Use of PC4 protein lys35 site as a target in preparation of tumor diagnosis and treatment products

Abstract

The application discloses application of PC4 protein Lys35 site as a target in preparation of tumor diagnosis and treatment products. The application provides application of PC4 protein Lys35 site as a target in preparation of tumor diagnosis products. Research shows that methylation of the Lys35 site of PC4 protein can promote cell cycle progression of tumor cells, promote proliferation and growth of tumor cells and malignant progression, and a scheme for inhibiting methylation of the site can be used for development of tumor treatment drugs. Therefore, the Lys35 site of PC4 protein can be used as a key target in development of tumor diagnosis products and targeted treatment drugs. The application further develops PC4 protein Lys35 methylation antigen peptides and antibodies capable of specifically recognizing the PC4 protein Lys35 methylation site, which can be applied to regulation of tumor cell proliferation or growth, can be used for development of clinical tumor disease diagnosis products and targeted treatment drugs, and has good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of the Lys35 site of the PC4 protein as a target in the preparation of tumor diagnostic and therapeutic products. Background Technology

[0002] Cancer, also known as malignant tumors, is one of the leading causes of death worldwide. Its increasing morbidity burden leads to a significant rise in mortality, making it a major public health challenge globally. Although significant progress has been made in cancer treatments (such as surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy) in recent years, highly effective and specific early diagnostic indicators and treatment methods are still lacking. Coupled with the tendency of tumors to recur and metastasize, the prognosis for most cancer patients is relatively poor. Therefore, there is an urgent need to conduct in-depth research into the mechanisms of malignant tumor development and to identify highly effective and specific early diagnostic indicators and treatment methods.

[0003] Human helper transcription factor 4 (PC4) is a potent and highly conserved nuclear protein located on chromosome 5p13.3, approximately 18.6 kb in length. The transcribed PC4 mRNA is approximately 3.5 kb in length, encoding a 127-amino acid protein of approximately 15 kDa. Our team was the first to report abnormally high expression of PC4 protein in models of malignant transformation of normal cells and in clinical specimens of various tumors, including lung cancer, liver cancer, and breast cancer, and its role in regulating tumor progression. However, PC4 protein regulates many molecular biological processes mediated by RNA polymerase II, such as basal transcription, chromatin remodeling, DNA replication, DNA repair, and mRNA stability. Developing therapeutic drugs targeting the entire PC4 protein presents poor targeting and could potentially have serious adverse effects on human health.

[0004] Therefore, exploring tumor-related specific sites on the PC4 protein is of great significance for developing safe and specific early diagnostic indicators and therapeutic drugs for tumors. Summary of the Invention

[0005] The present invention aims to provide a new approach for tumor diagnosis and targeted therapy.

[0006] The primary objective of this invention is to provide the application of the Lys35 site of the PC4 protein as a target in the preparation of tumor diagnostic products.

[0007] The second objective of this invention is to provide the application of a reagent for detecting the methylation level at the Lys35 site of the PC4 protein in the preparation of tumor diagnostic products.

[0008] A third objective of this invention is to provide the application of the PC4 protein Lys35 site as a target in the preparation of drugs for the prevention and / or treatment of tumors, or in the screening of drugs for the prevention and / or treatment of tumors.

[0009] The fourth objective of this invention is to provide the application of the PC4 protein Lys35 site as a target in the preparation of products that regulate tumor cell mitosis or proliferation.

[0010] The fifth objective of this invention is to provide the application of a reagent that inhibits methylation at the Lys35 site of the PC4 protein in the preparation of products for treating tumors.

[0011] The sixth objective of this invention is to provide the use of the SETDB1 inhibitor in the preparation of products that inhibit methylation modification at the Lys35 site of the PC4 protein, or in the preparation of products for treating tumors.

[0012] The seventh objective of this invention is to provide a PC4 protein Lys35 methylated (PC4-K35me2) antigenic peptide and its application.

[0013] The eighth objective of this invention is to provide an antibody that specifically recognizes the Lys35 methylation site of the PC4 protein.

[0014] The ninth objective of this invention is to provide the application of the above-mentioned antigenic peptide or the above-mentioned antibody.

[0015] The tenth objective of this invention is to provide a drug for treating tumors.

[0016] The above-mentioned objective of this invention is achieved through the following technical solution:

[0017] This invention reveals that the Lys35 site in the PC4 amino acid sequence is the site of protein methylation modification. Further research shows that methylation at the Lys35 site of the PC4 protein is expressed at higher levels in tumor cells or tissues than in normal cells or tissues. Furthermore, methylation at the Lys35 site of the PC4 protein can directly bind to and regulate CENPF mRNA stability, ensuring centromere protein expression balance and proper spindle assembly, ultimately driving mitosis and cell proliferation, and promoting malignant tumor progression. Therefore, Lys35 of PC4 can serve as a key target for developing tumor diagnostic products or targeted tumor therapies, and products for tumor screening, diagnosis, and monitoring of treatment efficacy can be developed targeting this site.

[0018] In addition, this invention has developed an antigenic peptide targeting the Lys35 methylation (PC4-K35me2) site of human PC4 protein, as well as an antibody that can specifically recognize the Lys35 methylation site of PC4 protein, which can be applied to the diagnosis, treatment and prognosis of diseases such as tumors.

[0019] Based on the research results of this invention, this invention claims protection for the following solutions:

[0020] Application of the PC4 protein Lys35 site as a target in the preparation of tumor diagnostic products.

[0021] Specifically, the tumor diagnostic product detects the methylation level at the Lys35 site of the PC4 protein.

[0022] Application of reagents for detecting methylation levels at the Lys35 site of PC4 protein in the preparation of tumor diagnostic products.

[0023] Application of the PC4 protein Lys35 site as a target in the preparation of drugs for the prevention and / or treatment of tumors.

[0024] Application of the PC4 protein Lys35 site as a target in screening drugs for the prevention and / or treatment of tumors.

[0025] Application of the PC4 protein Lys35 site as a target in the preparation of products that regulate tumor cell mitosis or proliferation.

[0026] Specifically, the regulation involves inhibiting tumor cell proliferation, growth, and malignant progression.

[0027] As an alternative implementation, the expression level of CENPF protein can be abnormally increased by inhibiting the methylation of PC4 protein at Lys35 site, leading to centromere protein expression imbalance and spindle assembly disorder, ultimately causing mitotic catastrophe, inhibiting tumor cell growth and malignant progression.

[0028] Application of reagents that inhibit methylation at the Lys35 site of PC4 protein in the preparation of tumor therapeutic products.

[0029] This invention also investigated the direct catalysis of methyltransferase SETDB1 on the methylation of PC4 at the Lys35 site, which regulates the stability of downstream centromere-related gene mRNAs, ensuring proper spindle assembly and mitosis, and ultimately promoting malignant tumor phenotypes. Therefore, protection is sought for:

[0030] The use of SETDB1 inhibitors in the preparation of products that inhibit methylation modification at the Lys35 site of PC4 protein, or in the preparation of products for treating tumors.

[0031] Application of SETDB1 in the preparation of products that can activate / promote methylation at the Lys35 site of the PC4 protein.

[0032] In addition, the present invention provides a PC4 protein Lys35 methylated antigenic peptide, the amino acid sequence of which is shown in SEQ ID NO.2.

[0033] Specifically, the antigenic peptide sequence is: QVAPE-(Dimethyl)K-PVKKQ, wherein the lysine residues are methylated. This antigenic peptide can not only solve the problem of effective research on PC4 protein methylation modification, but also be used for the diagnosis, treatment, and prognosis of diseases such as tumors.

[0034] The application of the above-mentioned antigenic peptide in the preparation of antibodies that specifically recognize the Lys35 methylation site of PC4 protein should also be within the scope of protection of this invention.

[0035] This invention provides an antibody that specifically recognizes the Lys35 methylation site of the PC4 protein, which is prepared from the above-mentioned antigenic peptide as an immunogen.

[0036] As an alternative implementation, the aforementioned antigenic peptide was used as an immunogen to immunize animals, thereby preparing a polyclonal antibody that specifically recognizes the Lys35 methylation site (PC4-K35me2) of the PC4 protein, named the PC4-K35me2 antibody. This antibody can not only solve the problem of effective research on PC4 protein methylation modification, but also be used for the diagnosis, treatment, and prognosis of diseases such as tumors.

[0037] The use of the aforementioned antigenic peptides or antibodies in the preparation of tumor detection / diagnosis / screening, targeted therapy, or prognosis products should also be within the scope of protection of this invention.

[0038] Antibodies are used in the development of diagnostic or targeted therapies, including linking antibodies to drugs for targeted cancer therapy and linking antibodies to radiolabels for radioimmunoassay to assist in cancer diagnosis.

[0039] The present invention also provides a medicament for treating tumors, comprising an agent that inhibits methylation at the Lys35 site of the PC4 protein.

[0040] Preferably, the drug for treating tumors further includes paclitaxel-based drugs; the paclitaxel-based drugs include paclitaxel and docetaxel.

[0041] As an alternative implementation, the drug for treating tumors includes an agent that inhibits methylation at the Lys35 site of the PC4 protein and a taxane drug; the taxane drug includes paclitaxel and docetaxel.

[0042] As an alternative implementation, the reagent that inhibits methylation at the Lys35 site of the PC4 protein includes a SETDB1 inhibitor.

[0043] The tumors described in this invention include liver cancer, cervical cancer, lung cancer, colorectal cancer, breast cancer, osteosarcoma, lymphoma, pancreatic cancer, prostate cancer, and bladder cancer, etc.

[0044] The present invention has the following beneficial effects:

[0045] (1) This invention has identified a specific site on the PC4 protein that is closely related to tumors, namely the lysine residue (Lys) at position 35 of the PC4 protein. Methylation modification at this site regulates the stability of downstream centromere-related gene mRNAs, ensures proper spindle assembly and mitosis, and promotes the malignant phenotype of tumors. Therefore, targeted antitumor drugs can be developed by inhibiting methylation at this site.

[0046] (2) The Lys35 site methylation of human PC4 protein provided by the present invention can be used as a tumor biomarker for screening, diagnosis and monitoring of the efficacy of treatment of clinical tumor diseases.

[0047] (3) The present invention also provides corresponding methylated antigen peptides and antibodies, which can be used as or for the development and preparation of tumor detection / diagnosis / screening products, as well as as or for the development and preparation of tumor targeted therapy or prognosis drugs, and have important application value for the prevention and treatment of tumors.

[0048] (4) The antigenic peptide and highly specific antibody against Lys35 site methylation of human PC4 protein provided by this invention can detect the methylation modification of human PC4 protein in practical applications. It can be used to detect the expression differences between normal cells / tissues and tumor cells / tissues by Western blot experiments, which helps to study the role of PC4 protein methylation modification in the occurrence and development of tumor diseases, and provides potential targets for the diagnosis or treatment of clinical tumor diseases. It can also be used to detect the methylation level of human PC4 protein by immunological experiments such as IHC and ELISA, and explore its relationship with systemic diseases such as tumors. It has broad clinical application prospects in disease diagnosis, treatment and prognosis. Attached Figure Description

[0049] Figure 1 This is the mass spectrum of the Lys35 site of the human PC4 protein.

[0050] Figure 2 The results of genome sequence comparison of PC4 protein Lys35 in humans, rats, mice, chimpanzees, macaques, cattle, and chickens.

[0051] Figure 3 Western blot results of PC4 protein Lys35 methylation expression level (Figure A shows the PC4 protein Lys35 methylation expression level at different cell cycles; Figure B shows PC4...). K35R (Results of methylation expression level detection of PC4 protein at Lys35 site in mutants).

[0052] Figure 4The CO-IP results for SETDB1 regulation of methylation modification at the Lys35 site of PC4 protein are shown in Figure A (CO-IP results for SETDB1 and PC4 proteins at different cell cycles; Figure B (CO-IP results for methylation modification at the Lys35 site of SETDB1 and PC4 proteins); and Figure C (CO-IP results for methylation modification at the Lys35 site of PC4 protein after knockdown of SETDB1).

[0053] Figure 5 Figure A shows the expression levels of CENPF protein in cells under different treatments (Figure A shows the qPCR results of CENPF mRNA expression levels in cells under different treatments; Figure B shows the Western blot results of CENPF protein expression levels in cells under different treatments).

[0054] Figure 6 The effect of inhibiting PC4 protein Lys35 site methylation modification on cell spindle assembly and mitosis (Figure A shows the immunofluorescence staining results of cells under different treatments; Figure B shows the proportion of cells with different mitotic abnormalities under different treatments).

[0055] Figure 7 To inhibit the increase in apoptosis levels caused by methylation modification of the PC4 protein at the Lys35 site (Figure A shows the flow cytometry results of apoptosis in cells under different treatments; Figure B shows the bar chart of apoptotic cells under different treatments).

[0056] Figure 8 To investigate the effect of PC4 protein Lys35 site methylation modification on paclitaxel-based drug sensitivity (Figure A shows the effect of paclitaxel on the survival rate of Huh7 cells under different treatments; Figure B shows the effect of docetaxel on the survival rate of Huh7 cells under different treatments).

[0057] Figure 9 The results show the effect of inhibiting methylation modification of PC4 protein at Lys35 site on tumor-bearing nude mice (Figure A is a schematic diagram of the experimental procedure; Figure B is a phenotypic diagram of tumor size under different treatments; Figure C is a tumor weight under different treatments; Figure D is a tumor volume under different treatments; Figure E is a body weight of tumor-bearing nude mice under different treatments).

[0058] Figure 10 The results of ELISA detection show the specificity of PC4-K35me2 antibody against methylated synthetic peptides at the Lys35 site.

[0059] Figure 11 The results of Dot blot analysis show the specificity of the PC4-K35me2 antibody against the methylated synthetic peptide at the Lys35 site.

[0060] Figure 12The results of Western blot analysis show the specificity of the PC4-K35me2 antibody against the methylated synthetic peptide at the Lys35 site.

[0061] Figure 13 Western blot results for the specific recognition of methylation modification at the Lys35 site of PC4 protein by the PC4-K35me2 antibody.

[0062] Figure 14 Results of PC4-K35me2 antibody detection of methylation levels at the Lys35 site of PC4 protein in normal cells / tissues and tumor cells / tissues (Figure A shows the Western blot results of PC4 protein Lys35 methylation levels in normal cells and tumor cells; Figure B shows the immunohistochemical results of PC4 protein Lys35 methylation levels in normal tissues and tumor tissues).

[0063] Figure 15 This diagram illustrates how methylation modification of the PC4 protein at the Lys35 site promotes tumor cell growth. Detailed Implementation

[0064] The following description, in conjunction with the accompanying drawings and specific embodiments, further elaborates and illustrates the regulatory role of methylation at the Lys35 site of human PC4 protein in tumor cells and the technical route for preparing specific antibodies provided by the present invention. However, the embodiments do not limit the present invention in any way.

[0065] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0066] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0067] The SETDB1 inhibitor is SETDB1-TTD-IN-1, CAS number: 2755823-12-0, and its structural formula is:

[0068] The amino acid sequence of human co-transcription factor 4 (PC4) is shown in SEQ ID NO.1. SEQ ID NO.1:

[0069] MPKSKELVSSSSSGSDSDSEVDKKLKRKKQVAPEKPVKKQKTGETSRALS SSKQSSSSRDDNMFQIGKMRYVSVRDFKGKVLIDIREYWMDPEGEMKPGRK GISLNPEQWSQLKEQISDIDDAVRKL.

[0070] The core sequence of the PC4 protein Lys35 methylated antigenic peptide is shown in SEQ ID NO.2, SEQ ID NO.2: QVAPEKPVKKQ, where the 6th lysine residue is methylated (Dimethyl).

[0071] The experimental materials used in the following examples are:

[0072] 1. Experimental cells

[0073] Human tumor cells Huh7, HepG2, SK-Hep-1, mcf7, and jurkat, and normal cells LO2 and LX2.

[0074] 2. Experimental drugs and antibodies

[0075] Fluorescently labeled antibody solution, 4% paraformaldehyde, PBS buffer, citrate buffer, hematoxylin staining solution, eosin staining solution, goat serum for blocking, immunohistochemical secondary antibody, DAB chromogenic solution, neutral resin, RPMI-1640 medium, DMEM high glucose medium, fetal bovine serum, trypsin, isopropanol, chloroform, TRIzol, qPCR kit, reverse transcription kit, SYBR, Annexin V / PI apoptosis detection kit, Actinomycin D, Western and IP cell lysis buffer, BCA protein concentration detection kit, protein marker, 5× protein loading buffer, SDS-PAGE gel preparation kit, Tris, glycine, SDS, PVDF membrane, HRP-labeled secondary antibody, Tween 20, Western blocking buffer, primary antibody dilution buffer, Beyoncé... ECL chemiluminescence solution, CCK-8 kit, PC4 antibody (Abcam, ab72132), SETDB1 antibody (Proteintec, 11231-1-AP), CENPF antibody (CST, 58982), Flag antibody (Sigma, F1804), GAPDH antibody (Bioss, bs-2188R), β-actin antibody (CST, 3700), etc.

[0076] 3. Experimental instruments and consumables

[0077] Microtome, electronic balance, microtome, inverted phase contrast microscope, immunohistochemistry humidifier, conventional PCR instrument, qPCR instrument, cell culture incubator, vortex shaker, constant temperature shaker, clean bench, centrifuge, ice maker, gel imaging system, vertical protein electrophoresis tank, ELISA reader, pipette, PVDF membrane, anti-detachment slides, coverslips, culture flasks, centrifuge tubes, pipette tips, 6-well plate, 96-well plate, etc.

[0078] The experimental methods used in the following examples are as follows:

[0079] The Huh7 cells were configured into the following treatment groups:

[0080] NC: Transfected with negative control shRNA lentivirus.

[0081] shPC4: PC4 stably knocked-down cell lines were constructed by transfecting PC4 shRNA lentivirus (introduced sequence: ACAGAGCAGCAGCGCAGATT).

[0082] shPC4+PC4 WT The above shPC4 group cells were re-transfected with PC4 wild-type (PC4) WT Overexpression of recombinant lentiviral vector. Construction of PC4 vectors containing lentiviral vectors. WT The sequence is as follows:

[0083] ATGGCTAAATCTAAGGAATTGGTGAGTAGTTCTAGTAGCGGTTCCGA

[0084] TTCAGATAGTGAAGTTGACAAAAAGCTCAAAAGGAAGAAACAAGTGGCA

[0085] CCCGAAAAACCAGTTAAGAAGCAGAAAACTGGAGAGACGAGCAGGGCTC

[0086] TGAGTTCTAGTAAGCAGAGCAGTTCTAGTAGAGACGATAATATGTTTCAA

[0087] ATAGGGAAGATGCGGTACGTTTCCGTAAGAGATTTCAAGGGAAAGGTTCT

[0088] TATTGACATTAGAGAGTATTGGATGGACCCCGAAGGCGAAATGAAGCCTG

[0089] GTCGAAAGGGCATCTCACTTAATCCAGAGCAGTGGAGTCAGCTGAAAGAG

[0090] CAAATCTCCGATATTGACGATGCTGTTAGGAAACTT (SEQ ID NO. 3).

[0091] shPC4+PC4 K35R The above shPC4 group cells were re-transfected with the PC4-K35 mutation (PC4 K35R Overexpression of recombinant lentiviral vector. Construction of PC4 vectors containing lentiviral vectors.K35R The sequence is as follows:

[0092] ATGGCTAAATCTAAGGAATTGGTGAGTAGTTCTAGTAGCGGTTCCGA

[0093] TTCAGATAGTGAAGTTGACAAAAAGCTCAAAAGGAAGAAACAAGTGGCA

[0094] CCCGAA AGA CCAGTTAAGAAGCAGAAAACTGGAGAGACGAGCAGGGCTC

[0095] TGAGTTCTAGTAAGCAGAGCAGTTCTAGTAGAGACGATAATATGTTTCAA

[0096] ATAGGGAAGATGCGGTACGTTTCCGTAAGAGATTTCAAGGGAAAGGTTCT

[0097] TATTGACATTAGAGAGTATTGGATGGACCCCGAAGGCGAAATGAAGCCTG

[0098] GTCGAAAGGGCATCTCACTTAATCCAGAGCAGTGGAGTCAGCTGAAAGAG

[0099] CAAATCTCCGATATTGACGATGCTGTTAGGAAACTTGGCTCCGGCGACTA

[0100] CAAAGACCATGAC(SEQ ID NO.4).

[0101] shPC4+PC4 WT +SETDB1-IN: The above shPC4+PC4 WT The cells were treated with SETDB1-IN (intravenous administration was used in animal experiments).

[0102] shPC4+PC4 K35R +SETDB1-IN: The above shPC4+PC4 K35R The cells were treated with SETDB1-IN (intravenous administration was used in animal experiments).

[0103] shPC4+PC4 WT +Paclitaxel: The above shPC4+PC4 WTThe cells were treated with Paclitaxel (intravenous administration was used in animal experiments).

[0104] shPC4+PC4 K35R +Paclitaxel: The above shPC4+PC4 K35R The cells were treated with Paclitaxel (intravenous administration was used in animal experiments).

[0105] shPC4+PC4 WT +SETDB1-IN+Paclitaxel: The above shPC4+PC4 WT The cells were treated with SETDB1-IN and Paclitaxel (intravenous administration was used in animal experiments).

[0106] shPC4+PC4 K35R +SETDB1-IN+Paclitaxel: The above shPC4+PC4 K35R The cells were treated with SETDB1-IN and Paclitaxel (intravenous administration was used in animal experiments).

[0107] 1. Flow cytometry detection of apoptosis

[0108] Each group (NC, shPC4, shPC4+PC4) WT and shPC4+PC4 K35R Huh7 cells were digested and then added to centrifuge tubes containing cell supernatant (the process should be gentle to avoid physical apoptosis). The cells were centrifuged at 1000 rpm for 3 minutes, and the cell pellet was collected. The cells were resuspended in PBS and centrifuged again at 1000 rpm for 3 minutes, and the cell pellet was collected once more. 4× Binding buffer was diluted to 1× Binding buffer with ultrapure water, and 200 μL of 1× Binding buffer was added to the cell pellet for resuspending. 5 μL of Annexin V was added to the cells, vortexed to mix, and incubated in the dark for 15 minutes. Then, 10 μL of PI solution (Annexin V / PI apoptosis detection kit) was added to the cells, vortexed to mix, and subsequent FACS analysis was performed.

[0109] 2. Cell cycle synchronization

[0110] To synchronize cells to G1 phase, cells were first treated with thymine (Sigma, T3763) for approximately 16-18 hours, then released and cultured in fresh medium for 8 hours. The cells were then treated with thymine again for 16-18 hours. This synchronized the cells to G1 phase.

[0111] For cells in S phase, release those arrested in G1 phase and culture them for 2 hours. Then, collect the cells for further research.

[0112] For both early and late G2 phase cells, cells arrested in the G1 phase are first released for 2 hours. Then, they are treated with the RO3306 inhibitor and cultured for 6 hours to obtain early G2 phase cells. A further 4 hours of culture yields late G2 phase cells.

[0113] For the M phase, cells in the late G2 phase can be obtained by releasing cells treated with the RO3306 inhibitor for 1 hour. Alternatively, cells can be cultured with nocodazole for 18 to 20 hours before collection.

[0114] Cells at each stage were collected through digestion and then subjected to Western blotting analysis.

[0115] 3. Immunofluorescence staining

[0116] 1) Cell fixation: First, Huh7 cells from each group (NC, shPC4, shPC4+PC4) were fixed. WT shPC4+PC4 K35R shPC4+PC4 WT +SETDB1-IN, shPC4+PC4 K35R Remove the culture medium (+SETDB1-IN), wash the cells twice with PBS. Add 4% paraformaldehyde fixative and incubate for 15-20 minutes.

[0117] 2) Cell membrane perforation: First, wash the cells once with PBS for 5 minutes. Then add 0.5% Triton-X100 and incubate for 10 minutes. Next, wash the cells again with PBS for 5 minutes.

[0118] 3) Blocking: Add 500 μL of immunostaining blocking solution and incubate for 30 minutes.

[0119] 4) Incubation with primary antibodies: Prepare primary antibodies diluted 1:200, namely ACA (Antibodies Incorporated, 15-235-0001) and β-Tubulin (BD, 556321). Add the primary antibodies to the sample and incubate overnight at 4°C. After incubation, wash with PBST (PBS containing 0.1% Triton-X100) for 5 minutes, repeating the washing process 3 times.

[0120] 5) Incubation of secondary antibody: Prepare secondary antibody diluted 1:1000, add the corresponding secondary antibody to the sample and incubate for 1 hour, wash with PBST for 5 minutes, and repeat the washing operation 3 times.

[0121] 6) Nuclear staining: Prepare a DAPI solution diluted 1:4000. Add DAPI to the sample and incubate for 5 minutes, shaking gently. Wash with PBST for 5 minutes, repeating 3 times. The sample can then be photographed.

[0122] 4. CCK-8 Detection

[0123] 1) Cell plating: shPC4+PC4 WT and shPC4+PC4 K35R Huh7 cells were digested, counted, and prepared into 2×10⁻⁶ cells. 4 Cell suspensions at a density of / mL were added to each well of a 96-well plate at 100μL. After adhesion, different concentrations (0.1, 1, 10, 100nM) of paclitaxel and docetaxel were added, along with DMSO or SETDB1-IN (divided into 4 groups: shPC4 + PC4). WT shPC4+PC4 K35R shPC4+PC4 WT +SETDB1-IN and shPC4+PC4 K35R +SETDB1-IN). Then incubate in a cell culture incubator at 37°C and 5% CO2.

[0124] 2) OD value detection and growth curve plotting: After 48 hours, remove the 96-well plate. Prepare working solution according to the number of culture wells to be tested each time, using a ratio of 10 μL CCK-8 reagent and 90 μL PBS. Aspirate the old culture medium, add 100 μL of the newly prepared CCK-8 working solution, and incubate at 37℃ for 2 hours. Then, use a microplate reader to detect the absorbance value at a wavelength of 450 nm, and finally plot the growth curve.

[0125] 5. Tumor-bearing nude mice

[0126] 1) Using the groups with good growth (shPC4+PC4) WT and shPC4+PC4 K35R Using Huh7 cells as a research model, cells from each group were digested, counted, and prepared into cells with a density of 2.5 × 10⁻⁶. 7 Prepare a cell suspension of / mL and store it in an ice box for later use.

[0127] 2) Purchase 5-6 week old male BALB / c nude mice from the Animal Center of the Third Military Medical University and divide them into 8 groups (shPC4+PC4). WT shPC4+PC4 K35R shPC4+PC4 WT +SETDB1-IN, shPC4+PC4 K35R+SETDB1-IN, shPC4+PC4 WT +Paclitaxel, shPC4+PC4 K35R +Paclitaxel, shPC4+PC4 WT +SETDB1-IN+Paclitaxel,shPC4+PC4 K35R +SETDB1-IN+Paclitaxel), 5 mice per group. 200 μL of cell suspension was injected into the back of each nude mouse, i.e., 5 × 10⁻⁶ cells per mouse. 6 Each cell.

[0128] 3) Nude mice were housed in an SPF animal facility. When the tumor grew to approximately 100 mm... 3 Mice were randomly assigned to groups and received drug treatment: paclitaxel (ip, 10 mg / kg), SETDB1-TTD-IN-1 (ip, 30 mg / kg), or a combination of paclitaxel and SETDB1-TTD-IN-1, for 6 cycles every 3 days. Tumor growth was observed, tumor size was measured, and mouse weight was assessed every 5 days. Tumor volume was calculated as (length × width). 2 ) / 2. At the end of the experiment, nude mice were euthanized and the tumors were removed, photographed, and weighed. Part of the tumor was frozen at -80°C, and the other part was fixed in 4% paraformaldehyde.

[0129] 6. qPCR detection of mRNA stability of cell cycle genes in each group

[0130] 1) Combine each group (NC, shPC4, shPC4+PC4) WT shPC4+PC4 K35R shPC4+PC4 WT +SETDB1-IN, shPC4+PC4 K35R After adding 5ug / mL Actinomycin D to Huh7 cells (+SETDB1-IN), samples were collected at 0h, 2h, 4h, and 6h. The culture medium in the culture flask was discarded, the cells were washed 1-2 times with PBS, and then 1mL Trizol was added. Cells from each group were scraped off with a cell scraper, collected, and transferred to 1.5mL EP tubes.

[0131] 2) RNA extraction: Add 0.2 mL of chloroform to each tube, vortex vigorously for 15 seconds, and let stand at room temperature for 15 minutes; centrifuge at 12000 rpm for 15 minutes at 4°C, until the liquid separates into 3 layers; carefully transfer 450 μL of supernatant to another 1.5 mL EP tube; add 0.5 mL of isopropanol, gently invert and mix for 30 seconds, and incubate at 4°C for about 10 minutes; centrifuge at 12000 rpm for 10 minutes at 4°C, discard the supernatant, and a white, pinhead-sized precipitate will be visible at the bottom of the tube; add 1 mL of 75% ethanol (prepared with DEPC water) / mL Trizol to each EP tube to wash the precipitate; centrifuge at 12000 rpm for 5 minutes at 4°C, and discard the supernatant; repeat the RNA washing once; air dry in a laminar flow hood (to prevent ethanol from inhibiting reverse transcriptase); dissolve the RNA at the bottom of the tube with an appropriate amount of RNase-removing DEPC water, measure the concentration, and balance the liquid;

[0132] 3) Remove genomic DNA and reverse transcribe RNA into cDNA;

[0133] 4) Real-time PCR: The system consisted of 20 μL (SYBR 10 μL, primer (top) 0.5 μL, primer (bottom) 0.5 μL, cDNA 1 μL, dH2O 8 μL), with a programmed annealing temperature of 59℃ and a cycle number of 40. The specific primer sequences are shown in Table 1.

[0134] 5) Statistically analyze the mRNA expression levels of each group and create a chart of mRNA stability for each gene.

[0135] Table 1. qPCR primer sequences for cell cycle genes

[0136]

[0137] 7. Western Blotting Detection

[0138] 1) Extraction of cell or tissue proteins: Collect cells or tissues from each group, add about 3 times the volume of cell lysis buffer containing 10% cocktail protease inhibitor, gently tap with your finger to fully lyse the cells, and place in an ice bath for 30 min; centrifuge at 12000 rpm for 10 min, and transfer the supernatant to a 1.5 mL EP tube.

[0139] 2) BCA method for protein concentration determination: Add 0.8 mL of protein standard preparation reagent to one tube of standard protein (20 mg BSA), dissolve completely to prepare a 25 mg / mL standard protein solution. Take 20 μL of the 25 mg / mL standard protein, add 980 μL of PBS, and dilute 50 times to obtain a concentration of 0.5 mg / mL. Prepare BCA working solution (the ratio of solution A to solution B is 50:1), and prepare fresh each time. Add 0.5 mg / mL standard protein to 96-well plates at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, then add 0.9% physiological saline or purified water to each well to a final volume of 20 μL. Add 2 μL of sample and 18 μL of PBS to each well, for a total volume of 20 μL. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the protein OD using a microplate reader. 562 Values. Plot a standard curve, calculate protein concentration, and balance each group using the lowest concentration as the benchmark. Take a certain volume of protein, add 1 / 5 volume of 6× loading buffer, and denature the protein at 100℃ for 5 min.

[0140] 3) SDS-PAGE electrophoresis: Prepare a 12% lower separating gel and a 5% upper stacking gel. Using a Bio-Rad electrophoresis apparatus, align and fix two clean Bio-Rad glass plates (8cm × 8cm, 1.5mm) on the rack. Pour the lower separating gel evenly to approximately 2 / 3 of the glass plate's height, and slowly add isopropanol as a sealing agent, moving it from side to side. Let it stand for approximately 30 minutes. When the boundary between the upper isopropanol and the lower separating gel is clear, wash with distilled water and discard the isopropanol. Pour the stacking gel, inserting the comb horizontally and slowly to prevent air bubble formation. Let it stand at room temperature for approximately 30 minutes. Place the prepared glass plates into the electrophoresis tank, and pour in 500mL of protein electrophoresis buffer. Note that the inner tank should use freshly prepared electrophoresis buffer, while the outer tank can use recycled electrophoresis buffer. Remove the protein, briefly centrifuge to collect the liquid from the tube wall, and use a microsyringe or pipette to transfer the sample protein. Add approximately 5μL of marker to both sides or other suitable locations. Electrophoresis at 80V for about 40 minutes. After the bromophenol blue has passed through the stacking gel, adjust the voltage to 120V and electrophoresis for about 1.5 hours. Stop electrophoresis when the bromophenol blue is about to reach the bottom edge of the separating gel (about 1 cm from the bottom of the gel).

[0141] 4) Transfer (wet transfer): Prepare transfer buffer, adding methanol during transfer preparation. Immerse clamps and filter paper in the buffer and pre-cool to 4°C. Using a comb as a control, cut approximately the same size PVDF membrane according to the number of wells, and immerse in methanol for about 10 minutes. Remove the glass plate from the electrophoresis tank and gently transfer the gel to the buffer. Cut the gel using a gel cutter according to the marker's indication of antibody location. Arrange the items in the transfer clamp according to the "black gel, white membrane" principle: black clamp, filter paper, gel, PVDF membrane, filter paper, white clamp. Carefully and gently remove air bubbles with a glass rod. Clamp the clamp and place it in the electrophoresis apparatus. Fill the inner white box with ice. Place the electrophoresis apparatus in an ice bath for transfer. Use a current of 250mA and electrophoresis for approximately 60-120 minutes, adjusting the time according to the protein molecular weight.

[0142] 5) Blocking: After electroporation, rinse the PVDF membrane once in TBST solution, place it in a clean incubation box, add Western blotting solution for blocking, place it on a shaker, and block at 37°C for about 2 hours.

[0143] 6) Incubation with primary and secondary antibodies: After blocking, rinse the PVDF membrane in TBST and place it in another clean incubation box. Dilute the antibody with primary antibody dilution buffer to a concentration of 1:1000 (PC4-K35me2) or 1:2000 (β-actin). Add the diluted antibody to the incubation box and incubate overnight at 4°C on a shaker. The next day, remove the PVDF membrane and place it in a transparent plastic washing box. Pour in approximately 20 mL of TBST to submerge the PVDF membrane and wash it three times at room temperature for approximately 10 minutes each time. Dilute the secondary antibody with Western blot secondary antibody dilution buffer (or TBST) to concentrations of goat anti-mouse (1:1000, anti-β-actin) and goat anti-rabbit (1:2000, anti-PC4-K35me2), respectively, and add it to the incubation box containing the PVDF membrane. Incubate the secondary antibody on a shaker at 37°C for approximately 2 hours. Wash the secondary antibody and place it on a shaker at room temperature for 3 washes, each lasting about 10 minutes.

[0144] 7) Chemiluminescence development: Mix 100 μL each of ECL chemiluminescence reagent solution A and solution B thoroughly and store in the dark. Lay the PVDF membrane flat on a cut plastic bag, and evenly drop the ECL chemiluminescence solution onto the PVDF membrane. Expose and image using a bio-rad gel imaging instrument.

[0145] 8. Immunohistochemistry

[0146] 1) Sampling and fixation: Label each sampling bottle and add an appropriate amount of 4% paraformaldehyde fixative. Place each tissue (tumor and normal control tissue) into the sampling bottle in sequence and fix for 24-72 hours.

[0147] 2) Tissue trimming and dehydration: Remove all tissue from the sampling bottle, trim each tissue individually to make them as flat as possible, place them in a dehydration basket, and carefully label them. Place them in a dehydrator and dehydrate at room temperature for 16 hours.

[0148] 3) Tissue embedding: Place the tissue to be embedded in liquid paraffin, arranging it neatly against the bottom surface, and make sure to mark it. Allow the paraffin block to solidify at room temperature (at least 0.5 hours).

[0149] 4) Reshaping: Remove the solidified wax block, shape it into a regular cuboid, and then re-stick the label.

[0150] 5) Sectioning: Half an hour before slicing, place the wax block at -20℃. The thickness of the slice is generally 5mm.

[0151] 6) Slides: Mark blank slides with a pencil in advance, and collect 5-6 slides of each type of tissue. Usually, 2-3 tissue samples are collected from each slide.

[0152] 7) Dewaxing: The sample is sequentially placed in xylene II, xylene I, 100% ethanol, 95% ethanol, and 80% ethanol for dewaxing, with residence times of 10 min, 10 min, 5 min, 5 min, and 5 min respectively. After dewaxing, the support is placed in a beaker and rinsed with water for 3 min.

[0153] 8) Sodium citrate repair: Add 300mL of 0.01mol sodium citrate solution to a beaker, then place the support in the beaker, and then place the beaker in a pre-boiled water pot. After the temperature in the beaker rises to above 93℃, start the repair time for 20 minutes, then remove the beaker and let it cool to room temperature of about 37℃.

[0154] 9) Hydrogen peroxide retrieval: Add 300 mL of PBS solution (or double-distilled water) to a beaker, immerse and rinse the scaffold in the beaker three times, 5 min each time. Directly drop 3% H2O2 onto the tissue surface of each slide, being careful not to touch the tissue, and leave at room temperature for 15 min.

[0155] 10) Serum blocking: Add 300mL of PBS solution (or double-distilled water) to a beaker, immerse the scaffold in the beaker and wash it 3 times, 5min each time. Add serum dropwise to the tissue surface of each slide to block some non-specific sites, then place the slide in a humidified chamber and incubate the humidified chamber at 37℃ for 30min.

[0156] 11) Add primary antibody: Add the pre-prepared primary antibody (PC4-K35me2 antibody diluted 1:100) to the surface of each slide (add PBS to the control tissue), and then place the slide in a humidified chamber and store it in a refrigerator at 4°C overnight.

[0157] 12) Add secondary antibody: Add 300mL of PBS solution (or double-distilled water) to a beaker, immerse and wash the scaffold in the beaker 3 times, 5min each time. Add the secondary antibody directly to the tissue surface of each slide, then place the slide in a humidified chamber, and then place the humidified chamber in a 37℃ incubator for 40min.

[0158] 13) Add chromogenic agent: Add 300mL of PBS solution (or double-distilled water) to a beaker, immerse the support in the beaker and wash it 3 times, 5min each time. Gently shake the slide dry, add the prepared DAB chromogenic solution to the tissue surface of each slide, develop the color for about 10s, quickly wash it off in water, and observe under a microscope. If the color development is insufficient, repeat the previous steps.

[0159] 14) Counterstaining: Place the developed slide back into the support and rinse with water for 3 minutes. Immerse in hematoxylin for 3 minutes (overstaining), then rinse with water for 3 minutes. Immerse in hydrochloric acid for 6 seconds (recovery), then rinse with water for 3 minutes. Observe under a microscope to see if counterstaining has occurred (if not, repeat this step; if the counterstaining is heavy, immerse in hydrochloric acid for 6 seconds, then rinse with water for 3 minutes).

[0160] 15) Drying and dehydration: Dry in an oven, then place the rack in xylene II for 20 minutes and air dry.

[0161] 16) Mounting: Mount the slide with neutral resin, air dry, observe and photograph under a microscope.

[0162] 9. Co-immunoprecipitation (CO-IP)

[0163] 1) Protein extraction: Wash cells twice with pre-cooled PBS, and aspirate the PBS after the last wash; add pre-cooled Lysis buffer; disperse the cells with a pipette, transfer the suspension to a 1.5 mL EP tube, and gently shake at 4°C for 15 min; centrifuge at 14000 rpm at 4°C for 15 min, and immediately transfer the supernatant to a new centrifuge tube.

[0164] 2) Removal of large protein molecules: Prepare agarose beads, wash the beads twice with Lysis buffer, and then prepare a 50% concentration using Lysis buffer. Add 100 μL of agarose beads to every 1 mL of total protein, and shake at 4°C for 10 min. Centrifuge at 14000 rpm for 15 min at 4°C, transfer the supernatant to a new centrifuge tube, and remove the agarose beads. Construct a protein curve and determine the protein concentration. Dilute the total protein to approximately 1 μg / μL with Lysis buffer.

[0165] 3) IP: Add 5 μg of specific primary antibody to 600 μL of total protein, and incubate the antigen-antibody mixture overnight at 4°C with gentle shaking.

[0166] 4) Magnetic bead capture: Take out 30 μL of Protein A / G magnetic beads, wash the beads twice with PBS, reconstitute the beads with 100 μL of Lysisbuffer and add the antigen-antibody complex, and slowly shake the antigen-antibody mixture at 4°C for 4 h.

[0167] 5) Cleaning the magnetic bead complex: Place the EP tube containing the magnetic bead antigen-antibody complex into the magnetic rack, remove the supernatant, and wash repeatedly 6 times with pre-cooled IP buffer;

[0168] 6) Suspend the magnetic bead antigen-antibody complex with 50 μL of IP buffer, add 10 μL of 6× loading buffer, and denature the protein at 100 °C for 5 min.

[0169] 10. Methylation modification mics (mass spectrometry analysis)

[0170] 1) Protein extraction: Samples were removed from -80℃, and each group of samples was lysed with 4 times the volume of lysis buffer (8M urea, 1% protease inhibitor, 1% ubiquitinase inhibitor) by sonication. The samples were then centrifuged at 12000g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube, and protein concentration was determined using a BCA kit.

[0171] 2) Trypsin hydrolysis: Equal volumes of protein from each sample were hydrolyzed, and the volumes were adjusted to be uniform using lysis buffer. 20% TCA was slowly added to a final concentration, vortexed, and incubated at 4°C for 2 hours. The mixture was centrifuged at 4500g for 5 minutes, the supernatant was discarded, and the precipitate was washed 2-3 times with pre-cooled acetone. After drying the precipitate, 200mM TEAB was added, and the precipitate was sonicated to disperse it. Trypsin was added at a ratio of 1:50 (protease: protein, m / m), and hydrolyzed overnight. Dithiothreitol (DTT) was added to a final concentration of 5mM, and the mixture was reduced at 56°C for 30 minutes. Iodoacetamide (IAA) was then added to a final concentration of 11mM, and the mixture was incubated at room temperature in the dark for 15 minutes.

[0172] 3) Modification and Enrichment: Dissolve the peptides in enrichment buffer (50% acetonitrile / 6% trifluoroacetic acid), transfer the supernatant to pre-washed IMAC material, and incubate gently with shaking on a rotary shaker. After incubation, wash the resin three times sequentially with 50% acetonitrile / 6% trifluoroacetic acid buffer and 30% acetonitrile / 0.1% trifluoroacetic acid buffer. Finally, elute the modified peptides with 10% ammonia, collect the eluent, and freeze-dry under vacuum. After drying, desalt according to the C18 ZipTips instructions, freeze-dry under vacuum, and then use for LC-MS / MS analysis.

[0173] 4) Liquid Chromatography-Mass Spectrometry (LC-MS): Peptides were dissolved in mobile phase A of the LC system and then separated using an EASY-nLC1000 ultra-high performance liquid chromatography (UHPLC) system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The LC gradient settings were: 0-86 min, 3%–17% B; 86-110 min, 17%–28% B; 110-115 min, 28%–80% B; 115-120 min, 80% B, with the flow rate maintained at 500 nL / min. After separation by the UHPLC system, the peptides were injected into an NSI ion source for ionization and then subjected to Q Exactive... TM Plus mass spectrometry was used for analysis. The ion source voltage was set to 2.2 kV, and high-resolution Orbitrap was used to detect and analyze peptide precursor ions and their secondary fragments. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 70,000; the secondary mass spectrometry scan range had a fixed starting point of 100 m / z, and the secondary scan resolution was set to 35,000. Data acquisition mode used a data-dependent scanning (DDA) procedure, that is, after the primary scan, the top 20 peptide precursor ions with the highest signal intensity were selected and sequentially entered into the HCD collision cell for fragmentation at 28% of the fragmentation energy, and then analyzed by secondary mass spectrometry in the same manner. To improve the efficiency of mass spectrometry, automatic gain control (AGC) was set to 1E5, the signal threshold was set to 2E4 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time for tandem mass spectrometry scans was set to 15 s to avoid repeated scanning of precursor ions.

[0174] 11. Preparation of specific polyclonal antibodies against PC4 protein Lys35 methylation.

[0175] (1) Rabbit Immunization: ① Preparation of immunization materials: Dilute the immunogen with physiological saline, and then mix it with the corresponding adjuvant at a 1:1 ratio. The antigen and adjuvant are completely mixed to form a stable emulsion. The antigen mixture is drawn with a syringe and injected subcutaneously at two points on both shoulders and muscle at two points on both hind legs of SPF experimental-grade New Zealand white rabbits. Each area contains approximately 1 / 4 volume of immunogen. This allows the immunogen to persist and thus enhance the immune response. ② Immunization: Each rabbit is immunized a total of 6 times, on days 1, 7, 14, 21, 28, and 40. ③ Blood collection: The first blood collection was on day 28, 2 mL of blood was collected and centrifuged. The supernatant was then sent to the laboratory for serum screening tests, including ELISA or Dot blot. The second, third and fourth blood collections were on days 35, 47 and 52, 30 mL of blood was collected each time. The supernatant was then centrifuged and sent to the laboratory for serum screening tests, including ELISA or Dot blot. The serum with positive test results was purified.

[0176] (2) Purification of PC4 protein Lys35 methylated antibody: ① Preparation of protein A affinity column: Usually, 5 mL or 10 mL of protein A packing material is selected. Equal volumes of packing material and PBS buffer solution are mixed and stirred, and air bubbles are removed from the packing material. The protein A packing material is slowly added to the glass column to prepare the chromatography column. The column should be kept from drying out during this process. After filling, the column is equilibrated with 10 times the volume of pre-cooled PBS buffer solution. ② Protein A affinity chromatography: After filtering the serum, it is loaded onto the equilibrated protein A chromatography column. To detect the binding efficiency between the antiserum and the packing material, the eluent should be retained. The column is washed with PBS buffer solution, and then eluted with 150 mM glycine buffer. The eluent is collected, and neutralization buffer solution is added to adjust the pH to 7. ③ Enrichment of target antibody: The crude pure IgG obtained after protein A purification is loaded onto the equilibrated antigen peptide affinity chromatography column to specifically enrich the target antibody. ④ Removal of non-specific antibodies: Load the target antibody obtained in the previous step onto an unmodified affinity chromatography column and collect the eluent (FT3) directly to remove non-specific antibody components. ⑤ Antibody preservation: Determine the protein content. Add 10% glycerol to preserve the antibody. After aliquoting the purified antibody, store it at -20°C.

[0177] (3) Quality control of PC4 protein Lys35 methylated antibody: The PC4-K35me2 antibody was purified using an affinity chromatography-affinity separation-affinity purification cycle. The antibody titer and recognition of methylated target antigen peptides and unmethylated synthetic peptides were detected by ELISA. Antibody identification was then performed using dot blot and Western blot experiments. Details are as follows:

[0178] 1) ELISA Detection: ① Antigen Coating: Dilute the antigen with coating buffer and add it sequentially to the ELISA plate at a rate of 50 μg / well. Incubate overnight at 4°C or incubate for 2 hours at 37°C. ② Washing: Remove the coated ELISA plate from the previous day and wash 3 times with 1×TBST. ③ Blocking: Add 1% BSA blocking buffer to the washed ELISA plate and incubate at 37°C for 1 hour, then wash 1-3 times. ④ Primary Antibody Incubation: Perform 3-fold serial dilutions of the antibody, starting at 1:1K. Adjust the dilution volume as needed. Add the antibody sequentially to the ELISA plate and incubate at 37°C for 1.5 hours, then wash 1-3 times. ⑤ Secondary Antibody Incubation: Dilute the secondary antibody to 1:10K with 1% BSA blocking buffer and incubate at room temperature or 37°C for 45 minutes, then wash 1-3 times. ⑥ Color Development: Add TMB colorimetric solution for 5-10 minutes, then stop the reaction with 1M sulfuric acid. Read the data using an ELISA reader.

[0179] 2) Dot blot detection: ① Spotting: Spot uncrosslinked antigen peptides onto the PVDF membrane in gradients of 1 ng, 4 ng, 16 ng, and 64 ng. ② Blocking: After the membrane surface dries, add blocking buffer and block at room temperature for 60 min. ③ Washing: Wash with 1×TBST for 10 min. ④ Primary antibody incubation: Dilute the antibody with 2.5-5% skim milk powder, incubate at room temperature for 2 h, and then wash three times with 1×TBST for 5-10 min each time. ⑤ Secondary antibody incubation: Select the corresponding mouse or rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a 1:10K dilution ratio, incubate at room temperature for 45 min-1 h, and then wash three times with 1×TBST for 5-10 min each time. ⑥ Expose the washed membrane to the chromogenic substrate.

[0180] 3) Western blot detection: The specific method is the same as in 7.

[0181] The following examples illustrate the experimental results and analysis:

[0182] Example 1: Identification of methylation sites in human PC4 protein

[0183] Firstly, mass spectrometry analysis using methylmics was performed. Figure 1 The study discovered that Lys35 is a methylation site in the human PC4 amino acid sequence. Protein sequence alignment showed that Lys35 is highly conserved in the genomes of humans, rats, mice, chimpanzees, macaques, cattle, and chickens. Figure 2 ).

[0184] CO-IP experimental results (using PC4 antibody to capture and enrich the protein) confirmed the presence of PC4 protein methylation modification in Huh7 cells. Figure 3 (Figure A). Next, PC4 without methylation is constructed. K35R Mutant vector (specific sequence experimental grouping).

[0185] Transfecting Huh7 cells with the Flag-PC4 recombinant vector overexpressing the Flag tag. WT and Flag-PC4 K35R The results of the CO-IP experiment (using Flag antibody to capture and enrich proteins) are as follows: Figure 3 As shown in Figure B, the results indicate that, compared to PC4 WT Group comparison, PC4 K35R PC4 methylation disappeared in the mutant group. These results ultimately identified the specific methylation site Lys35 of the human PC4 protein.

[0186] Example 2: Methyltransferase SETDB1 mediates Lys35 methylation of PC4

[0187] Previous research by our group has shown that PC4 is an atypical RNA-binding protein that can directly bind to the mRNA of cell cycle-related genes, thereby controlling their mRNA stability and protein expression levels. Therefore, PC4 protein plays an important role in the coordinated control of cell cycle progression.

[0188] To investigate the modifying enzymes of PC4 Lys35 methylation, the lysine methyltransferase SETDB1 was identified by analyzing the PC4 interaction proteome. Huh7 cells at different stages of the cell cycle were collected after cell cycle synchronization, and IP experiments (using PC4 antibody to capture and enrich proteins) confirmed that SETDB1 has a strong binding affinity to PC4 protein in the G2 phase. Figure 4 (Figure A).

[0189] Subsequently, this invention constructed a HA-tagged SETDB1 plasmid for transfection, and IP experiments (using PC4 antibody to capture and enrich the protein) confirmed that wild-type SETDB1 could increase Lys35 methylation of PC4 protein. Figure 4 (Figure B).

[0190] Simultaneously, SETDB1 was knocked down using Lipo3000 transfection with siRNA. Specifically, Huh7 cells were transfected with siRNA from the NC group (sequence: UUCUCCGAACGUGUCACGUTT), the SETDB1-1 group (sequence: GGCCAAAUCAACCCUAACUTT), and the SETDB1-2 group (sequence: CCGAGGCUUUGCUCUUAAATT). CO-IP assays (using PC4 antibody to capture and enrich the protein) showed that, compared to the NC group, inhibiting SETDB1 expression reduced Lys35 methylation of PC4 protein. Figure 4 (Figure C).

[0191] The above results confirm that the methyltransferase SETDB1 regulates the methylation of Lys35 in PC4.

[0192] Next, this invention constructs a Flag-tagged PC4 mutant vector (overexpressing the recombinant vector Flag-PC4). WT and overexpression of the recombinant vector Flag-PC4 K35R Cells were transfected with different PC4 mutant vectors or treated with a combination of SETDB1 inhibitors (NC, shPC4, shPC4+PC4). WT shPC4+PC4 K35R shPC4+PC4 WT +SETDB1-IN, shPC4+PC4 K35R +SETDB1-IN).

[0193] The expression of CENPF protein in cells under different treatments was detected. The results showed that inhibiting the methylation modification of PC4 protein at the Lys35 site (PC4) K35R Mutations or SETDB1-IN increase the stability of downstream CENPF mRNA. Figure 5 Figure A shows that the expression level of CENPF protein is abnormally increased. Figure 5 (Figure B) This leads to an imbalance in centromere protein expression and impaired spindle assembly, ultimately causing mitotic catastrophe. Figure 6 These results confirm that Lys35 methylation is crucial for PC4 to regulate the stability of downstream centromere-related gene mRNAs, ensuring proper spindle assembly and mitosis.

[0194] Example 3: PC4 protein Lys35 methylation promotes malignant phenotype of tumors.

[0195] This invention evaluates the role of Lys35 methylation of the PC4 protein in tumor malignancy and tumor treatment.

[0196] First, flow cytometry was used to detect stable PC4 after endogenous PC4 knockdown. WT and PC4 K35R Regarding the effect on Huh7 cell apoptosis, the experimental results showed that compared to shPC4+PC4... WT Processing group, shPC4+PC4 K35R The treatment group significantly increased the level of apoptosis in Huh7 cells. Figure 7 ).

[0197] Secondly, as a classic chemotherapy drug for tumors, paclitaxel disrupts microtubule polymerization by isolating tubulin monomers and blocking mitosis. However, common drug resistance limits its clinical efficacy. Given that blocking PC4-K35me2 leads to abnormal spindle assembly, this invention further demonstrates its effect on the sensitivity of paclitaxel-based chemotherapy. The results of the effect of paclitaxel on the survival rate of Huh7 cells under different treatments are as follows: Figure 8 Figure A shows the effect of docetaxel on the survival rate of Huh7 cells under different treatments. Figure 8 As shown in Figure B, in vitro cell experiments indicate that inhibiting the methylation modification of PC4 protein at the Lys35 site (PC4 K35R Mutants or SETDB1 inhibitors can increase the sensitivity of tumor cells to paclitaxel-based drugs (paclitaxel and docetaxel).

[0198] The results of the nude mouse tumor-bearing experiment also confirmed that inhibiting the methylation modification of PC4 protein at the Lys35 site (PC4) K35RMutants or SETDB1 inhibitors can inhibit tumor growth and increase the tumor's sensitivity to paclitaxel treatment. Figure 9 ).

[0199] The above experimental results confirm that Lys35 methylation modification of PC4 can regulate the malignant phenotype of tumors and drug treatment response, and this site can be used as a key target for the development of anti-tumor drugs.

[0200] Example 4: Preparation of a specific synthetic peptide (antigen peptide) targeting the Lys35 methylation site of PC4.

[0201] To further investigate the function of PC4 Lys35 methylation (hereinafter simplified as PC4-K35me2) in regulating tumor cell growth, this invention prepared a specific PC4-K35me2 antigenic peptide. First, the antigenicity and homology of this site were analyzed, and a corresponding antigenic peptide sequence was designed. Centered on the Lys35 site of the human PC4 protein, with an N-terminal QVAPE and a C-terminal PVKKQ, a methylated C-terminus was added to the Lys35 amino acid. The core sequence of the designed synthetic peptide (antigenic peptide) is: QVAPE-(Dimethyl)K-PVKKQ (SEQ ID NO.2). Finally, two modified antigenic peptides A / B and two modified control peptides C / D were designed and synthesized using peptide synthesis technology, and coupled to the carrier protein hemocyanin (KLH) for animal immunization, purification, and detection. Simultaneously, one unmodified control peptide was designed and synthesized, and coupled to the carrier protein hemocyanin (KLH) for purification and detection. The peptide mass spectrometry results were all satisfactory. Specific peptide information is shown in Table 2.

[0202] Table 2 Peptide Information

[0203]

[0204]

[0205] Example 5: Preparation of polyclonal antibodies that specifically recognize the Lys35 methylation site of PC4

[0206] To further investigate the function of PC4-K35me2 in regulating tumor cell growth, this invention prepares a polyclonal antibody that specifically recognizes PC4-K35me2.

[0207] First, all PC4-K35me2 antigenic peptides from Example 4 were conjugated with KLH carrier protein to prepare a complete antigen. This complete antigen was then combined with an adjuvant to immunize SPF-grade rabbits via intradermal injection at multiple sites. The immunization cycle included one initiating injection, three to four booster injections, and a final direct antigen injection. The titer of the antiserum against the target synthetic peptide was determined by ELISA. Next, affinity chromatography was used to purify the PC4-K35 site-specific methylated antibody through an affinity separation-affinity purification cycle, naming it the PC4-K35me2 antibody. The titer of the purified antibody and its recognition of methylated and unmethylated synthetic peptides were again detected by ELISA. Finally, the antibody was identified using dot blot and Western blot experiments.

[0208] ELISA test results as follows Figure 10 As shown, the results indicate that the Ab1 and Ab2 antibodies have a recognition ability for modified peptides that is no less than that at a 1:50K dilution, and are more than 10 times stronger than their ability to recognize unmodified negative peptides.

[0209] Dot blot results are as follows Figure 11 As shown, the results indicate that the detection limit of the antibody for modified antigen peptides reaches 1 ng, and the hybridization signal of the antibody recognizing modified peptides is 10 times stronger than that recognizing unmodified peptides.

[0210] Western blot results are as follows Figure 12 As shown, the results indicate that a distinct band of approximately 15 kDa can be detected in the Jurkat lysis buffer. MCF cells also show a weaker band at this location. This band represents the target protein PC4-K35me2, and the band size is accurate.

[0211] Example 6: Application of PC4 protein Lys35 site methylation antibody in tumors

[0212] To further verify that the PC4-K35me2 antibody can specifically recognize the Lys35 methylation modification site of the PC4 protein in cells, this invention transfected Huh7 cells with the recombinant vector Flag-PC4. WT or overexpression of the recombinant vector Flag-PC4 K35R Cell proteins were extracted 24 hours later. In the immunoprecipitation Flag product, the PC4-K35me2 antibody could recognize PC4 protein with methylation at Lys35, but could not recognize PC4 protein with unmethylation at Lys35. Figure 13 This demonstrates that the methylated antibody has good specificity.

[0213] Further, using a PC4-K35me2 antibody, the level of PC4-K35me2 in hepatocellular carcinoma cells / tissues was detected by immunohistochemistry and Western blot. Compared with paired normal cells / tissues, the level of PC4-K35me2 in tumor cells / tissues was significantly decreased (…). Figure 14 This indicates that Lys35 methylation of PC4 plays an important role in tumor development and progression, and has potential diagnostic and therapeutic value.

[0214] In summary, PC4-K35 methylation promotes tumor cell growth by regulating mitosis / cell cycle. Figure 15 The preparation of antibodies that specifically recognize PC4-K35me2 can be applied in practical applications to detect the post-transcriptional methylation modification of human PC4 protein in immunological experiments such as WB, ELISA, and immunohistochemistry, thereby exploring the significance of human PC4 protein methylation modification in tumor-related diseases.

[0215] PC4 is an RNA-binding protein (RBP) that plays an important role in early embryonic development, stem cell differentiation / reprogramming, tumorigenesis, and metabolism in mouse embryos.

[0216] This invention is the first to demonstrate that methylation at the PC4-Lys35 site directly binds to and regulates the mRNA stability and expression level of the centromere-related gene CENPF, maintaining proper spindle assembly and mitosis, thereby promoting cell cycle progression, growth, and malignant progression of tumor cells.

[0217] This invention also discovered PC4 K35R After mutation, K35 methylation disappears, which in turn increases the stability of downstream CENPF mRNA and abnormally increases protein expression levels, leading to centromere protein expression imbalance and spindle assembly disorder, ultimately causing mitotic catastrophe and cell death, and inhibiting the malignant phenotype of tumor cells.

[0218] More importantly, the methylation level of PC4-K35me2 is significantly increased in a variety of human malignant tumors, and this increase in methylation level is positively correlated with the malignant phenotype of the tumor.

[0219] In summary, this invention provides information demonstrating that methylation at the Lys35 site of the RNA-binding protein PC4 can promote the malignant progression of tumors. Specifically, this invention prepares polyclonal antibodies targeting this methylation site. On the one hand, this helps to explore the effects of Lys35 methylation modification of the PC4 protein on specific biological events such as mitosis / cell proliferation and its role in the development and progression of diseases such as tumors. On the other hand, it allows for the linking of antibodies to drugs for targeted tumor therapy and the linking of antibodies to radiolabeled markers for radioimmunoassay to assist in tumor diagnosis.

[0220] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of a reagent for detecting the methylation level at the Lys35 site of PC4 protein in the preparation of tumor diagnostic products, wherein the tumor is liver cancer.

2. The use of the SETDB1 inhibitor in the preparation of a product that inhibits methylation modification at the Lys35 site of the PC4 protein, wherein the SETDB1 inhibitor is SETDB1-TTD-IN-1.

3. A PC4 protein Lys35 methylated antigenic peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

4. The use of the antigenic peptide of claim 3 in the preparation of polyclonal antibodies for tumor detection / diagnosis / screening, wherein the tumor is liver cancer.

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