Use of polypeptides interfering with the binding of tia1 to eif4a3 in the preparation of a medicament for treating hepatocellular carcinoma

Abstract

The application discloses application of a polypeptide interfering with TIA1 and EIF4A3 combination in preparation of a hepatocellular carcinoma treatment drug, and relates to the technical field of biological medicine.The amino acid sequence of the polypeptide is shown as SEQ ID NO.1 or SEQ ID NO.2.The application first discloses a new mechanism that TIA1 is combined with an EIF4A3 / ALYREF complex through a Q domain, promotes MYC mRNA nuclear export, and provides a brand-new drug target, TIA1-EIF4A3 interaction interface, for HCC treatment.The application obtains, through virtual screening, a polypeptide specifically interfering with the interaction, the polypeptide can effectively block the protein-protein interaction between TIA1 and EIF4A3, inhibit MYC mRNA nuclear export, reduce MYC protein expression, and significantly inhibit the malignant biological behavior of HCC cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of peptides that interfere with the binding of TIA1 and EIF4A3 in the preparation of drugs for treating hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor, ranking sixth in global incidence and third in mortality, with over 700,000 deaths annually, posing a significant public health challenge. HCC is insidious in onset and highly aggressive, with approximately 60% of patients already having metastasized at initial diagnosis, primarily through intrahepatic dissemination and distant extrahepatic metastases, which are key reasons for treatment failure and high postoperative recurrence rates. Early-stage HCC can be cured through surgical resection and liver transplantation, with a five-year survival rate of 60%-80%; however, treatment options for metastatic patients are limited, with a five-year survival rate of less than 10%. Current treatments such as surgery, transplantation, and local ablation have limited efficacy against widespread metastatic lesions. First-line targeted drugs and immune checkpoint inhibitors have low objective response rates and are prone to drug resistance, only offering slight extensions of survival. This clinical predicament highlights the urgent need to elucidate the molecular mechanisms of HCC metastasis and identify key targets to provide a theoretical basis for developing intervention strategies to block HCC metastasis.

[0003] TIA1 is a functionally important member of the RNA-binding protein family, named after its initial discovery in T cells and its significant role in stress granule assembly. This protein contains three RNA recognition motifs (RRMs) that enable it to specifically recognize and bind to RNA sequences, thereby influencing processes such as RNA alternative splicing, stability, and translation efficiency, thus participating in tumor progression. However, the mechanism of action of TIA1 in HCC is not fully elucidated, particularly whether it participates in HCC progression through non-canonical pathways, especially by regulating RNA nuclear export; this has not yet been reported.

[0004] RNA nuclear export is a crucial step in the regulation of gene expression in eukaryotic cells. After transcription and splicing in the nucleus, mRNA must be transported to the cytoplasm via the nuclear pore complex to be translated into proteins. This process is synergistically accomplished by multiple nuclear export-related factors, among which EIF4A3 and ALYREF are important components of the RNA nuclear export complex. Specifically, during mRNA splicing, EIF4A3, as a core component of the exon joiner complex (EJC), is recruited and deposited at the exon join sites of mRNA, forming a stable EJC complex. Subsequently, ALYREF, as a key RNA nuclear export aptamer, is recruited to the mRNA through interaction with EIF4A3 and other components in the EJC complex. Finally, ALYREF further recruits the TREX complex and the nuclear export receptor NXF1, thereby mediating mRNA nuclear export.

[0005] Abnormal RNA nuclear export can lead to the overexpression of oncogenes. MYC is one of the most important driver genes in HCC, and the efficiency of its mRNA nuclear export directly affects MYC protein levels, thereby regulating tumor cell proliferation, metabolism, and apoptosis. Currently, direct targeted therapy against MYC faces many challenges, while intervening in its mRNA nuclear export process may be an effective alternative strategy.

[0006] In summary, whether TIA1 utilizes the EIF4A3 / ALYREF nuclear export complex to regulate MYC mRNA nuclear export and participate in HCC progression, and whether effective HCC treatment strategies can be developed based on this mechanism, have not been reported in the existing technology. Summary of the Invention

[0007] The purpose of this invention is to provide the application of a polypeptide that interferes with the binding of TIA1 and EIF4A3 in the preparation of drugs for treating hepatocellular carcinoma, thereby addressing the problems existing in the prior art. This polypeptide can effectively block the protein-protein interaction between TIA1 and EIF4A3, inhibit MYC mRNA nuclear export, and reduce MYC protein expression, thereby significantly inhibiting the malignant biological behavior of HCC cells.

[0008] To achieve the above objectives, the present invention provides the following solution: The present invention provides a polypeptide that interferes with the binding of TIA1 and EIF4A3, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0009] The present invention also provides a method for preparing the above-mentioned polypeptide, comprising the step of synthesizing the polypeptide by solid-phase synthesis.

[0010] The present invention also provides the use of the above-mentioned polypeptide in the preparation of drugs for treating hepatocellular carcinoma.

[0011] Furthermore, the hepatocellular carcinoma treatment drug exerts its therapeutic effect on hepatocellular carcinoma by inhibiting the proliferation, migration, and / or invasion of liver cancer cells.

[0012] Furthermore, the hepatocellular carcinoma treatment drug inhibits the proliferation, migration, and / or invasion of liver cancer cells by interfering with the binding of TIA1 to EIF4A3.

[0013] Furthermore, the hepatocellular carcinoma drug includes pharmaceutically acceptable excipients.

[0014] The present invention also provides a drug for treating hepatocellular carcinoma, wherein the active ingredient includes the aforementioned polypeptide.

[0015] Furthermore, the hepatocellular carcinoma drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

[0017] Furthermore, the dosage form of the drug is powder, tablet, granule, capsule, pill, or oral liquid.

[0018] The present invention discloses the following technical effects: This invention reveals for the first time a novel mechanism by which TIA1 promotes the nuclear export of MYC mRNA by binding to the EIF4A3 / ALYREF complex through its Q domain. This breakthrough overturns the previous understanding that TIA1 regulates tumors only through alternative splicing and RNA stability, and provides a completely new drug target for the treatment of HCC—the TIA1-EIF4A3 interaction interface.

[0019] Based on this novel target, this invention obtained peptides that specifically interfere with this interaction through virtual screening. Experiments demonstrated that these peptides effectively blocked the protein-protein interaction between TIA1 and EIF4A3, inhibited MYC mRNA nuclear export, reduced MYC protein expression, and significantly suppressed the malignant biological behavior of HCC cells. Therefore, this invention provides an HCC treatment strategy based on a novel mechanism and target, with promising clinical application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The following figures illustrate the experimental results to verify the binding of TIA1 to the EIF4A3 / ALYREF complex and its regulation of MYC mRNA nuclear output. A shows the Western blot electrophoresis result of the co-immunoprecipitation assay detecting the interaction between TIA1 and EIF4A3 / ALYREF; B shows the GST pull-down electrophoresis result verifying in vitro protein-protein interactions; C shows the qRT-PCR quantitative results of the RNA-binding protein immunoprecipitation assay detecting TIA1 binding to MYC mRNA; and D shows the quantitative statistical graph of the cytoplasmic and nuclear distribution of MYC mRNA in each group, detected by the nucleocytoplasmic separation assay. Figure 2Figure 1 shows the experimental results of blocking peptides to inhibit TIA1-EIF4A3 binding and downregulate MYC expression. A is a schematic diagram of molecular docking of the peptides targeting the TIA1 Q domain obtained through virtual screening; B is a Western blot electrophoresis image of TIA1-EIF4A3 binding levels detected by immunoprecipitation after peptide intervention; C is a quantitative statistical diagram of MYC mRNA distribution in the nucleus and cytoplasm after peptide treatment; D is a Western blot electrophoresis image of MYC protein expression levels in cells after peptide intervention. Figure 3 The diagrams show the functional validation of the peptide's in vitro inhibition of the malignant phenotype of liver cancer cells. A represents the quantitative staining results of the liver cancer cell colony formation assay after peptide treatment; B is a line graph showing the effect of the peptide on the proliferation activity of liver cancer cells at different time points as detected by the CCK-8 assay; C shows the Transwell assay results of the peptide's ability to inhibit the migration and invasion of liver cancer cells; and D is the cell count statistics for C. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1: Interaction between TIA1 and the EIF4A3 / ALYREF complex and its regulation of MYC mRNA nuclear export. 1. Immunoprecipitation assay Immunoprecipitation was used to verify the interaction between TIA1 protein and the EIF4A3 / ALYREF complex in the human hepatocellular carcinoma cell line MHCC97H, and the key functional domains of binding were identified, confirming that the Q domain of TIA1 is the core region for binding the EIF4A3 / ALYREF complex.

[0028] 1.1 Experimental Procedure Logarithmic growth phase MHCC97H cells were seeded into cell culture dishes. When the cell confluence reached 70%~80%, plasmid transfection was performed according to the following groups: (1) Negative control group (Vector group): transfected with an equal amount of empty vector plasmid; (2) Full-length TIA1 overexpression group (TIA1 group): transfected with Flag-TIA1 overexpression plasmid; (3) Q domain deletion mutant group (TIA1-ΔQ group): transfected with Flag-TIA1-ΔQ overexpression plasmid. After transfection, cells were cultured in a 37℃, 5% CO2 incubator for 48 h, and then collected for subsequent experiments.

[0029] Total protein extraction: Discard the cell culture medium, wash the cells three times with pre-cooled PBS; add RIPA lysis buffer containing protease inhibitors, and lyse on ice for 20 min; collect the lysis buffer, centrifuge at 4℃ and 12000 rpm for 5 min, take the supernatant to determine the protein concentration, and take an equal amount of total protein from each group for subsequent precipitation.

[0030] Immunoprecipitation: Take equal amounts of total protein supernatant from each group, add Flag antibody-coated magnetic beads that have been equilibrated with pre-cooled lysis buffer, and incubate overnight on a shaker at 4°C. After incubation, use a magnetic rack to separate the magnetic beads, discard the supernatant, and wash the magnetic beads 4 times with pre-cooled lysis buffer to thoroughly remove non-specific binding proteins.

[0031] Protein detection: SDS loading buffer was added to the washed magnetic beads, and the protein was denatured in a metal bath for 10 min to dissociate the protein. After separation by SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 2 h. EIF4A3 and ALYREF primary antibodies were added and incubated overnight at 4℃. After washing the membrane, the corresponding secondary antibodies were added and incubated at room temperature for 2 h. After adding ECL reagent, the luminescence image was acquired.

[0032] 1.2 Experimental Results Experimental results showed that specific bands of EIF4A3 and ALYREF were clearly detected in the precipitate of the TIA1 group, while no obvious binding bands were found in the Vector group and the TIA1-ΔQ group. Figure 1 (A)

[0033] 2. Pull Down Experiment The interaction mode of TIA1 with EIF4A3 and ALYREF was verified in the purified recombinant protein system using in vitro GST pull-down technology, elucidating the binding mode of the TIA1-EIF4A3 / ALYREF complex.

[0034] 2.1 Experimental Procedure Take equal amounts of GST-tagged protein agarose beads pre-equilibrated with incubation buffer, add the corresponding proteins according to the following groups, and construct a co-incubation reaction system. Add incubation buffer to each group to the same volume: (1) Negative control group: GST empty vector protein + His-EIF4A3 + Strep-ALYREF; (2) ALYREF-only group: GST-TIA1 fusion protein + Strep-ALYREF; (3) Double protein co-incubation group: GST-TIA1 fusion protein + His-EIF4A3 + Strep-ALYREF.

[0035] After thoroughly mixing each reaction system, incubate overnight on a 4°C rotating shaker to ensure complete binding of the GST fusion protein, the test protein, and the agarose magnetic beads. After incubation, separate the proteins using a magnetic rack and discard the supernatant. Wash the magnetic beads five times with pre-cooled washing buffer to thoroughly remove unbound free proteins and non-specifically bound proteins.

[0036] SDS protein loading buffer was added to the washed magnetic beads, and the mixture was heated in a boiling water bath for 10 min to denature the protein complexes bound to the magnetic beads. The eluted samples were then separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blocked with 5% skim milk at room temperature for 2 h. The membrane was then incubated overnight at 4°C with primary antibodies against Strep, His, and GST, respectively. After washing, the membrane was incubated with the corresponding secondary antibodies at room temperature for 2 h. Finally, ECL chemiluminescence reagent was added, and protein band images were acquired.

[0037] 2.2 Experimental Results Western blot results showed that no His-EIF4A3 and Strep-ALYREF bands were detected in the negative control group; in the group with only Strep-ALYREF added, GST-TIA1 could not produce an ALYREF band; while in the group with both His-EIF4A3 and Strep-ALYREF added, specific bands of EIF4A3 and ALYREF could be detected simultaneously. Figure 1 (B)

[0038] 3. RNA-binding protein immunoprecipitation assay Using RNA-binding protein immunoprecipitation, the interaction between TIA1 protein and MYC mRNA was verified in human hepatocellular carcinoma Huh7 cells, confirming that TIA1 can act as an RNA-binding protein to target and bind to MYC mRNA.

[0039] 3.1 Experimental Procedure Immunoprecipitation experiments were performed on lysates of Huh7 cells overexpressing Flag-TIA1. The following groups were set up, and a portion of the lysate was retained as an Input positive control: (1) IgG control group: normal IgG was added as a negative control antibody. (2) Flag antibody group: anti-Flag specific antibody was added. The cells were incubated overnight at 4°C to allow the intracellular TIA1-RNA complex to be captured by the specific antibody.

[0040] After incubation, the magnetic beads were washed several times with the kit's washing solution to remove non-specific binding components; proteinase K digestion solution was added to digest the binding proteins, releasing the co-precipitated RNA, and then the total RNA was purified and recovered.

[0041] The RNA from each group was simultaneously reverse transcribed into cDNA along with the RNA from the Input sample. The content of MYC mRNA was detected by real-time quantitative PCR, and the enrichment fold was calculated with the Input sample as a reference.

[0042] 3.2 Experimental Results qRT-PCR results showed that, compared with the IgG control group, the enrichment level of MYC mRNA in the Flag antibody group was significantly increased. Figure 1 (C)

[0043] 4. Nucleocytoplasmic separation experiment By using nuclear-cytoplasmic component separation technology, we detected the distribution changes of MYC mRNA in subcellular structures, verified the regulatory role of TIA1 in the nuclear export process of MYC mRNA, and clarified whether this regulatory effect depends on the mediation of the EIF4A3 / ALYREF complex.

[0044] 4.1 Experimental Procedure Log-phase Huh7 cells were seeded and cultured. When the confluence reached 70%, they were transfected into the following groups: (1) Ctrl group; (2) TIA1 overexpression group; (3) TIA1 overexpression + EIF4A3 knockdown group; (4) TIA1 overexpression + ALYREF knockdown group. Each group was set up with 3 replicates. Cells were collected 48 h after transfection.

[0045] Collect cells from each group, wash with pre-chilled PBS, resuspend cells in pre-chilled Cell Fractionation Buffer, and incubate on ice for 10 min; centrifuge at 4°C and 500×g for 5 min, carefully aspirate the supernatant as the cytoplasmic fraction; the precipitate is the nuclear fraction, add pre-chilled Cell Disruption Buffer and vortex thoroughly to lyse.

[0046] Add equal volumes of 2×Lysis / Binding Solution to the cytoplasmic and nuclear components, mix well, and then purify and extract total RNA from both components according to the kit procedure.

[0047] RNA from each group was simultaneously reverse transcribed into cDNA, and MYC mRNA levels were detected using real-time quantitative PCR. Cytoplasmic samples were standardized using GAPDH as an internal control, and nuclear samples were standardized using U6 as an internal control. The cytoplasmic-nuclear abundance of MYC mRNA was calculated to reflect its nuclear export level.

[0048] 4.2 Experimental Results The results showed that compared with the Ctrl group, the cytoplasmic level of MYC mRNA in the TIA1 overexpression group was significantly increased and the nuclear level was decreased; while knocking down EIF4A3 or ALYREF simultaneously blocked TIA1-mediated nuclear export of MYC mRNA. Figure 1 (D).

[0049] Example 2 Virtual screening and synthesis of peptides Based on the molecular mechanism by which TIA1 binds to EIF4A3 protein through its Q domain, computer-aided drug design technology was used to screen for peptides that can specifically bind to the TIA1 Q domain, thereby blocking the TIA1-EIF4A3 interaction through competitive binding.

[0050] 1. Experimental Procedure The three-dimensional structure of the TIA1Q domain and EIF4A3 was constructed using AlphaFold 3, and the complex conformation was obtained by ZDOCK protein docking. By combining molecular dynamics and MM-PBSA free energy decomposition, the key binding residues of the TIA1Q domain (Asn-315, Gln-318, Met-334, Pro-356, Tyr-358) were identified, and the core targeting pocket was determined.

[0051] Using the core pocket as the target, 1,000 15-30 aa peptide backbones were designed using RFdiffusion, and a total of 10,000 candidate sequences were obtained after ProteinMPNN sequence optimization; then, high-affinity peptides were retained through AutoDock Vina molecular docking preliminary screening.

[0052] The complex structure was predicted using AlphaFold 3, and 543 high-confidence peptides were screened based on ipTM > 0.8 and ranking_score > 0.85. The binding energy was then evaluated using Rosetta, and 9 high-affinity candidate peptides were obtained.

[0053] Molecular dynamics simulations were performed on nine candidate peptides to evaluate the conformational stability of the complexes, and finally, the optimal peptide with the most stable binding was selected.

[0054] 2. Experimental Results After multiple rounds of virtual screening and kinetic verification, a blocking peptide (amino acid sequence shown in SEQ ID NO.1) that can specifically bind to the TIA1Q domain was obtained, which can competitively inhibit the TIA1-EIF4A3 interaction. Figure 2 (A)

[0055] The membrane-penetrating peptide and the localizing peptide were linked to the screened blocking peptide, and the peptide F-TN-TIA1 was synthesized using a solid-phase synthesis method. Its amino acid sequence is shown in SEQ ID NO.2. The synthesized peptide met the purity and structure requirements and can be used for subsequent functional verification.

[0056] SEQ ID NO. 1: AEEEARLAEIQGLLNALRIEMEE.

[0057] SEQ ID NO.2: YGRKKRRQRRRAEEEARLAEIQGLLNALRIEMEEPKKKRKV; wherein, amino acids 1-11 are transmembrane peptides; amino acids 12-34 are blocking peptides; and amino acids 35-41 are nuclear localization signal peptides.

[0058] Further, a 6-aminocaproic acid flexible spacer arm (Acp) was attached to the N-terminus of the peptide F-TN-TIA1, and then the FITC fluorescent group was coupled to prepare a fluorescently labeled peptide to meet the subsequent fluorescence detection requirements.

[0059] Fluorescently labeled peptide: Fitc-Acp-YGRKKRRQRRRAEEEARLAEIQGLLNALRIEMEEPKKKRKV.

[0060] Example 3: Peptide blocks TIA1-EIF4A3 binding and inhibits MYC nucleus export. To verify the competitive blocking effect of the blocking peptide targeting the TIA1Q domain on the TIA1-EIF4A3 protein interaction, and to clarify the regulatory role of this peptide in the nuclear export of MYC mRNA and the expression of MYC protein.

[0061] 1. Experimental Methods Logarithmic growth phase MHCC97H cells were divided into 3 groups for treatment: (1) DMSO solvent group (DMSO): an equal amount of DMSO was added as a blank solvent control; (2) Empty vector control group (F-TN): 40 μM empty vector peptide F-TN was added; (3) Targeting peptide group (F-TN-TIA1): 40 μM targeting peptide F-TN-TIA1 was added. Each group was incubated in a 37℃, 5% CO2 cell culture incubator for 24 h.

[0062] Total protein was extracted from cells in each group, and anti-TIA1 antibody was added and incubated overnight at 4°C. Protein A / G magnetic beads were added to capture the antibody-protein complex. After thorough washing, the bound protein was eluted, and the content of EIF4A3 in the precipitated complex was detected by Western blot to evaluate the binding blocking effect of the peptide.

[0063] Cells from each group were collected, washed with pre-cooled PBS, and incubated on ice with cytoplasmic separation buffer. After centrifugation, the supernatant was the cytoplasmic fraction, and the precipitate was treated with nuclear lysis buffer to obtain the nuclear fraction. Total RNA was extracted from both fractions, reverse transcribed into cDNA, and MYC mRNA levels were detected by qRT-PCR. Cytoplasmic samples were standardized using GAPDH as an internal control, and nuclear samples were standardized using U6 as an internal control. The cytoplasmic / nuclear abundance ratio of MYC mRNA was calculated to reflect its nuclear export level.

[0064] Total protein was extracted from cells in each group, and after SDS-PAGE electrophoresis, membrane transfer, and blocking, the cells were incubated with MYC primary and secondary antibodies in sequence. ECL chemiluminescence imaging was then performed to detect the expression level of MYC protein in the cells.

[0065] 2. Experimental Results Immunoprecipitation results showed that clear EIF4A3-specific bands were detected in the TIA1 precipitate complex in both the DMSO group and the empty vector control group; however, almost no EIF4A3 bands were detected in the precipitate complex in the targeted peptide treatment group. Figure 2 (B)

[0066] Nuclear-cytoplasmic separation results showed that, compared with the DMSO group and the empty vector control group, the level of MYC mRNA in the cell nucleus of the targeted peptide group was significantly increased. Figure 2 (C)

[0067] Western blot results showed that there was no significant difference in MYC protein expression levels between the DMSO group and the empty vector control group; however, the intracellular MYC protein expression level was significantly downregulated in the targeted peptide group. Figure 2 (D).

[0068] Example 4 Cell Experiment To verify the regulatory effect of the TIA1Q domain-targeting blocking peptide on the proliferation, colony formation, migration, and invasion of human hepatocellular carcinoma cells, and to evaluate the in vitro antitumor activity of the peptide.

[0069] 1. Experimental Methods 1.1 Cloning experiment Logarithmic growth phase MHCC97H cells were seeded at a low density (1000 cells / well) into 6-well plates and divided into 3 groups, including (1) DMSO solvent group (DMSO): an equal amount of DMSO was added to the culture medium as a blank solvent control; (2) empty vector control group (F-TN): the empty vector peptide F-TN was added to the culture medium at a final concentration of 40 μM; (3) target peptide group (F-TN-TIA1): the target peptide F-TN-TIA1 was added to the culture medium at a final concentration of 40 μM.

[0070] Each group was cultured for 14 days until visible clones were formed, at which point the culture was terminated. After fixation with paraformaldehyde and staining with crystal violet, the clone formation was calculated.

[0071] 1.2 Cell proliferation CCK-8 assay and migration assay Logarithmic growth phase MHCC97H cells were divided into 3 groups for treatment: (1) DMSO solvent group (DMSO): an equal amount of DMSO was added as a blank solvent control; (2) Empty vector control group (F-TN): 40 μM empty vector peptide F-TN was added; (3) Targeting peptide group (F-TN-TIA1): 40 μM targeting peptide F-TN-TIA1 was added. Each group was incubated in a 37℃, 5% CO2 cell culture incubator for 24 h, and cell proliferation CCK-8 assay and migration experiment were performed respectively. Cell proliferation CCK-8 assay: Cells from each group were seeded into 96-well plates at the same density. CCK-8 reagent was added to the corresponding wells at 0, 24, 48, 72, and 96 h of culture. After incubation for 1 h, the absorbance at 450 nm was measured using an ELISA reader. Cell proliferation curves were plotted to assess changes in cell proliferation capacity.

[0072] Migration assay: Cells from each group, resuspended in serum-free medium, were seeded into the upper chamber of a Transwell cell line, while serum-containing complete culture medium was added to the lower chamber. After 24 hours of culture, the chambers were removed, and any cells that had not penetrated the membrane from the upper chamber were wiped away. After fixation and crystal violet staining, the number of cells that had penetrated the membrane was counted under a microscope. Invasion assay: The upper chamber was pre-coated with Matrigel, and the remaining procedures were the same as for the migration assay. Changes in cell invasion ability were then detected.

[0073] 2. Experimental Results Clonogenic assays showed no significant difference in clone formation between the DMSO group and the empty vector control group, but the number of clones in the targeted peptide group was significantly reduced, and clone formation was substantially decreased. Figure 3 (A)

[0074] CCK-8 results showed that compared with the DMSO group and the empty vector control group, the absorbance value of the targeted peptide group was significantly reduced, and cell proliferation was significantly slowed down. Figure 3 (B)

[0075] Transwell experiments showed that there was no significant difference in the number of cells that crossed the chamber between the DMSO group and the empty control group, while the number of cells that crossed the membrane was significantly reduced in the targeted peptide group, and both migration and invasion abilities were significantly inhibited. Figure 3 Medium CD).

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A polypeptide that interferes with the binding of TIA1 to EIF4A3, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A method for preparing the polypeptide as described in claim 1, characterized in that, The method includes the step of synthesizing the polypeptide using a solid-phase synthesis method.

3. The use of the polypeptide as described in claim 1 in the preparation of a drug for treating hepatocellular carcinoma.

4. The application according to claim 3, characterized in that, The drug for treating hepatocellular carcinoma exerts its therapeutic effect by inhibiting the proliferation, migration, and / or invasion of liver cancer cells.

5. The application according to claim 4, characterized in that, The drug for treating hepatocellular carcinoma inhibits the proliferation, migration, and / or invasion of liver cancer cells by interfering with the binding of TIA1 to EIF4A3.

6. The application according to claim 3, characterized in that, The hepatocellular carcinoma drug includes pharmaceutically acceptable excipients.

7. A drug for treating hepatocellular carcinoma, characterized in that, The active ingredient includes the polypeptide described in claim 1.

8. The drug for treating hepatocellular carcinoma according to claim 7, characterized in that, The hepatocellular carcinoma drug also includes pharmaceutically acceptable excipients.

9. The drug for treating hepatocellular carcinoma according to claim 8, characterized in that, The excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

10. The drug for treating hepatocellular carcinoma according to claim 9, characterized in that, The dosage form of the drug is powder, tablet, granule, capsule, pill, or oral liquid.

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