ID No.2, including a 25bp 5′-untranslated sequence, a 408bp open reading frame, and a 559bp 3′-untranslated sequence. The start codon is ATG, the stop codon is TGA, and the polyadenylation signal is AATAAA.

[0038] CTTATCTCAGCAGAAGTGGTACAAA ATGCAGTGGAAACCATTGTTCATCGTGTTCATTTTCCTGTTGGCTCTGTTTATCAACAATTACGTCGAAGGAGGTCGGAAAGATAAAGAAGGATAAGCCCGGCAAGTGCCCCACACCGACAGGCGCTAGTGCTTGTGTAGAACTATGTTCCTCAGACCGAGACTGTACTGGGAAAATGAAATGTTGCGTTAATAGCTGCGGC GGACATATTTGTACGCTCCCTGTGGATACCTGTCAACTTCCGAAAGTGGTAGGACCATGCCTTGCAAGCAAGAGATATTACTTCAACAAGGAAACGGGAAAATGCCGTGCATTTTACTATGGGGGTTGTTTCGGCAACGAAAATAATTTCAGATCGCGAAGGAGCTGTCGAAAACGCTGCGAAAAAGAAGGATGAC TGA AGTCACCAGACTATGAAAAGCCAGTATTCAAGGATTATGTTGACCAGTTTACATTTTGGAAATCAGCGTGACTTTTATCACTCTAGTTGTTTATGTCACCAATTTCCGAATTTTCTAAGAATATGGAATAAGAAGTTAAAAATCAGGATGCAAAACATTGAATAGAATATGGAGCTTACAAAGTCGGAACATTTTATTTTTCAATACTCGAATGTTTAAAGTGGTCATATTCATTCTTTTCAATATTGAGATTCCTCAAATCAGTTTGAGAAAATAACATTACCATATATCGTATGCAAAATATCATCCGGGTTATCTCTCAATATAAAAATAATGAACGTAACACATTTCGTTTGATGTTTCTTTGAAATATATTTCTTTGTCAAAGAATATTCCAAGAGAAAAGAAATTTGATAATAGTTTCAAACTTGAATATGCTTGCATTTACATAATTAGATAATGTATGAGTCCATAAAATTGGTCAAATTTACTTACTGAAGAAACGCTG AATAAA GTTTGAGAACTAAATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID No.2)。

[0039] Main reagents:

[0040] DMEM medium (No.: 12491015) and premium fetal bovine serum (No.: A5669701) were purchased from BBI Pharmaceuticals, USA; 0.25wt% trypsin digestion solution (EDTA and phenol red-free, No.: A510829), sterile phosphate buffer (PBS solution, No.: ZAX0DP3), and serum-free medium (model E670081) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; penicillin-streptomycin solution (No.: C0222), enhanced CCK-8 assay kit (No.: C0041), Annexin V-FITC / PI apoptosis assay kit (No.: C1062S), reactive oxygen species assay kit (No.: S0033S), cell cycle assay kit (No.: C1052), matrix gel (No.: C0371-1ml), Transwell chambers, Trizol reagent (No.: C90205), Tween-20, hematoxylin, D AB chromogenic solution (item number: ST2067-5g), goat serum blocking solution (item number: AB3016-50μl), and hydrochloric acid ethanol differentiation solution (item number: C0161S) were all purchased from Beyotime Biotechnology Co., Ltd.; TaKara reagent kit was purchased from BioNTech (Beijing) Co., Ltd., catalog number: 9765; SYBR Premix ExTaq™ II was purchased from Nearshore Protein (Shanghai) Technology Co., Ltd., catalog number: ZBA004S; Rainbow 180 broad-spectrum protein marker was purchased from Beijing Solarbio Technology Co., Ltd., catalog number: PR1910; eosin was purchased from Shanghai Gefan Biotechnology Co., Ltd.; rabbit monoclonal antibody P53 was purchased from Wuhan Proteintech Co., Ltd., catalog number: ZCP0GWN; rabbit monoclonal antibody VEGFD was purchased from Abcam (USA), catalog number: ZCP02RW; and goat anti-rabbit IgG-HRP was purchased from Hangzhou Baoke Biotechnology Co., Ltd., catalog number: ZB654EN.

[0041] Example 1: Effect of Eppin protein on human hepatocellular carcinoma cells Huh-7

[0042] 1.1 Cell resuscitation

[0043] (1) Take a cryopreservation tube of human liver cancer Huh-7 cells stored in a -80°C freezer and place it in a 37°C constant temperature water bath and shake it continuously until the contents are completely thawed within 1 minute.

[0044] (2) Once the contents of the cryovial have completely thawed (usually when only a small piece of ice crystal remains), immediately remove it from the water bath, wipe the outer wall of the cryovial dry with sterile gauze or paper towel, then spray it with 75% (v / v) alcohol for thorough disinfection, and then quickly move it into the clean bench.

[0045] (3) In a clean bench, transfer the cell suspension in the cryopreservation tube to a new 15 mL sterile centrifuge tube, slowly add 1 mL of DMEM complete medium (that is, the medium after adding fetal bovine serum and antibiotic solution to DMEM medium, the content of fetal bovine serum is 10 wt%, the content of antibiotic solution is 1 wt% (the antibiotic solution is a penicillin-streptomycin mixture, the final concentration of streptomycin in DMEM complete medium is 0.1 mg / mL, and the final concentration of penicillin is 100 U / mL), mix well by pipetting, and then add 4 mL of DMEM complete medium; centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0046] (4) Add preheated DMEM complete medium at 37°C to the cell pellet, gently pipette to resuspend the cells and form a single cell suspension; inoculate the single cell suspension into a culture flask, add sufficient DMEM complete medium, and culture in a 37°C, 5% CO2 incubator as usual.

[0047] 1.2 Cell passage

[0048] (1) After the cells have reached 90% confluence (or between 80% and 90%), discard the old culture medium in the original culture flask, add preheated PBS solution, gently shake to rinse the entire cell layer, and then aspirate.

[0049] (2) Add 0.25wt% trypsin digestion solution to the culture flask (enough to cover the bottom of the flask, such as 1-2mL for T25 flasks), gently shake to ensure that the digestion solution evenly covers the cells, and place in a 37℃ incubator for 2 minutes (or 1-3 minutes).

[0050] (3) When the cells are observed to detach slightly and the cell morphology changes slightly, the digestion is complete. Add DMEM complete culture medium with 2 times the volume of 0.25wt% trypsin digestion solution to the culture flask to stop the digestion. Gently and repeatedly blow the flask wall with a pipette to detach all the adherent cells and form a single cell suspension. Collect the suspension into a 15mL centrifuge tube and centrifuge at 1000rpm for 5min.

[0051] (4) Discard the supernatant and gently pipette 1 mL of DMEM complete medium to form a cell suspension; dispense the cell suspension into new culture flasks (divided into 2-6 portions according to the cell growth rate) and replenish with fresh DMEM complete medium; place in a constant temperature incubator at 37°C and 5% CO2 for continued culture.

[0052] 1.3 CCK Cell Proliferation Toxicity Assay

[0053] (1) Cell plating: Digest Huh-7 cells that are growing well and in the logarithmic growth phase with 0.25wt% trypsin digestion solution and prepare cell suspension with DMEM complete medium; seed the cell suspension into 96-well plates at a concentration of 5000 Huh-7 cells per well, 100μL per well; incubate at 37℃ and 5%CO2 for 24 hours (or 12-24h) to allow the cells to adhere.

[0054] (2) After the cells adhere to the culture medium, remove the original medium and add fresh DMEM complete medium containing different concentrations of Eppin protein (0, 50, 100, 200, 400, 800, 1600 ng / mL) to each well, and continue culturing for 48 h. (3) After the drug treatment is complete, discard the medium, add 90 μL of DMEM complete medium and 10 μL of CCK-8 reagent to each well, gently shake to mix (avoid generating air bubbles), and return to the incubator at 37℃ and 5% CO2 in the dark for 3 h (or 1-4 h). Use a microplate reader to measure the absorbance at 450 nm wavelength and calculate the cell viability.

[0055] The results of Huh-7 cell viability assays after treatment with different concentrations of Eppin protein are shown below. Figure 2 As shown, the morphology of Huh-7 cells treated with different concentrations of Eppin protein is shown in the figure. Figure 3 As shown in the figure. The results indicate that the effect of Eppin protein on Huh-7 cells is dose-dependent and has a significant inhibitory effect; under the treatment of high concentrations of Eppin protein (800 μg / mL and 1600 μg / mL), the growth viability of Huh-7 cells is significantly inhibited, and the cell morphology under an optical microscope clearly shows an apoptotic state.

[0056] 1.4 Flow cytometry assay for apoptosis (Annexin V-FITC / PI double staining)

[0057] (1) Cell plating: Digest Huh-7 cells with good cell growth with 0.25wt% trypsin digestion solution, prepare cell suspension with DMEM complete medium, seed into 6-well culture plates, and culture until the cell density reaches 80% (or culture until the cell density reaches 70-80%).

[0058] (2) Remove the original culture medium and add DMEM complete culture medium containing different concentrations of Eppin protein (Eppin protein concentrations are 0, 50, 100, 200 and 400 ng / mL, respectively), and continue to culture at 37℃ and 5% CO2 for 48 h.

[0059] (3) Discard the culture medium, gently digest with 0.25wt% trypsin digestion solution (avoid excessive cell damage), add DMEM complete culture medium to stop digestion, centrifuge (1000rpm, 5min) to collect cells, and resuspend the cells in 195μL of Annexin V-FITC / PI apoptosis kit binding buffer (adjust cell concentration to 1×10⁻⁶). 6 (cells / mL).

[0060] (4) Add 5 μL Annexin V-FITC staining solution, mix gently, and incubate at room temperature in the dark for 10 minutes (or 10-15 minutes); add 5 μL LPI staining solution and incubate at room temperature in the dark for 5 minutes; immediately after staining, add 200 μL binding buffer, mix well, and test within 1 hour.

[0061] The flow cytometry detection conditions are as follows:

[0062] Excitation wavelength: 488nm (excitation light for FITC and PI); Detection channels: FITC (Annexin V) → FL1 channel (green fluorescence), PI → FL2 or FL3 channel (red fluorescence); Adjustment voltage and compensation: Adjust fluorescence compensation using a single-stain tube (Annexin V or PI only) to avoid signal overlap.

[0063] Figure 4 The apoptosis rate of Huh-7 cells treated with different concentrations of Eppin protein is shown. Figure 5 The figures show apoptosis in Huh-7 cells treated with different concentrations of Eppin protein. From the bottom left, clockwise, each figure represents normal cells, necrotic cells, early apoptotic cells, and late apoptotic cells. The results showed that at a concentration of 50 μg / mL Eppin protein, the apoptosis rate was not different from the normal control group. However, with increasing Eppin protein concentration, the apoptosis rate of Huh-7 cells gradually increased to 21.73%, 27.2%, and 31.7%, respectively, indicating that Eppin protein can promote apoptosis in Huh-7 cells in a concentration-dependent manner.

[0064] 1.5 Flow Cytometry for Cell Cycle Detection

[0065] (1) Well-grown Huh-7 cells were digested with 0.25 wt% trypsin digestion solution, and after being suspended in DMEM complete medium, they were seeded into 6-well plates (100 μL per well, with the cell concentration adjusted to 1 × 10⁻⁶ cells per well). 6 After the cells adhered to the wells, fresh DMEM complete medium containing different concentrations of Eppin protein (Eppin protein concentrations of 0, 50, 100, 200, and 400 ng / mL) was added to each well, and the cells were cultured at 37°C and 5% CO2 for 48 hours.

[0066] (2) Discard the culture medium, digest the cells and collect them in a centrifuge tube, resuspend and wash with 1 mL PBS solution, centrifuge for 5 min, discard the PBS, add 250 μL PBS solution to resuspend, and transfer to a new 1.5 mL centrifuge tube.

[0067] (3) After the cells are resuspended into single cells, 0.75 mL of pre-cooled anhydrous ethanol is added dropwise while gently vortexing for fixation (final concentration of anhydrous ethanol is 75%), and the cells are incubated overnight at 4°C.

[0068] (4) After fixing overnight, wash the cells twice with pre-cooled PBS solution, centrifuge (8500 rpm, 5 min) to remove PBS; wash once more with PBS solution, add 1 mL of Rnase A working solution to resuspend the cells; digest in a 37°C water bath in the dark for 30 min, add 0.5 mL of propidium iodide staining solution to resuspend the cells, centrifuge to remove the supernatant, add PI dye (final concentration 50 μg / mL), stain in the dark at 4°C for 30 min, wash twice with PBS solution, filter through a 200 mesh cell filter, and perform flow cytometry analysis.

[0069] The results of testing the effects of different concentrations of Eppin protein on the cell cycle of human hepatocellular carcinoma Huh-7 cells are as follows: Figure 6 As shown in Table 1, after treatment with Eppin protein at low to medium concentrations (50-200 μg / mL), the proportion of cells in the G0 / G1 phase slightly increased, indicating that cells were arrested in the G1 phase and inhibited from entering the S phase. However, at the highest concentration of 400 μg / mL, the G0 / G1 ratio decreased to 42.5%, lower than the control group, indicating increased cell death or altered cell cycle progression at high concentrations. The proportion of cells in the S phase was lower in all treatment groups than in the control group, especially at 200 μg / mL (18.7%), indicating that Eppin protein significantly inhibited DNA synthesis. This suggests that Eppin protein inhibits cell cycle progression by suppressing DNA replication.

[0070] Table 1

[0071]

[0072] 1.6 Reactive Oxygen Species (ROS) Level Detection Experiment

[0073] First, Huh-7 cells in the division phase and growing well were digested with 0.25wt% trypsin digestion solution and then made into a cell suspension in DMEM complete medium. The cell suspension was seeded into 6-well plates and cultured until the cell density reached 70%, then the DMEM complete medium was replaced. Eppin protein was added to different wells at different concentrations (0, 50, 100, 200, and 400 ng / mL, respectively), and the cells were cultured at 37℃ and 5% CO2 for 48 h. The medium was then discarded, and the cells were digested with 0.25wt% trypsin digestion solution. The digestion was stopped by adding DMEM complete medium, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. Add diluted DCFH-DA (diluted DCFH-DA 1:1000 with serum-free culture medium to a final concentration of 10 μmol / L) to the cell pellet, incubate at 37°C for 20 min, inverting and mixing every 3-5 minutes to ensure the probe is in full contact with the cells; centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 1 mL PBS and wash once, centrifuge for 5 min, resuspend in 500 μL PBS solution, filter, and then perform flow cytometry analysis.

[0074] The results of ROS level detection in Huh-7 cells treated with different concentrations of Eppin protein are as follows: Figure 7 As shown in the figure. The results showed that the ROS level was not different from the control group under the treatment of 50 μg / mL Eppin protein. The ROS level increased under the treatment of 100 μg / mL, 200 μg / mL and 400 μg / mL Eppin protein concentrations, and reached its peak at 200 μg / mL. It can be seen that Huh-7 cells undergo oxidative stress response under the treatment of Eppin protein.

[0075] 1.7 Cell Plate Cloning Assay

[0076] Huh-7 cells in logarithmic growth phase were digested with 0.25 wt% trypsin digestion solution, resuspended in DMEM complete medium, and seeded into 6-well plates at a concentration of 1000 cells / well for each experimental group. Cells were cultured for 14 days at 37°C and 5% CO2 in fresh DMEM complete medium containing different concentrations of Eppin protein (0, 50, 100, 200, and 400 ng / mL), with the medium changed every 3 days. After cloning, each well was fixed with 1 mL of 4 wt% paraformaldehyde fixative for 30 min, washed three times with PBS, and then stained with 1 mL of 0.1 wt% crystal violet solution for 20 min, washed three times with PBS, air-dried, and photographed with a digital camera.

[0077] The effects of different concentrations of Eppin protein on Huh-7 cell clones are as follows: Figure 8As shown, the number of cell colonies decreased with increasing Eppin protein concentration. The number of colonies with more than 50 cells was counted using ImageJ software, and the colony area was statistically analyzed. The results showed that compared with the control group (0 μg / mL Eppin protein concentration), the colony formation rates of the 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL treatment groups were 29%, 8%, 6%, and 1%, respectively, with statistically significant differences among the groups (p<0.05). Furthermore, the average size of the colonies also decreased significantly with increasing Eppin protein concentration. These results indicate that Eppin protein can irreversibly and persistently inhibit the proliferation and self-renewal potential of hepatocellular carcinoma Huh-7 cells. The decreased colony-forming ability means that fewer cells can survive and continue to divide and form offspring, directly demonstrating that Eppin protein has a potent scavenging or inhibitory effect on tumor stem cell-like populations or highly proliferating cell subpopulations.

[0078] 1.8 Cell Invasion (Transwell Assay) Experiment

[0079] (1) Matrix gel plating: Thaw the matrix gel overnight at 4°C, and pre-cool the consumables for later use; dilute the matrix gel with serum-free culture medium at a volume ratio of 1:8, take an appropriate amount of diluted gel solution (100μL) and add it to the bottom of the upper chamber of the Transwell chamber, incubate at 37°C for 2h to allow the gel to polymerize and form a matrix membrane.

[0080] (2) Hydration and equilibration: Remove excess liquid from the upper chamber, add serum-free culture medium (100 μL) to the upper chamber, add serum-containing culture medium (10% fetal bovine serum added to the serum-free culture medium as a chemotactic agent) to the lower chamber, and incubate at 37°C for 1 h to equilibrate the chamber environment.

[0081] (3) Preparation of cell suspension: Huh-7 cells in the logarithmic growth phase were digested with 0.25 wt% trypsin digestion solution, and the cells were resuspended in serum-free culture medium to adjust the cell concentration of the cell suspension to 5 × 10⁻⁶. 5 Cells / mL, different concentrations of Eppin protein were added to the cell suspension to obtain cell suspensions containing different concentrations of Eppin protein (Eppin protein concentrations were 0, 50, 100, 200, and 400 ng / mL).

[0082] (4) Inoculate cells: Aspirate the equilibration solution from the upper chamber and gently add 200 μL of cell suspension containing the corresponding concentration of Eppin protein into the upper chamber; replenish the lower chamber with serum-containing culture medium.

[0083] (5) Invasion culture: Place the Transwell plate in a 37°C, 5% CO2 incubator and incubate for 48 hours.

[0084] (6) Termination and fixation: Remove the chamber, gently rinse with PBS solution, wipe the surface of the upper chamber membrane with a cotton swab to completely remove uninvaded cells, and fix the chamber in 4wt% paraformaldehyde for 30 min.

[0085] (7) Staining: After rinsing the fixed chambers with PBS solution, stain with 0.1wt% crystal violet solution for 30 min, then rinse with PBS solution to remove excess dye; air dry the chambers and observe the cells that have invaded the lower chamber membrane surface under an electron microscope.

[0086] The results of the test on the effect of different concentrations of Eppin protein on Huh-7 cell invasion are as follows: Figure 9 As shown in the figure, the invasive ability of Huh-7 cells decreases with increasing Eppin protein concentration, indicating a concentration-dependent effect. Eppin protein has a significant inhibitory effect on the invasive ability of Huh-7 cells.

[0087] 1.9 Cell Scratch Assay

[0088] (1) Cell seeding: Inoculate well-grown Huh-7 cells evenly into 6-well plates, ensuring 90% confluence, and culture overnight to allow the cells to adhere firmly to the plate.

[0089] (2) Create scratches: Use a sterile pipette tip to scrape vertically across the cell layer, and gently rinse with PBS 2-3 times to remove detached cell debris.

[0090] (3) Change the culture medium: Add DMEM complete medium containing low concentrations of fetal bovine serum (1%) but different concentrations of Eppin protein (Eppin protein concentrations of 0, 50, 100, 200, and 400 ng / mL). Locate the scratched area under a microscope and record the initial width of the scratch (0 h). Return the 6-well plate to a 37°C, 5% CO2 incubator for further incubation. At 24 h and 48 h, remove the plate and take pictures at the same marked location. Analyze the results using image software.

[0091] The results of the test on the effect of different concentrations of Eppin protein on Huh-7 cell migration are as follows: Figure 10 As shown, the migration ability of Huh-7 cells decreased significantly after being treated with Eppin protein, indicating that Eppin protein has a significant inhibitory effect on the migration ability of Huh-7 cells.

[0092] 1.10 Total RNA extraction from cells expressing related genes after Eppin protein treatment

[0093] Well-grown Huh-7 cells were digested with 0.25 wt% trypsin and resuspended in DMEM complete medium. The cells were then seeded into 6-well plates (100 μL per well, cell concentration adjusted to 1 × 10⁻⁶ cells per well).6 After cell adhesion, fresh DMEM complete medium containing different concentrations of Eppin protein (0, 50, 100, 200, and 400 ng / mL) was added to different wells, and the cells were cultured at 37°C and 5% CO2 for 48 h. Total RNA was extracted from Huh-7 cells treated with different concentrations of Eppin protein using the Trizol method. The specific steps are as follows:

[0094] (1) Take Huh-7 cells from 6-well plates treated with the corresponding concentration of Eppin protein, remove the culture medium on ice, and wash three times with PBS solution. Then add 1 mL of Trizol reagent to each well, collect the cells with a cell scraper into a 1.5 mL centrifuge tube, and lyse on ice for 20 min.

[0095] (2) Add 200 μL of chloroform to the centrifuge tube, shake it up and down, and let it stand on ice for 10 min; centrifuge at 4°C and 12000 rpm for 20 minutes.

[0096] (3) After centrifugation, carefully remove the EP tube and aspirate 300 μL of the supernatant into a new EP tube (be careful not to aspirate the white membrane); add an equal volume of isopropanol, gently invert 5 times, and let stand on ice for 10 min; then centrifuge at 4°C and 12000 rpm for 10 minutes.

[0097] (4) Discard the supernatant, add 1 mL of 75% ethanol (knock up the precipitate, it is white and feathery), centrifuge at 4°C and 8000 rpm for 5 minutes.

[0098] (5) Discard the supernatant, add 500 μL of anhydrous ethanol, and centrifuge at 4°C and 8000 rpm for 3 minutes.

[0099] (6) Discard the supernatant and air dry for 10 minutes (the RNA will be semi-transparent when dried).

[0100] (7) Dissolve the RNA in 10-20 μL of sterile water according to the amount of RNA precipitate. Measure the RNA concentration.

[0101] 1.11 Reverse transcription

[0102] In this experiment, the total RNA extracted was reverse transcribed using the TaKara kit. The specific procedure is as follows:

[0103] (1) Add the gDNA removal reaction system (10 μL system) reagent shown in Table 2 to the tube, mix well, centrifuge; incubate at 42°C for 2 minutes, and then immediately place on ice.

[0104] Table 2

[0105]

[0106] (2) Add the reverse transcription reaction reagents shown in Table 3 directly to the above reaction tube, mix well, centrifuge briefly, and run according to the following program: 37°C, 15min, 85°C, 5s, 4°C, ∞.

[0107] Table 3

[0108]

[0109] 1.12 Real-time quantitative PCR analysis

[0110] Preparation of the reaction system: The total volume is 20 μL (including SYBR Premix Ex Taq). TM II(2X) 10 μL, upstream and downstream primers (see Table 4) 0.8 μL, template (cDNA) 2 μL, and the remainder ddH2O 6.4 μL), 3 technical replicates.

[0111] Program setup and instrumentation: After setting up the PCR program, place the sealed PCR plate into a centrifuge and centrifuge at 2000 rpm for 2 minutes to allow any liquid that may be adhering to the well walls to fall back to the bottom. After centrifugation, place the PCR plate into the PCR instrument and run the program. The program settings are shown in Table 5.

[0112] Table 4 qRT-PCR primers

[0113]

[0114] Table 5 qRT-PCR program

[0115]

[0116] 1.13 Transcriptome sequencing (RNA-seq)

[0117] Huh-7 cells treated with 200 μg / mL Eppin protein were selected, and transcriptome sequencing was performed by Shanghai Sangon Biotech Co., Ltd. The simplified procedure is as follows:

[0118] (1) Cells were cultured according to the experimental design and treated with drugs. Then TRIzol was added to collect the cells into EP tubes, which were stored on dry ice and sent to Shanghai Sangon Biotech Co., Ltd.

[0119] (2) Quality Control 1-RNA Quality Inspection: Purity: The A260 / A280 (~2.0) and A260 / A230 (>2.0) ratios were detected using NanoDrop to ensure the absence of protein and organic contamination. Integrity: RNA integrity was detected using Agilent Bioanalyzer or agarose gel electrophoresis. A RIN value >8.0 (or a 28S / 18S ratio of ~2:1) is the gold standard for subsequent experiments. Concentration: Accurate quantification was performed using a fluorometer such as Qubit.

[0120] (3) mRNA enrichment and fragmentation: magnetic beads with Oligo(dT) are used to specifically bind to the 3' PolyA tail of eukaryotic mRNA, thereby separating the mRNA. This method can effectively remove rRNA.

[0121] (4) cDNA synthesis and library construction: First-strand cDNA synthesis: Using mRNA fragments as templates, first-strand cDNA is synthesized under the action of reverse transcriptase using random hexamer primers or Oligo(dT) primers. Second-strand cDNA synthesis: Using first-strand cDNA as templates, double-stranded cDNA is synthesized. End repair and addition of "A" tail: The ends of the double-stranded cDNA are repaired to blunt ends, and an adenine (A) is added to the 3' end.

[0122] (5) Library quality testing and quantification: The fragment size distribution of the library was tested using the Agilent Bioanalyzer to ensure it met expectations. Accurate quantification was performed using qPCR or Qubit to ensure accurate molar concentrations for sequencing.

[0123] (6) Sequencing.

[0124] (7) Differential gene expression was analyzed using DEseq. The criteria for screening differentially expressed genes were: |log2FoldChange|>1 and significance P-value<0.05.

[0125] Differential gene analysis results as follows Figure 11 As shown, the results indicated that after treatment with 200 ug / mL of Eppin protein, 1189 genes were upregulated and 3205 genes were downregulated.

[0126] 1.14 Effect of Eppin protein on gene expression levels in human hepatocellular carcinoma cell line Huh-7

[0127] Transcriptome sequencing analysis was used to further detect changes in the expression of genes such as BBC3 (pro-apoptotic gene), TP53 (tumor suppressor gene), and cell cycle-related protein (CDK1) in human hepatocellular carcinoma Huh-7 cells after 48 hours of Eppin protein treatment at the gene level using qPCR. GAPDH was used as an internal reference gene, and relative quantification was performed using the 2^-ΔΔCt method. All data were from three independent replicate experiments, and results are expressed as mean ± standard error. Statistical analysis was performed using the Student's test. P<0.05, P<0.01). The results are as follows: Figure 12 As shown, after treatment with Eppin protein, the BBC3 and TP53 genes in Huh-7 liver cancer cells were upregulated, while the CDK1 and VEGFD genes were downregulated, which is consistent with previous cell cycle and invasion experiments.

[0128] Example 2: Study on the inhibitory effect of Eppin protein on hepatocellular carcinoma in mice

[0129] 2.1 Construction of a mouse liver cancer model

[0130] The hepatocellular carcinoma mouse model used in this study was purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The specific process for constructing the hepatocellular carcinoma mouse model involved routinely culturing the human hepatocellular carcinoma cell line Huh-7 in DMEM complete medium, then collecting cells in the logarithmic growth phase, resuspending them in sterile PBS solution (or mixing them 1:1 with matrix gel), and adjusting the cell concentration to 5 × 10⁶ cells / day. 8 cells / mL. 4-6 week old NCG-bearing rats were anesthetized with isoflurane and subcutaneously inoculated with 200 μL of cell suspension (containing 1 × 10⁻⁶ cells / mL) under the right axilla. 8 (1 cell); after inoculation, the mice were observed regularly, and the tumor volume was measured using calipers. The tumor volume was allowed to grow to approximately 100 mm². 3 At that time, the tumor-bearing mice were randomly divided into groups for subsequent experimental interventions. All mice were housed in a specific pathogen-free animal room with a constant temperature (22±2℃), constant humidity (50±10%), and a 12-hour light / dark cycle environment; the mice had free access to sterile feed and drinking water.

[0131] 2.2 Animal grouping and treatment

[0132] Hepatocellular carcinoma model mice were randomly divided into four groups: control group, low-concentration group, medium-concentration group, and high-concentration group, with five mice in each group. All drugs were administered via intraperitoneal injection every three days. The low-concentration, medium-concentration, and high-concentration groups were injected with Eppin protein solution (using physiological saline as a solvent) at doses of 50 mg / kg, 100 mg / kg, and 200 mg / kg, respectively, while the control group received an equal volume of physiological saline. The experimental period was one month. At the end of the experiment, mice were euthanized by cervical dislocation, and tumor tissue was quickly dissected and removed. A portion of the tumor tissue from each group was frozen at -80°C (the same number of tumor tissues were used in each group), while another portion was gently rinsed with pre-cooled PBS solution to remove surface blood, and then fixed in 10% formalin fixative (20 times the volume of the tumor tissue or 10-20 times the volume of the tissue) for 48 hours for subsequent analysis.

[0133] 2.3 Hematoxylin-Eosin (H&E) staining

[0134] The tumor tissues of each group of mice were stained separately. The specific steps were as follows: (1) The fixed liver tissue was removed from the fixation solution, the required part was selected, and a tissue block with a length and width of 1 cm was cut with a scalpel. The thickness was kept at 2.5 mm (or 2-3 mm). The tissue block was placed in a dehydration box and marked. It was then rinsed thoroughly with running water.

[0135] (2) Dehydration, clearing, paraffin infiltration, and embedding: The tissue blocks were dehydrated using an alcohol gradient as follows: 75% ethanol → 85% ethanol → 95% ethanol I → 95% ethanol II → 100% ethanol I → 100% ethanol II, with each step requiring 1.5 hours of soaking time (or 1-2 hours). Clearing was then performed using xylene (the tissue was sequentially immersed in multiple portions of xylene until it became clear, with each immersion lasting 1 hour). Afterward, the cleared tissue was placed in melted paraffin (in a 60°C constant temperature incubator), with the paraffin being replaced 3 times, for a total infiltration time of 3 hours (optionally, the paraffin was replaced 2-3 times, for a total infiltration time of 3-4 hours). Finally, the tissue was placed in an embedding cassette, filled with paraffin, and allowed to cool and solidify into a paraffin block.

[0136] (3) Slicing and spreading: After trimming the wax block with a paraffin microtome, cut it into continuous slices with a thickness of 5μm (or 4-6μm); transfer the continuous slices to warm water at 40-45°C, and after they are fully flattened, pick them up with a glass slide.

[0137] (4) Baking the slides: Place the slides in a 60°C oven and bake for 1 hour to ensure that the tissue adheres tightly to the slides and prevent them from falling off later.

[0138] (5) Dewaxing and hydration: Place the sections in the following reagents in sequence: xylene I (10 min) → xylene II (10 min) → 100% ethanol I (5 min) → 100% ethanol II (5 min) → 95% ethanol (3 min) → 85% ethanol (3 min) → 70% ethanol (3 min) → rinse with running water for 2 minutes to remove paraffin and allow water to enter the tissue.

[0139] (6) Hematoxylin staining: Immerse the slides in 1% hematoxylin staining solution (with alcohol as solvent) for 10 minutes (or 5-10 minutes), rinse with running water for 1 minute to remove the surface stain; dip the slides in 1% hydrochloric acid ethanol solution (prepared with 70% alcohol) several times; rinse again with running water; then immerse the slides in 0.1% ammonia water (or warm water or other weakly alkaline water) for several minutes until the cell nuclei turn bright blue; rinse thoroughly with running water.

[0140] (7) Eosin staining: Immerse the blue-returned sections in a 1% eosin staining solution (with alcohol as the solvent) for 60 seconds (or between 30 and 90 seconds); after removing them, rinse them quickly with running water for a few seconds to stop the staining.

[0141] (8) Dehydration and clearing: Pass the slices through the following reagents in sequence: 70% ethanol (1 min) → 85% ethanol (1 min) → 95% ethanol (1 min) → 100% ethanol I (1 min) → 100% ethanol II (1 min) → xylene I (3 min) → xylene II (3 min).

[0142] (9) Mounting: Remove the slide from the xylene, let it air dry in a ventilated place for a while, but do not let the tissue dry out. Add a drop of neutral resin to the center of the tissue, gently cover it with a coverslip to avoid air bubbles, and let it air dry at room temperature.

[0143] 2.4 Total RNA extraction from tissues

[0144] (1) Remove the mouse liver tissue stored at -80℃ and thaw it. After thawing, cut 0.2g of the tissue and put it into a 1.5mL centrifuge tube. Add 500μL of Trizol reagent to the tube and grind the liver tissue into a relatively uniform paste using a disposable grinding stick and an electric grinder. The grinding process must be carried out on ice throughout, and the electric grinder should be kept running at a low speed to prevent the temperature from rising and damaging the RNA. Add another 500μL of Trizol reagent to the tube and mix well. Rinse the tissue from the grinding stick into the tube to prevent tissue loss. Place the tube on ice for lysis for 15min.

[0145] (2) Perform total RNA extraction according to steps (2)-(7) of 1.10.

[0146] 2.5 Real-time quantitative PCR analysis

[0147] Perform the procedure as described in steps “1.11 Reverse Transcription” and “1.12 Real-time Quantitative PCR Analysis”. The qRT-PCR primers are shown in Table 6.

[0148] Table 6

[0149]

[0150] 2.6 Immunohistochemistry

[0151] (1) Prepare sections according to steps (1)-(5) of “2.3 Hematoxylin-Eosin (H&E) staining”.

[0152] (2) Antigen retrieval: Using deionized water as a solvent, add 3g of trisodium citrate and 0.4g of citric acid to a final volume of 1L of antigen retrieval solution and heat to boiling. Place the slides into the antigen retrieval solution and microwave at 98℃ for 15 minutes, checking the solution level occasionally to prevent the slides from drying out. After completion, allow to cool naturally, remove the slides, and wash them three times with PBST for 5 minutes each time.

[0153] (3) Blocking: In order to eliminate the peroxidase contained in the tissue and prevent it from producing background staining during subsequent DAB staining, 3% hydrogen peroxide was used for blocking. The tissue was incubated at room temperature in the dark for 20 min, and then washed three times with PBST for 5 min each time.

[0154] (4) Blocking: Blot off excess PBS on the slide with filter paper, then draw a circle with a histochemical pen, about 0.5 cm away from the tissue. Add 5% normal goat serum to the tissue to completely cover it, and incubate at room temperature for 1 hour.

[0155] (5) Primary antibody incubation: Discard the blocking serum (do not wash), and directly add the primary antibody working solution (P53 antibody 1:500) prepared with antibody dilution buffer, ensuring complete coverage of the tissue. Place the slide horizontally in a humidified chamber and incubate overnight (approximately 16 hours) at 4°C.

[0156] (6) Secondary antibody incubation: Remove the humidified chamber from the refrigerator and allow it to warm to room temperature for 15 minutes. Wash the slides three times with PBST for 5 minutes each time to completely remove unbound primary antibody. Blot dry the PBST, add HRP-labeled secondary antibody working solution (goat anti-rabbit IgG-HRP) to completely cover the tissue, and incubate at room temperature for 1 hour. After incubation, rinse the slides three times with PBST for 5 minutes each time to remove unbound secondary antibody.

[0157] (7) DAB staining: According to the DAB kit instructions, mix DAB substrate solutions A and B in the specified ratio before use. Add the mixed DAB solution dropwise onto the tissue, ensuring complete coverage. Determine the staining time based on the specific antibody. When the positive signal (brownish-yellow) is clearly visible and the background staining is very light (usually 30 seconds to 5 minutes), immediately immerse the slide in running water to stop the reaction.

[0158] (8) Counterstaining: Counterstain the cell nuclei with hematoxylin for 5 minutes. Then rinse with running water. Differentiate with 1% hydrochloric acid for 30 seconds, then return to blue with running water.

[0159] (9) Dehydration and clearing: Pass the slices through the following steps in sequence: 75% ethanol (1 min) → 85% ethanol (1 min) → 95% ethanol (1 min) → 100% ethanol I (1 min) → 100% ethanol II (1 min) → xylene I (3 min) → xylene II (3 min).

[0160] (10) Mounting: Remove the section from the xylene, blot off excess liquid, add a drop of neutral resin to the center of the tissue, cover with a coverslip to avoid air bubbles. Let it air dry horizontally at room temperature.

[0161] Results analysis:

[0162] (a) Analysis of the effect of Eppin protein treatment on the growth of hepatocellular carcinoma xenografts in mice

[0163] Table 7 shows the body weight, tumor weight, and tumor size ratio of mice in each group at the end of the experiment. The transplanted tumors dissected from each group of mice are shown in Table 7. Figure 13 The results showed that the average tumor weight removed from the control group was 3.34±0.06g, while the average tumor weights in the low, medium, and high concentration Eppin protein treatment groups were 2.91±0.1g, 2.67±0.15g, and 2.48±0.08g, respectively. Therefore, the tumor weights in the medium and high concentration Eppin treatment groups were significantly lower than those in the control group (p<0.05). Simultaneously, the tumor-to-body weight ratio in the treatment groups was also significantly lower than that in the control group, demonstrating its tumor-suppressing effect. The body weight of the mice in the control group increased from 21.75±0.6g at the start of the experiment to 25.15±1.1g. The weight gain trend in each concentration Eppin protein treatment group was basically consistent with that in the control group, with no statistically significant difference between the groups. This indicates that within the concentration and treatment period set in this experiment, Eppin protein treatment did not cause significant systemic toxicity or weight loss in mice, suggesting good safety.

[0164] Table 7

[0165]

[0166] (b) Pathological analysis of Eppin protein on hepatocellular carcinoma xenografts in mice

[0167] Histopathological observation of mouse xenograft tumor tissue is shown in the figure. Figure 14 In the control group, the tumor tissue exhibited invasive growth; tumor cells were densely packed, with large, deeply stained nuclei and an increased nucleus-to-cytoplasm ratio, and numerous pathological mitotic figures were observed. Small focal necrosis (approximately <5%) was visible in the central part of the tumor, and a small number of lymphocytes infiltrated the stroma. Compared to the control group, the treatment group showed extensive areas of coagulative necrosis within the tumor tissue, accounting for approximately 60-70% of the total cut surface area; surviving tumor cells were mainly distributed at the tumor periphery, with significantly increased apoptosis phenomena such as nuclear pyknosis and fragmentation, and rare mitotic figures; numerous lymphocytes and macrophages infiltrated the necrotic areas and the tumor stroma. HE staining results indicated that Eppin protein promoted apoptosis in the tumor tissue.

[0168] (c) Analysis of the effect of Eppin protein on gene expression levels in hepatocellular carcinoma xenografts in mice

[0169] The results of the analysis on the effect of Eppin protein on gene expression levels in hepatocellular carcinoma xenografts in mice are shown in the table below. Figure 15 The results showed that the relative expression levels of BBC3, Caspase-3, Caspase-9, and TP53 genes were upregulated, while the expression level of VEGFD gene was downregulated. This indicates that Eppin protein leads to tumor cell death by activating the p53 pathway and inducing mitochondrial apoptosis.

[0170] (d) Analysis of the effect of Eppin protein on the expression level of xenograft proteins in hepatocellular carcinoma mice

[0171] Immunohistochemical map showing the effect of Eppin protein on p53 protein expression level is shown below. Figure 16 As shown, p53 protein is located in the cell nucleus and appears as brownish-yellow granular signals. In the control group, p53 protein showed only sporadic weak positive or negative expression. In contrast, in the tumor tissue of the drug treatment group, p53 protein expression was significantly upregulated, manifested by significantly deeper nuclear staining intensity and a significantly increased proportion of positive tumor cells. Furthermore, p53-positive cells were mostly distributed around the periphery of tumor necrosis areas or in areas with poor blood supply, suggesting that the drug may activate the p53 signaling pathway by inducing DNA damage stress.

[0172] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. The application of Eppin protein in the preparation of drugs for treating liver cancer, characterized in that, The amino acid sequence of the Eppin protein is shown in SEQ ID No.

1.

2. The application of the Eppin protein according to claim 1 in the preparation of a liver cancer treatment drug, characterized in that, The cDNA sequence of the Eppin protein is shown in SEQ ID No.

2.

3. The application of the Eppin protein according to claim 1 in the preparation of a liver cancer treatment drug, characterized in that, The Eppin protein is a Kunitz-type serine protease inhibitor extracted from pearl powder.

4. The application of the Eppin protein according to claim 1 in the preparation of a liver cancer treatment drug, characterized in that, The liver cancer treatment drug mentioned is a drug that inhibits the proliferation of liver cancer cells.

5. The application of the Eppin protein according to claim 1 in the preparation of a liver cancer treatment drug, characterized in that, The liver cancer treatment drug mentioned is a drug that inhibits the migration of liver cancer cells.

6. The application of the Eppin protein according to claim 1 in the preparation of a liver cancer treatment drug, characterized in that, The liver cancer treatment drug mentioned is a drug that inhibits the invasion of liver cancer cells.

7. The application of the Eppin protein according to claim 1 in the preparation of a liver cancer treatment drug, characterized in that, The liver cancer treatment drug mentioned is a drug that induces apoptosis in liver cancer cells.

8. The application of the Eppin protein according to claim 1 in the preparation of a liver cancer treatment drug, characterized in that, The liver cancer treatment drug mentioned is a drug that inhibits the cell cycle of liver cancer cells.

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