Biomarker related to acetylation of K147 site of histone H1 and application of biomarker in liver cancer treatment

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a biomarker related to acetylation of a K147 site of histone H1 and application of the biomarker in liver cancer treatment. The invention relates to a biomarker related to acetylation of a K147 site of histone H1. According to the biomarker, the histone H1 is subjected to acetylation modification at the K147 site. Bioinformatics analysis shows that the high expression state of acetyltransferase ELP3 is closely related to the occurrence and development of hepatocellular carcinoma, ELP3 can directly regulate and control acetylation modification of histone H1, then the core catalytic site of ELP3 is verified through site mutation, and the K147 acetylation site located on the histone H1 is precisely determined. Test results show that intervention of the ELP3-H1-K147 pathway can significantly inhibit proliferation and migration ability of liver cancer cells, and can provide research and development direction and theoretical support for development of novel liver cancer treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomarker related to the acetylation of histone H1 at the K147 site and its application in the treatment of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver and is one of the leading causes of cancer death and morbidity worldwide. According to data from the Asian Pacific Association for the Study of the Liver (APSL) and the European Association for the Study of the Liver (EASL), liver cancer accounts for approximately 5.4% of all cancer cases, with patients in the Asia-Pacific region accounting for 75%-80% of global cases. The main causes of liver cancer include chronic infection with hepatitis B or hepatitis C virus, consumption of food contaminated with aflatoxin, alcoholism, being overweight, type 2 diabetes, and smoking. Chronic viral hepatitis infection is a major risk factor leading to liver fibrosis, cirrhosis, and ultimately, hepatocellular carcinoma. Currently, the main treatment for HCC is targeted therapy; however, most HCC cases are diagnosed at an advanced stage, resulting in limited treatment efficacy. Therefore, there is an urgent clinical need to find effective treatments and interventions for HCC.

[0003] Traditional treatments for liver cancer include surgical intervention, radiotherapy, chemotherapy, and immunotherapy. However, liver cancer cells are characterized by unlimited proliferation, detachment, and easy metastasis. Currently, the main clinical treatments for early-stage liver cancer are radical surgical resection, local ablation, and liver transplantation. However, due to the insidious onset of early-stage liver cancer, approximately 60% of patients are asymptomatic or have already progressed at the time of diagnosis, missing the optimal window for radical surgery and resulting in poor prognosis. Advanced-stage patients can receive adjuvant therapies such as radiotherapy and chemotherapy, but issues such as drug resistance and patient intolerance remain. Furthermore, advanced-stage liver cancer cells may have already metastasized to other tissues, leading to a very high recurrence rate. Although many chemotherapy drugs are already in clinical use, existing targeted therapies are limited. In the development and progression of liver cancer, in addition to gene mutations, epigenetic dysregulation, particularly the imbalance of histone modifications, plays a crucial role in driving malignant proliferation and metastasis of cancer cells. Therefore, screening key genes related to the proliferation and differentiation of liver cancer cells and exploring the biological characteristics of liver cancer are of great significance for finding new molecular targets and effective treatment strategies. Summary of the Invention

[0004] To address the shortcomings of existing technologies, one objective of this invention is to provide a biomarker associated with acetylation at the K147 site of histone H1.

[0005] A second objective of this invention is to provide a biomarker related to the acetylation of histone H1 at the K147 site for use in the preparation of drugs for the treatment of liver cancer.

[0006] A third objective of this invention is to provide the application of biomarkers in screening drugs for the treatment of liver cancer.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A biomarker associated with acetylation at the K147 site of histone H1, wherein the biomarker is an acetylation modification at the K147 site of histone H1.

[0008] Furthermore, the amino acid sequence of histone H1 is shown in SEQ ID NO.3.

[0009] Furthermore, the biomarker is used in the treatment of liver cancer.

[0010] Furthermore, acetylation modification was performed using the acetyltransferase ELP3.

[0011] The above-mentioned biomarkers are used in the preparation of drugs for the treatment of liver cancer.

[0012] The above-mentioned biomarkers are used in the preparation of inhibitors, antibodies or nanomedicines for the treatment of liver cancer.

[0013] Furthermore, the target site of the inhibitor, antibody, or nanomedicine is the K147 acetylation site of histone H1.

[0014] The above-mentioned biomarkers are used in screening drugs for the treatment of liver cancer.

[0015] Furthermore, the target of the drug for screening liver cancer treatment is the K147 acetylation site of histone H1.

[0016] Compared with the prior art, the main advantages of the present invention are as follows: (1) This invention, through bioinformatics and molecular biology experiments, reveals for the first time that the acetyltransferase ELP3 can directly regulate the acetylation modification of histone H1. Furthermore, by verifying its core catalytic site through site mutation, it was precisely located at the K147 acetylation site of histone H1. The experimental results show that intervention in the "ELP3-H1-K147" pathway can significantly inhibit the proliferation and migration of liver cancer cells, providing research direction and theoretical support for the development of novel liver cancer therapeutic drugs.

[0017] (2) This invention is the first to propose histone H1 K147 acetylation level as a specific biomarker, providing a new and more accurate molecular detection indicator for early diagnosis, prognosis assessment and efficacy monitoring of hepatocellular carcinoma.

[0018] (3) This invention innovatively establishes the “ELP3-H1-K147” pathway as a precise target for drug intervention. Based on this, small molecule inhibitors and antibodies targeting ELP3 can be screened or designed, or gene / protein therapeutic drugs based on H1 K147R mutants can be developed, providing a clear direction and direct target for developing novel targeted therapies for liver cancer that are highly effective and low in toxicity. Attached Figure Description

[0019] Figure 1 Bioinformatics analysis of ELP3 and histone H1 in hepatocellular carcinoma; among which, Figure 1 (a) Results showing the association between ELP3 and hepatocellular carcinoma; Figure 1 (b) To preliminarily screen the interaction between histone H1 and ELP3; Figure 2 The results of plasmid construction; among them, Figure 2 (a) shows the results of double enzyme digestion of the pBobi vector; Figure 2 (b) Colony PCR results for the H1 and ELP3 genes; Figure 3 The results are from Western blotting analysis using immunoprecipitation; among them, Figure 3 (a) ELP3 can be coprecipitated by H1 in HEK293T cells; Figure 3 (b) In HEK293T cells, H1 can be coprecipitated by ELP3; Figure 4 These are the results of an immunofluorescence experiment; Figure 5 The image shows the Western blot results of the acetylation catalysis of histone H1 by ELP3; where, Figure 5 (a) Both Flag-H1 and HA-ELP3 are expressed normally; Figure 5 (b) ELP3 can regulate the acetylation of histone H1; Figure 6 The image shows the results of screening key sites for ELP3-catalyzed histone H1 acetylation; among them, Figure 6 (a) Prediction of histone H1 acetylation sites; Figure 6 (b) is a graph showing the decrease in acetylation level after mutation at the K147 site of histone H1; Figure 7 The figure shows the effect of key sites in ELP3-catalyzed histone H1 acetylation on the proliferation ability of liver cancer cells; among them... Figure 7(a) Microscopic images showing cell migration results from scratches in different experimental groups; Figure 7 (b) is a graph showing the results of quantitative analysis of cell migration ability; compared with the HA group, *P<0.05; compared with the H1K147R-ELP3 group, *P<0.05; compared with the H1K147R-ELP3 group, ***P<0.001. Figure 8 This refers to the results of Western Blot analysis. Figure 9 The figure shows the effect of key sites of ELP3-catalyzed histone H1 acetylation on the migration ability of hepatocellular carcinoma cells; compared with the H1-ELP3 group, *P<0.05; at 48 h, compared with the HA group, **P<0.01, and compared with the H1-ELP3 group, *P<0.05; at 72 h, compared with the HA group, **P<0.01, and compared with the H1-ELP3 group, ****P<0.0001. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commonly available channels.

[0021] Example 1 To investigate the mechanism of action of ELP3 in hepatocellular carcinoma (HCC) and screen its key interacting proteins in HCC cells, the specific experimental steps are as follows: Transcriptome data of HCC and adjacent normal tissues were downloaded from the Cancer Genome Database (TCGA). Bioinformatics analysis was used to differentiate the mRNA expression levels of ELP3 between HCC tissues and adjacent normal tissues. The results are as follows: Figure 1 As shown in (a). Target proteins interacting with ELP3 were initially screened using mass spectrometry identification and protein network analysis, followed by Western blotting. The results are shown in (a). Figure 1 As shown in (b).

[0022] The results are as follows Figure 1 The figure shows a bioinformatics analysis of ELP3 and histone H1 in hepatocellular carcinoma. As can be seen from the figure, compared with adjacent normal liver tissue, the expression level of ELP3 mRNA in hepatocellular carcinoma tissue is significantly upregulated (…). Figure 1(a) indicates that high ELP3 expression is closely related to the development and progression of hepatocellular carcinoma. Mass spectrometry identification and protein network results show that histone H1 is one of the candidate proteins with the highest enrichment and strongest correlation among ELP3 interacting proteins. Figure 1 (b) indicates that ELP3 may participate in regulating the biological processes of liver cancer cells through direct interaction with nuclear chromatin structural protein H1.

[0023] Example 2 To clarify the relationship between ELP3 and histone H1, this study constructed related plasmids such as pBobi-N-HA-ELP3 and pBobi-N-Flag-H1 to further explore the interaction between ELP3 and protein H1 and their impact on liver cancer cells. The specific experimental steps are as follows: (1) Construction of recombinant expression plasmid Using ELP3 and histone H1 genes as templates, specific primers were designed to amplify the ELP3 target gene fragment (SEQ ID NO.1) of approximately 1644 bp and the H1 target gene fragment (SEQ ID NO.2) of approximately 648 bp, respectively, using PCR. The above fragments and the pBobi empty vector were double-digested with restriction endonucleases EcoRI and Not I, respectively. After purification and recovery, they were ligated using T4 DNA ligase and transformed into competent *E. coli* cells. The pBobi vector was identified by restriction endonucleases EcoRI and Not I, and the construction of the pBobi-N-HA-ELP3 expression plasmid and the pBobi-N-Flag-H1 expression plasmid was verified by colony PCR. The results are as follows: Figure 2 As shown.

[0024] The results are as follows Figure 2 The image shows the construction results of the pBobi-N-HA-ELP3 and pBobi-N-Flag-H1 expression plasmids. Figure 2 It can be seen that the pBobi-N-HA-ELP3 expression plasmid and the pBobi-N-Flag-H1 expression plasmid were successfully constructed.

[0025] Table 1 Sequence List Table 2 Sequence List (2) Immunoprecipitation was used to verify the protein-protein interaction between ELP3 and histone H1. pBobi-N-HA-ELP3 and pBobi-N-Flag-H1 plasmids were transfected into HEK293T cells using PEI transfection reagent. 48 h after transfection, cells were collected and washed once with pre-chilled 1×PBS. 1 mL of weak lysis buffer was added, and the cells were gently pipetted and transferred to centrifuge tubes. The cell suspension was sonicated at 30% power (5 seconds per sonication, repeated twice). The sample was then centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected as total protein lysate. 100 μL of the supernatant was used as a positive control. The remaining supernatant was transferred to new centrifuge tubes, and 20 μL of Protein A / G agarose beads and 0.1 μg of mouse IgG were added to each tube. The tubes were incubated at 4°C for 1 h. The sample was then centrifuged at 4,000 rpm for 3 minutes, and the supernatant was transferred to another centrifuge tube containing 20 μL of Protein A / G agarose beads and 0.1 μg of mouse anti-Flag antibody. The tube was incubated overnight at 4°C. After incubation, the beads were washed three times with pre-chilled lysis buffer. Finally, the beads were resuspended in 40 μL of lysis buffer, and 10 μL of 5× SDS protein loading buffer was added. After mixing, the sample was boiled at 100°C for 10 minutes to obtain the immunoprecipitated sample. The protein sample was then separated by SDS-PAGE and transferred to a PVDF membrane. Western blotting was performed using antibodies against histone H1 (IP:H1) and anti-ELP3 (IP:ELP3), respectively. Non-specific IgG antibody (IP:IgG) was used as a negative control to verify the interaction. The results are as follows: Figure 3 As shown.

[0026] The results are as follows Figure 3 The image shows the results of Western blotting analysis using immunoprecipitation. Figure 3 It can be seen that in HEK293T cells, when immunoprecipitation is performed using an anti-histone H1 antibody (IP:H1), ELP3 protein can be detected in the precipitation complex. Figure 3 a). In HEK293T cells, H1 protein was also detected in the precipitation complex when immunoprecipitation was performed using anti-ELP3 antibody (IP:ELP3). Figure 3 b). The results of this two-way immunoprecipitation experiment demonstrate that there is a specific direct protein-protein interaction between exogenously expressed ELP3 and histone H1 in HEK293T cells.

[0027] (3) Immunofluorescence assay to analyze the subcellular localization of ELP3 and histone H1 Immunofluorescence assays were performed on HEK293T cells by transfecting plasmids pBobi-N-HA-ELP3 and pBobi-N-Flag-H1. The specific steps of the immunofluorescence assay were as follows: HEK293T cells were cultured by placing coverslips at the bottom of a 6-well plate. After transfecting HEK293T cells with plasmids pBobi-N-HA-ELP3 and pBobi-N-Flag-H1, the cells were cultured for another 48 h. The culture medium was discarded, and the cells grown on the coverslips were gently washed three times with PBS. The cells were then fixed with 4% paraformaldehyde solution for 15 min. Subsequently, the cells were perforated with 0.3% Triton X-100 solution for 20 min. After perforation, the cells were gently washed with PBS. The coverslips were then blocked by immersing them in 1% BSA solution and incubated at room temperature for 1 h. After blocking, the antibody was diluted to an appropriate concentration in 1% BSA solution and added to the coverslips. The coverslips were incubated overnight at 4°C to ensure adequate binding of the antibody to the cellular antigen. After washing with PBS to remove unbound primary antibody, the corresponding secondary antibody conjugated with Alexa Fluor 594 was added, and the slides were incubated at room temperature in the dark for 1 hour. After washing three times with PBS, a DAPI-containing anti-fluorescence quenching mounting medium was added to the slides, and the cell side of the coverslips was placed face down. Fluorescence images were observed and acquired using a Leica DMRE confocal laser scanning microscope. The results are shown below. Figure 4 As shown.

[0028] The results are as follows Figure 4 The image shows the results of an immunofluorescence experiment. Figure 4 It was found that ELP3 protein is distributed in both the cytoplasm and the nucleus, but its fluorescence signal is stronger in the cytoplasm and relatively weaker in the nucleus. Histone H1 is completely localized in the nucleus of eukaryotes, forming chromosomes together with DNA; therefore, histone H1 is uniformly distributed in the nucleus. These results indicate that although ELP3 is mainly located in the cytoplasm, its portion entering the nucleus has a strong positive localization with histone H1 in the nucleus, suggesting a cellular basis for their interaction within the nucleus.

[0029] Example 3 To investigate whether ELP3 catalyzes histone H1 acetylation modification, we performed an in vivo acetylation experiment. The specific experimental steps are as follows: The expression vector pBobi-N-HA-ELP3, alone or co-transfected with the pBobi-N-Flag-H1 plasmid, was transfected into HEK293T cells. Forty-eight hours after transfection, cells were collected and lysed to obtain total protein. Immunoprecipitation was performed on the cell lysates using an anti-Flag antibody to specifically enrich Flag-H1 protein and its interacting complexes. Subsequently, the obtained immunoprecipitated complexes and the total protein lysates (input) used as controls were analyzed by Western blotting. The following antibodies were used for detection: anti-Flag antibody was used to detect Flag-H1 protein expression (WB:Flag-H1), anti-HA antibody was used to detect HA-ELP3 protein expression (WB:HA-ELP3), and anti-acetylated lysine antibody Ac was used to directly detect changes in histone H1 acetylation modification levels (WB:Ac). The results are shown below. Figure 5 As shown.

[0030] The results are as follows Figure 5 The image shows the Western blot results of the acetylation catalysis of histone H1 by ELP3. The input group shows that both Flag-H1 and HA-ELP3 are normally expressed. In the IP group, the first three lanes are for single-transfer Flag-H1, and the last three lanes are for co-transfer of Flag-H1 and HA-ELP3. Figure 5 It can be seen that Flag-H1 protein was normally expressed in total cell lysate, regardless of whether it was transfected alone or co-transfected; HA-ELP3 protein was also successfully expressed in the co-transfected group, indicating that the experimental system was successfully constructed. Figure 5 a). In the control group transfected with Flag-H1 alone, only a very weak acetylation signal was observed at the location corresponding to the H1 protein (approximately 33 kDa); while in the experimental group co-transfected with Flag-H1 and HA-ELP3, a significantly enhanced and clear acetylation signal band appeared at the same location. Figure 5 (b) The above results indicate that co-expression of ELP3 significantly increased the acetylation level of histone H1. This suggests that ELP3, as an acetyltransferase, directly catalyzes the acetylation modification of histone H1 in vivo, revealing a novel mechanism by which ELP3 regulates chromatin structure.

[0031] Example 4 To determine the key domains of the ELP3 protein interaction with histone H1 and to screen for key sites on ELP3-catalyzed histone H1 acetylation, the specific experimental steps are as follows: (1) A series of ELP3 truncated expression plasmids carrying different functional domains were constructed using molecular cloning technology. Each truncated plasmid was co-transfected into HEK293T cells with the full-length pBobi-N-Flag-H1 plasmid. Cell lysates were collected 48 hours after transfection, and immunoprecipitation was performed using anti-Flag antibody. The co-precipitation of each ELP3 truncated plasmid with H1 was then detected by Western blotting to identify the key structural domains mediating the interaction.

[0032] (2) To screen key sites for H1 acetylation catalyzed by ELP3, we used bioinformatics software to analyze the amino acid sequence of histone H1 (SEQ ID NO.3) and predict its potential acetylation modification lysine sites. Based on the prediction results, we successfully constructed histone H1 mutant plasmids H1K129R, H1K138R, H1K140R, and H1K147R using site-directed mutagenesis.

[0033] SEQ ID NO.3: MSETVPPAPAASAAPEKPLAGKKAKKPAKAAAASKKKPAGPSVSELIVQAASSSKERGGVSLAALKKALAAAGYDVEKNNSRIKLGIKSLVSKGTLVQTKGTGASGSF KLNKKASSVETKPGASKVATKTKATGASKKLKKATGASKKSVKTPKKAKKPAATRKSSKNPKKPKTVKPKKVAKSPAKAKAVKPKAAKARVTKPKTAKPKKAAPKKK.

[0034] (3) To screen key acetylation sites catalyzed by ELP3, we performed an in vivo acetylation experiment based on immunoprecipitation. The specific steps are as follows: The expression vector pBobi-N-HA-ELP3 and pBobi-N-Flag-H1 Flag-H1 (wild-type) or each site-directed mutant plasmid (Flag-H1K129R, H1K138R, H1K140R, H1K147R) were co-transfected into HEK293T cells using PEI transfection reagent. Forty-eight hours after transfection, the cells were washed with pre-cooled PBS, weak lysis buffer was added, and the cells were fully lysed by sonication (30% power, twice, 5 seconds each time). The lysis buffer was centrifuged at 12,000 ×g for 10 minutes at 4°C, and the supernatant was collected as the total protein lysis buffer. A portion of the lysis buffer was used as an input control. The remaining lysis buffer was incubated overnight at 4°C with agarose beads pre-coated with anti-Flag antibody to immunoprecipitate Flag-H1 protein and its interaction complex. The immunoprecipitated complex was washed three times with pre-chilled lysis buffer, then 1× SDS loading buffer was added, and the mixture was boiled at 100°C for 10 minutes to elute the protein. The eluted sample and input sample were separated by SDS-PAGE and then analyzed by Western blotting. The acetylation modification level of H1 protein (WB:Ac) was detected using an anti-acetylated lysine antibody. Simultaneously, anti-Flag antibody (WB:Flag) and anti-HA antibody (WB:HA) were used to verify the precipitation efficiency of H1 and the co-precipitation of ELP3, respectively, to ensure the effectiveness of the experimental system. The results are as follows: Figure 6 As shown.

[0035] The results are as follows Figure 6 As shown, in vivo acetylation experiments revealed that lysine 147 of histone H1 is the most critical site for ELP3-catalyzed acetylation modification in all H1 point mutants. This discovery pinpoints the regulation of H1 by ELP3 to a specific amino acid residue, providing a core target for the subsequent development of precise intervention strategies.

[0036] Example 5 To investigate the effect of different potential acetylation sites of histone H1 catalyzed by ELP3 on the migration ability of liver cancer cells, the specific experimental steps are as follows: (1) Cell scratch migration assay To investigate the effect of different potential acetylation sites of histone H1 catalyzed by ELP3 on the migration ability of hepatocellular carcinoma cells, we constructed a transient transfection model in HepG2 cells for comparison: HA group (control group): transfected with empty pBobi-N-HA plasmid.

[0037] H1 group: transfected with pBobi-N-Flag-H1 plasmid. H1+ELP3 group: co-transfected with pBobi-N-Flag-H1 and pBobi-N-HA-ELP3 plasmids. H1 point mutation co-expression group: pBobi-N-HA-ELP3 plasmid was co-transfected with four pBobi-N-Flag-H1 mutant plasmids (K129R, K138R, K140R, K147R) into HepG2 cells to obtain H1K129R-ELP3, H1K138R-ELP3, H1K140R-ELP3, and H1K147R-ELP3. Forty-eight hours after transfection, uniform scratches were created on a HepG2 cell monolayer using a sterile pipette tip. At 0 and 24 hours post-scratching, images were taken under a microscope showing the cells migrating to the fixed positions of the scratches. The results are as follows: Figure 7 As shown in (a). By calculating the percentage of the healed area in the scratch region, the cell migration ability was quantitatively analyzed, and the results are as follows. Figure 7 As shown in (b).

[0038] The results are as follows Figure 7 Figures a and 7b show the effects of key sites in ELP3-catalyzed histone H1 acetylation on the proliferation ability of liver cancer cells; among them... Figure 7 (a) Microscopic image of cell migration results after scratching; Figure 7 (b) is a graph showing the quantitative analysis results of cell migration ability. (From...) Figure 7 It was found that co-expression of wild-type ELP3 and H1 significantly promoted the migration of HepG2 cells compared with H1 expression alone. In the context of ELP3 co-expression, comparison of the four H1 point mutation groups revealed that the K147R mutant group had significantly lower cell migration ability than the other three mutant groups (K129R, K138R, and K140R). These results indicate that co-expression of ELP3 and H1 can promote the migration of liver cancer cells, and that ELP3 positively regulates the migration ability of liver cancer cells by catalyzing the K147 site of histone H1 protein.

[0039] Example 6 To investigate the effect of ELP3 on the proliferation of liver cancer cells through acetylation modification of histone H1, the specific experimental steps are as follows: (1) Verify the acetylation regulation of H1 by ELP3 in hepatocellular carcinoma cells. The human hepatocellular carcinoma cell line HepG2 was divided into four groups for transfection: HA group (control group): transfected with the empty pBobi-N-HA plasmid; H1 group: transfected with the pBobi-N-Flag-H1 plasmid; ELP3 group: transfected with the pBobi-N-HA-ELP3 plasmid; H1+ELP3 group: co-transfected with both pBobi-N-Flag-H1 and pBobi-N-HA-ELP3 plasmids. After transfection, the Flag-H1 protein complex was enriched using the same immunoprecipitation method as in Example 3 (using anti-Flag antibody), followed by Western blotting analysis. The acetylation modification of H1 was verified using anti-Flag antibody, anti-HA antibody, and anti-acetylated lysine antibody, respectively. The results are as follows: Figure 8 As shown.

[0040] The results are as follows Figure 8 The image shows the results of Western blotting. Figure 8 It was found that co-transfection of HA-ELP3 and Flag-H1 in HepG2 cells led to a significant increase in the acetylation level of H1 protein. This result confirms that ELP3 also has the function of catalyzing H1 acetylation in liver cancer cells.

[0041] (2) Cell proliferation capacity detection The transfected HepG2 cells were divided into groups of 5 × 10⁶ cells per well. 3 Cells were seeded into 96-well plates and cultured at 37°C and 5% CO2. At 24, 48, and 72 hours post-transfection, 10 μL of LTT solution was added to each well, and incubation continued for 4 hours at 37°C and 5% CO2. Subsequently, the supernatant was carefully aspirated from each well, and 100 μL of formazan was added to each well. The plates were then shaken at 300 rpm for 10 minutes. Finally, the absorbance (OD value) of each well was measured at 570 nm using a microplate reader to assess cell proliferation viability. The results are shown below. Figure 9 As shown.

[0042] The results are as follows Figure 9 The image shows the effect of histone H1 on the proliferation of HepG2 cells. Figure 9 It can be seen that H1 overexpression alone can significantly inhibit the proliferation of HepG2 cells at 24, 48 and 72 hours; co-expression of ELP3 (H1+ELP3 group) will interfere with the inhibitory effect of H1 on HepG2 proliferation, and the cell proliferation capacity will recover to a level close to or higher than that of the control group at different time points.

[0043] In summary, histone H1 itself possesses antitumor functions in hepatocellular carcinoma (HCC) cells, inhibiting cell proliferation and migration. However, the acetyltransferase ELP3 can effectively antagonize the antitumor effects of H1 by catalyzing its acetylation modification, thereby promoting the malignant progression of HCC cells. This reveals the crucial role of the "ELP3-H1 acetylation" regulatory axis in driving the development and progression of HCC; that is, ELP3 modifies H1 to deactivate its antitumor function, providing a direct theoretical basis for targeting this pathway in the treatment of HCC.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A biomarker associated with acetylation at the K147 site of histone H1, characterized in that, The biomarker is acetylation modification at the K147 site of histone H1.

2. The biomarker according to claim 1, characterized in that, The amino acid sequence of histone H1 is shown in SEQ ID NO.

3.

3. The biomarker according to claim 1, characterized in that, The biomarkers are used in the treatment of liver cancer.

4. The biomarker according to claim 1, characterized in that, Acetylation modification was performed using the acetyltransferase ELP3.

5. The use of the biomarker according to claim 1 in the preparation of a drug for the treatment of liver cancer.

6. The use of the biomarker according to claim 1 in the preparation of inhibitors, antibodies or nanomedicines for the treatment of liver cancer.

7. The application according to claim 6, characterized in that, The target site of the inhibitor, antibody, or nanomedicine is the K147 acetylation site of histone H1.

8. The application of the biomarker according to claim 7 in screening drugs for the treatment of liver cancer.

9. The application according to claim 8, characterized in that, The target of the drugs selected for the treatment of liver cancer is the K147 acetylation site of histone H1.

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