Application of Lys35 site of PC4 protein as target spot in preparation of tumor diagnosis and treatment products

Abstract

The invention discloses application of a Lys35 site of a PC4 protein as a target spot in preparation of tumor diagnosis and treatment products. The invention provides application of a Lys35 site of a PC4 protein as a target spot in preparation of a tumor diagnosis product. The research shows that the Lys35 site methylation of the PC4 protein can promote the cell cycle progress of tumor cells and promote the proliferation growth and malignant progression of the tumor cells, and the scheme for inhibiting the site methylation can be used for developing tumor treatment drugs. Therefore, the Lys35 site of the PC4 protein can be used as a key target to be applied to development of tumor diagnosis products and targeted therapy drugs. The invention further develops the PC4 protein Lys35 methylation antigen peptide and the antibody capable of specifically recognizing the PC4 protein Lys35 methylation site, and the PC4 protein Lys35 methylation antigen peptide and the antibody can be applied to regulation and control of tumor cell proliferation or growth, can be used for developing diagnostic products and targeted therapy drugs for clinical tumor diseases, and have good clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of PC4 protein Lys35 site as a target in the preparation of tumor diagnosis and treatment products. Background Art

[0002] Cancer, also known as malignant tumors, is one of the leading causes of death worldwide. Its incidence burden continues to increase, leading to a significant increase in mortality, and is one of the major public health challenges facing the world. Although great progress has been made in cancer treatment methods (such as surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy) in recent years, there is still a lack of efficient and specific early diagnostic indicators and treatment methods. In addition, tumors are prone to recurrence and metastasis, so the prognosis of most tumor patients is relatively poor. Therefore, it is urgent to conduct in-depth research on the mechanism of occurrence and development of malignant tumors and identify some efficient and specific early diagnostic indicators and treatment methods.

[0003] Human Positive Cofactor 4 (PC4) is a powerful and highly conserved nuclear protein located on chromosome 5p13.3, with a total length of approximately 18.6 kb. The transcribed PC4 mRNA is approximately 3.5 kb in length, and the encoded PC4 protein contains 127 amino acids and is approximately 15 kDa in size. Our team was the first to report that PC4 protein is abnormally highly expressed in models of normal cell malignant transformation and in clinical specimens of various tumors such as lung cancer, liver cancer, and breast cancer, and regulates the malignant progression of tumors. However, PC4 protein can regulate many molecular biological processes such as RNA polymerase II-mediated basal transcription, chromatin remodeling, DNA replication, DNA repair, and mRNA stability. The development of tumor treatment drugs using the entire PC4 protein as a target has poor targeting and is likely to cause serious effects on human health.

[0004] Therefore, exploring the specific tumor-related sites on the PC4 protein is of great significance for the development of early tumor diagnostic indicators and therapeutic drugs with high safety and specificity. Summary of the invention

[0005] The present invention aims to provide a new solution that can be used for tumor diagnosis and targeted therapy.

[0006] The first purpose of the present invention is to provide the use of PC4 protein Lys35 site as a target in the preparation of tumor diagnosis products.

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

[0008] The third object of the present invention is to provide the use of the Lys35 site of PC4 protein as a target in the preparation of drugs for preventing and / or treating tumors, or in the screening of drugs for preventing and / or treating tumors.

[0009] The fourth objective of the present invention is to provide the use of PC4 protein Lys35 site as a target in the preparation of products for regulating tumor cell mitosis or proliferation.

[0010] The fifth object of the present invention is to provide an application of a reagent for inhibiting methylation of Lys35 site of PC4 protein in the preparation of a product for treating tumors.

[0011] The sixth object of the present invention is to provide the use of a SETDB1 inhibitor as or in the preparation of a product for inhibiting the methylation modification of the Lys35 site of PC4 protein, or in the preparation of a product for treating tumors.

[0012] The seventh objective of the present invention is to provide a PC4 protein Lys35 methylation (PC4-K35me2) antigen peptide and its application.

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

[0014] The ninth objective of the present invention is to provide the use of the above antigen peptide or the above antibody.

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

[0016] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0017] The present invention has found that the Lys35 site in the amino acid sequence of PC4 is a site of protein methylation modification. Further studies have shown that the expression level of Lys35 site methylation of PC4 protein in tumor cells or tissues is higher than that in normal cells or tissues, and the methylation of Lys35 site of PC4 protein can directly bind to and regulate the stability of CENPF mRNA, ensure the balance of centromere protein expression and the correct assembly of spindles, and ultimately drive mitosis and cell proliferation, promoting the malignant progression of tumors. Therefore, Lys35 of PC4 can be used as a key target for the development of tumor diagnostic products or tumor targeted therapeutic drugs, and tumor screening, diagnosis and treatment efficacy monitoring products can be developed for this site.

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

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

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

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

[0022] Application of a reagent for detecting the methylation level of Lys35 site of PC4 protein in the preparation of tumor diagnostic products.

[0023] The application of PC4 protein Lys35 site as a target in the preparation of drugs for preventing and / or treating tumors.

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

[0025] The application of PC4 protein Lys35 site as a target in the preparation of products for regulating tumor cell mitosis or proliferation.

[0026] Specifically, the modulation is the inhibition of tumor cell proliferation, growth and malignant progression.

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

[0028] Application of reagents for inhibiting methylation of Lys35 site of PC4 protein in the preparation of tumor treatment products.

[0029] The present invention also studies that the methyltransferase SETDB1 directly catalyzes the methylation of the Lys35 site of PC4, which regulates the stability of the downstream centromere-related gene mRNA, ensures the correct spindle assembly and mitosis, and ultimately promotes the malignant phenotype of the tumor, and therefore claims protection for:

[0030] The use of a SETDB1 inhibitor as or in the preparation of a product for inhibiting methylation modification of the Lys35 site of PC4 protein, or in the preparation of a product for treating tumors.

[0031] Use of SETDB1 as or in the preparation of a product capable of activating / promoting methylation of Lys35 site of PC4 protein.

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

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

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

[0035] The invention provides an antibody which specifically recognizes the Lys35 methylation site of PC4 protein, and is prepared by using the above antigen peptide as an immunogen.

[0036] As an optional implementation scheme, the above antigen peptide is used as an immunogen to immunize animals to prepare a polyclonal antibody that specifically recognizes the Lys35 methylation site of PC4 protein (PC4-K35me2), named 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 above antigenic peptides or the above antibodies as or in the preparation of tumor detection / diagnosis / screening, targeted therapy or prognosis determination products should also be within the scope of protection of the present invention.

[0038] Among them, antibodies are used in the development of diagnostic or targeted therapeutic drugs, including linking antibodies to drugs for tumor targeted therapy and linking antibodies to radioactive markers for radioimmunoassay to assist in the diagnosis of tumors.

[0039] The invention also provides a drug for treating tumors, comprising a reagent for inhibiting the methylation of Lys35 site of PC4 protein.

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

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

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

[0043] The tumors described in the scheme of the present 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) The present invention has obtained a specific site on the PC4 protein that is closely related to tumors, namely the 35th lysine residue (Lys) of the PC4 protein. Methylation modification of this site will regulate the stability of downstream centromere-related gene mRNA, ensure correct spindle assembly and mitosis, and promote the malignant phenotype of tumors. Therefore, targeted anti-tumor drugs can be developed by inhibiting the methylation of this site.

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

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

[0048] (4) The antigen peptide and highly specific antibody against the methylation of Lys35 site of human PC4 protein provided by the present invention can detect the methylation modification of human PC4 protein in practical applications, and can be used to detect the expression difference between normal cells / tissues and tumor cells / tissues by Western blot experiments, which is helpful to study the role of methylation modification of PC4 protein in the occurrence and development of tumor diseases, and provide potential targets for the diagnosis or treatment of clinical tumor diseases. In addition, immunological related experiments such as IHC and ELISA can be used to detect the methylation level of human PC4 protein and explore its relationship with systemic diseases such as tumors. It has broad clinical application prospects in disease diagnosis, treatment and prognosis determination. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0050] Figure 2 Comparison results of PC4 protein Lys35 in the genome sequences of humans, rats, mice, orangutans, macaques, cattle and chickens.

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

[0052] Figure 4These are the CO-IP detection results of SETDB1 regulating the methylation modification of Lys35 site of PC4 protein (Figure A shows the CO-IP detection results of SETDB1 protein and PC4 protein in different cell cycles; Figure B shows the CO-IP detection results of SETDB1 protein and methylation modification of Lys35 site of PC4 protein; Figure C shows the CO-IP detection results of methylation modification of Lys35 site of PC4 protein after knocking down SETDB1 protein).

[0053] Figure 5 Figure 2 shows the expression of CENPF protein in cells with different treatments (Figure A shows the qPCR detection results of CENPF mRNA expression levels in cells with different treatments; Figure B shows the Western blot detection results of CENPF protein expression levels in cells with different treatments).

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

[0055] Figure 7 To inhibit the methylation modification of Lys35 site of PC4 protein, which leads to increased cell apoptosis level (Figure A is the flow cytometry detection result of apoptosis of cells with different treatments; Figure B is the bar chart of apoptotic cells with different treatments).

[0056] Figure 8 To inhibit the effect of methylation modification at the Lys35 site of PC4 protein on the sensitivity of paclitaxel treatment (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] Fig. 9 The results of inhibiting the methylation modification of Lys35 site of PC4 protein on tumor-bearing nude mice (Figure A is a schematic diagram of the experimental process; Figure B is the tumor size phenotype of different treatments; Figure C is the tumor weight of different treatments; Figure D is the tumor volume of different treatments; Figure E is the body weight of tumor-bearing nude mice with different treatments).

[0058] Fig.10 This is the ELISA test result of the specificity of PC4-K35me2 antibody to synthetic peptide methylated at Lys35 site.

[0059] Fig.11 Dot blot test results show the specificity of PC4-K35me2 antibody to synthetic peptide methylated at Lys35 site.

[0060] Fig.12Western blot test results of the specificity of PC4-K35me2 antibody to synthetic peptide methylated at Lys35 site.

[0061] Fig.13 Western blot detection results showing that the PC4-K35me2 antibody specifically recognizes the methylation modification of the Lys35 site of the PC4 protein.

[0062] Fig.14 Results of the PC4-K35me2 antibody detection of the methylation level of the Lys35 site of the PC4 protein in normal cells / tissues and tumor cells / tissues (Figure A is the Western blot detection result of the methylation level of the Lys35 site of the PC4 protein in normal cells and tumor cells; Figure B is the immunohistochemistry result of the methylation level of the Lys35 site of the PC4 protein in normal tissues and tumor tissues).

[0063] Fig.15 Schematic diagram of how methylation modification of the Lys35 site of PC4 protein promotes tumor cell growth. DETAILED DESCRIPTION

[0064] The following is a further description and explanation of the technical route for the regulation of the methylation of Lys35 site of human PC4 protein in tumor cells and the preparation of its specific antibody, in combination with the drawings and specific examples of the specification. However, the examples do not limit the present invention in any form.

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

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

[0067] The SETDB1 inhibitor is SETDB1-TTD-IN-1, CAS No.: 2755823-12-0, and the structural formula is:

[0068] The amino acid sequence of human Positive Cofactor 4 (PC4) is shown in SEQ ID NO.1:

[0069] MPKSKELVSSSSSGSDSDSEVDKKLKRKKQVAPEKPVKKQKTGETSRALS SSKQSSSSRDDNMFQIGKMRYVSVRDFKGKVLIDIREYWMDPEGEMKPGRK GISLNPEQWSQLKEQISDIDDAVRKL.

[0070] The core sequence of the PC4 protein Lys35 methylated antigen peptide is shown in SEQ ID NO.2, SEQ ID NO.2: QVAPEKPVKKQ, wherein the 6th lysine 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, 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, blocking goat serum, immunohistochemistry secondary antibody, DAB colorimetric solution, neutral gum, 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 lysate, 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 solution, primary antibody diluent, Beyo ECL luminescent liquid, 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] Slicer, electronic balance, slicer, inverted phase contrast microscope, immunohistochemical humidification box, ordinary PCR instrument, qPCR instrument, cell culture incubator, vortex oscillator, constant temperature shaker, clean bench, centrifuge, ice maker, gel imager, vertical protein electrophoresis tank, microplate reader, pipette, PVDF membrane, anti-slip slide, cover glass, culture bottle, centrifuge tube, gun tip, 6-well plate, 96-well plate, etc.

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

[0079] Huh7 cells were set up in the following treatment groups:

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

[0081] shPC4: Transfect PC4 shRNA lentivirus (introduced sequence: ACAGAGCAGCAGCGCAGATT) to construct a PC4 stable knockdown cell line.

[0082] shPC4+PC4 WT : The above shPC4 group cells were then transfected with PC4 wild type (PC4 WT ) overexpression of recombinant lentiviral vector. PC4 constructed by lentiviral vector 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 then transfected with PC4-K35 site mutation (PC4K35R ) overexpression of recombinant lentiviral vector. PC4 constructed by lentiviral vector 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: above shPC4+PC4 WT The cells were treated with SETDB1-IN (the animal experiments were intravenous administration).

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

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

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

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

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

[0107] 1. Detection of cell apoptosis by flow cytometry

[0108] Each group (NC, shPC4, shPC4+PC4 WT and shPC4+PC4 K35R ) and then add the cells to the corresponding centrifuge tube containing the cell supernatant culture medium (the process needs to be gentle to avoid physical apoptosis of the cells). Centrifuge at 1000rpm for 3 minutes to collect the cell pellet. Add PBS to resuspend the cells, centrifuge again at 1000rpm for 3 minutes, and collect the cell pellet. Use ultrapure water to dilute 4×Binding buffer to 1×Binding buffer, and add 200μL 1×Binding buffer to the cell pellet to resuspend. Add 5μL annexin V to the cells, shake and mix, and stand in the dark for 15 minutes. Then add 10μL PI solution (Annexin V / PI Cell Apoptosis Detection Kit) to the cells, shake and mix, and perform subsequent FACS analysis.

[0109] 2. Cell cycle synchronization

[0110] To synchronize cells to the G1 phase, first treat the cells with thymidine (Sigma, T3763) for about 16-18 hours, then release the cells and culture them in new medium for 8 hours. Next, treat the cells with thymidine again for 16-18 hours. In this way, the cells will be synchronized to the G1 phase.

[0111] For S phase, cells arrested in G1 phase were released and cultured for 2 hours. Then, cells were collected for subsequent studies.

[0112] For the early and late G2 phases, cells arrested in the G1 phase were first released for 2 hours. Then, they were treated with the RO3306 inhibitor and cultured for 6 hours to obtain cells in the early G2 phase. Then, they were cultured for another 4 hours to obtain cells in the late G2 phase.

[0113] For M phase, cells in M ​​phase were released from late G2 phase by treatment with the inhibitor RO3306 for 1 hour, or cells were incubated with nocodazole for 18 to 20 hours before collection.

[0114] Cells at each stage were collected by digestion and then analyzed by Western Blotting.

[0115] 3. Immunofluorescence staining

[0116] 1) Cell fixation: First, the Huh7 cells of each group (NC, shPC4, shPC4+PC4 WT ,shPC4+PC4 K35R ,shPC4+PC4 WT +SETDB1-IN、shPC4+PC4 K35R +SETDB1-IN) remove the culture medium, 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. Then 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 antibody: Prepare primary antibodies with a dilution ratio of 1:200, namely ACA (Antibodies Incorporated, 15-235-0001) and β-Tubulin (BD, 556321). Add the primary antibody to the sample and incubate overnight at 4 degrees. After incubation, wash with PBST (PBS containing 0.1% Triton-X100) for 5 minutes, and repeat the washing operation 3 times.

[0120] 5) Incubation with secondary antibody: prepare secondary antibody with a dilution ratio of 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 DAPI (1:4000) at a dilution of 1:4000, add DAPI to the sample and incubate for 5 minutes with gentle shaking. Wash with PBST for 5 minutes and repeat 3 times. After the end, you can take pictures on the machine.

[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 4 / mL cell suspension was added to each well of a 96-well plate at a density of 100 μL. After adherence, different concentrations of paclitaxel (0.1, 1, 10, 100 nM) and docetaxel (0.1, 1, 100 nM) were added, combined 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, the cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0124] 2) OD value detection and growth curve drawing: After 48 hours, take out the 96-well plate, and prepare the working solution according to the ratio of 10μL CCK-8 reagent and 90μL PBS according to the number of culture wells to be tested each time. Aspirate the old culture medium, add 100μL of the newly prepared CCK-8 working solution, put it back in the 37℃ cell culture incubator for 2 hours, and then use an enzyme reader to detect the absorbance value at a wavelength of 450nm, and finally draw a growth curve.

[0125] 5. Nude mice bearing tumors

[0126] 1) The well-growing groups (shPC4+PC4WT and shPC4+PC4 K35R ) Huh7 cells were used as the research model. The cells in each group were digested, counted and prepared into a density of 2.5×10 7 / mL of cell suspension was placed in an ice box for later use.

[0127] 2) Male BALB / c nude mice aged 5-6 weeks were purchased from the Animal Center of the Third Military Medical University and divided 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 in each group. 200 μL of cell suspension was injected into the back of nude mice, that is, 5×10 6 cells.

[0128] 3) The nude mice were kept in an SPF animal room. When the tumor grew to about 100 mm 3 At 14:1, mice were randomly divided into groups and received drug treatment, namely paclitaxel (ip, 10 mg / kg), SETDB1-TTD-IN-1 (ip, 30 mg / kg), or the combination of paclitaxel and SETDB1-TTD-IN-1, for 6 cycles every 3 days. The tumor growth was observed every 5 days, the tumor size was measured, and the mice were weighed. Tumor volume = (length × width 2 ) / 2. At the end of the experiment, the nude mice were killed and the tumors were taken out for photographing and weighing. 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) Each group (NC, shPC4, shPC4+PC4 WT ,shPC4+PC4 K35R ,shPC4+PC4 WT +SETDB1-IN、shPC4+PC4 K35RAfter adding 5ug / mL of Actinomycin D to Huh7 cells (+SETDB1-IN), samples were collected at 0h, 2h, 4h, and 6h, i.e., the medium in the culture flask was poured out, the cells were washed 1-2 times with PBS, and 1mL of Trizol was added. The cells of each group were scraped off with a cell scraper, collected, and transferred to a 1.5mL EP tube;

[0131] 2) Extract RNA: Add 0.2mL chloroform to each tube, shake vigorously for 15s, and let stand at room temperature for 15 minutes; centrifuge at 4℃, 12000rpm, 15 minutes, and the liquid can be seen to be divided into 3 layers; carefully take 450μL supernatant to another 1.5mL EP tube; add 0.5mL isopropanol, gently invert and mix for 30s, and let it stand at 4℃ for about 10min; centrifuge at 4℃, 12000rpm, 10min, discard the supernatant, and a white needle-sized precipitate can be seen at the bottom of the tube; add 1mL 75% ethanol (prepared with DEPC water) / mL Trizol to each EP tube to wash the precipitate; centrifuge at 4℃, 12000rpm, 5min, and discard the supernatant; repeat the RNA cleaning; dry in the clean bench (to prevent ethanol from inhibiting reverse transcriptase); dissolve the RNA at the bottom of the tube with an appropriate amount of RNase-free DEPC water, measure the concentration and balance;

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

[0133] 4) Real-time PCR: system 20 μL (SYBR 10 μL, primer (upper) 0.5 μL, primer (lower) 0.5 μL, cDNA 1 μL, dH2O 8 μL), program annealing temperature 59°C, cycle number 40. Specific primer sequences are shown in Table 1.

[0134] 5) The mRNA expression levels of each group were statistically analyzed, and a graph of the mRNA stability of each gene was prepared.

[0135] Table 1 qPCR primer sequences of cell cycle genes

[0136]

[0137] 7. Western Blotting

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

[0139] 2) Determination of protein concentration by BCA method: Take 0.8mL of protein standard preparation agent and add it to a tube of standard protein (20mg BSA). After complete dissolution, a 25mg / mL standard protein solution is prepared. Take 20μL of 25mg / mL standard protein, add 980μL PBS, dilute 50 times, and the concentration is 0.5mg / mL. Prepare BCA working solution (the ratio of A solution and B solution is 50:1) and prepare it before use. Add 0.5mg / mL standard protein to a 96-well plate according to 0, 1, 2, 4, 8, 12, 16, and 20μL, and then add 0.9% saline or purified water to each well to make up to 20μL. Add 2μL of sample and 18μL PBS to a 96-well plate, with a total volume of 20μL. Add 200μL of BCA working solution to each well and let stand at 37℃ for 30min. Determine protein OD with an enzyme reader 562 Draw a standard curve, calculate the protein concentration, and balance each group based on the lowest concentration. Take a certain volume of protein, add 1 / 5 volume of 6× loading buffer, and denature the protein at 100℃ for 5min.

[0140] 3) SDS-PAGE electrophoresis: prepare 12% lower separation gel and 5% upper concentration gel. Take the electrophoresis device produced by Bio-Rad, align and fix two clean Bio-Rad glass plates (8cm×8cm, 1.5mm) on the rack. Evenly pour the lower separation gel to about 2 / 3 of the height of the glass plate, slowly add isopropanol sealing glue while moving left and right, and let it stand for about 30 minutes. When the boundary between the upper isopropanol and the lower separation gel is obvious, wash with distilled water and discard the isopropanol. Pour the concentration gel and slowly insert the comb horizontally to prevent the formation of bubbles. Let it stand at room temperature for about 30 minutes. Put the glass plate with the gel into the electrophoresis tank and pour in 500mL of protein electrophoresis buffer. Note that the inner tank uses the newly configured electrophoresis solution, and the outer tank can use the recycled electrophoresis solution. Take out the protein, centrifuge briefly to collect the liquid on the wall of the tube, use a microinjector or pipette to load the sample protein, and add about 5μL marker on both sides or other suitable positions. Run electrophoresis at 80V for about 40min. When bromophenol blue has run through the concentrated gel, adjust the voltage to 120V and run electrophoresis for about 1.5h. Stop electrophoresis when bromophenol blue is about to run to the lower edge of the separation gel (about 1cm from the bottom of the gel).

[0141] 4) Transfer (wet transfer): Prepare transfer buffer. Methanol is added when preparing for transfer. Soak the clamp and filter paper in the buffer and pre-cool at 4°C. According to the number of sample wells, use a comb as a control to cut PVDF membranes of approximately the same size and soak them in methanol for about 10 minutes. Remove the glass plate from the electrophoresis tank and gently transfer the gel to the buffer. Use a gel cutting board to cut the gel according to the location of the antibody indicated by the marker. Place the items in the transfer clamp according to the principle of "black gel and white membrane", from bottom to top: black clamp, filter paper, gel, PVDF membrane, filter paper, white clamp. Use a glass rod to carefully and gently drive away bubbles. Clamp the clamp and place it in the electrotransfer device. Fill the built-in white box with ice cubes. Place the electrotransfer device in an ice bath for transfer. The current is 250mA, and the electrotransfer is about 60min-120min. Adjust the time according to the molecular weight of the protein.

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

[0143] 6) Incubate primary and secondary antibodies: Rinse the blocked PVDF membrane in TBST and place it in another clean incubation box. Dilute the antibody to a concentration of 1:1000 (PC4-K35me2) or 1:2000 (β-actin) with the primary antibody diluent, add the diluted antibody to the incubation box, place it on a shaker, and incubate overnight at 4°C. Take out the PVDF membrane the next day, place it in a transparent plastic box for washing the membrane, pour about 20mL TBST so that it can immerse the PVDF membrane, and place it on a shaker to wash 3 times at room temperature, each time for about 10 minutes. Dilute the secondary antibody with western secondary antibody diluent (or TBST) to the concentrations of goat anti-mouse (1:1000, anti-β-actin) and goat anti-rabbit (1:2000, anti-PC4-K35me2), and add it to the incubation box containing the PVDF membrane. Place it on a shaker and incubate the secondary antibody at 37°C for about 2 hours. Wash the secondary antibody 3 times at room temperature on a shaker, each time for about 10 minutes.

[0144] 7) Chemiluminescence development: Take 100 μL of ECL chemiluminescence reagent A and B, mix them evenly, and place them away from light. Spread the PVDF membrane on the cut plastic bag, drop the ECL chemiluminescence solution evenly on the PVDF membrane, and use the bio-rad gel imaging instrument for exposure imaging.

[0145] 8. Immunohistochemistry

[0146] 1) Sample collection 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 turn and fix for 24-72 hours.

[0147] 2) Tissue trimming and dehydration: Take out all tissues in the sampling bottle, trim them one by one, try to make each tissue flat, put them into the dehydration basket, and make sure to mark them. Put them into the dehydrator and dehydrate them at room temperature for 16 hours.

[0148] 3) Tissue embedding: Place the tissue to be embedded in liquid paraffin and place it neatly against the bottom surface, making sure to mark it. Allow the wax block to solidify at room temperature (more than 0.5h).

[0149] 4) Trimming: Take out the solidified wax block, trim it into a regular rectangular block, and then re-iron the label.

[0150] 5) Slicing: Place the wax block at -20℃ half an hour before slicing. The thickness of the slices is generally 5mm.

[0151] 6) Collecting slides: Mark blank slides with pencil in advance, collect 5-6 slides for each tissue, and usually collect 2-3 tissues on each slide.

[0152] 7) Dewaxing: Dewaxing was performed by placing the stent in xylene II, xylene I, 100% ethanol, 95% ethanol, and 80% ethanol in sequence, with the dwell time being 10 min, 10 min, 5 min, 5 min, and 5 min, respectively. After dewaxing, the stent was placed in a beaker and rinsed with clean water for 3 min.

[0153] 8) Sodium citrate repair: Add 300mL of 0.01mol sodium citrate solution to a beaker, then place the stent into the beaker, and then place the beaker into a pot of pre-boiled water. When the temperature in the beaker rises to above 93°C and the repair is timed for 20 minutes, take out the beaker and cool it to about 37°C at room temperature.

[0154] 9) Hydrogen peroxide repair: Add 300 mL of PBS solution (or double distilled water) to a beaker, and place the stent in the beaker for immersion and cleaning for 3 times, 5 minutes each time. Directly drop 3% H2O2 on the tissue surface of each slide, being careful not to touch the tissue, and place it in the room for 15 minutes.

[0155] 10) Serum blocking: Add 300 mL of PBS solution (or double distilled water) to a beaker, and place the stent in the beaker for immersion and cleaning for 3 times, 5 minutes each time. Add serum drops on the tissue surface of each slide to block some non-specific sites, then place the slide in a humidified box, and then place the humidified box in a 37°C incubator for 30 minutes.

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

[0157] 12) Add secondary antibody: Add 300 mL PBS solution (or double distilled water) to a beaker, and soak the scaffold in the beaker for 3 times, 5 minutes each time. Add the secondary antibody directly onto the tissue surface of each slide, and then place the slide in a humidified box, and then place the humidified box in a 37°C incubator for 40 minutes.

[0158] 13) Add color developer: Add 300 mL PBS solution (or double distilled water) to a beaker, and soak the stent in the beaker for 3 times, 5 minutes each time. Gently shake the slide dry, and drop the prepared DAB color developer on the tissue surface of each slide. Let the color develop for about 10 seconds, then quickly wash it in water and observe it under a microscope. If the color development is not enough, repeat the previous steps.

[0159] 14) Re-staining: Place the developed slide back into the holder and rinse with water for 3 minutes. Soak in hematoxylin for 3 minutes (overstaining), rinse with water for 3 minutes. Place in hydrochloric acid for 6 seconds (recovery), rinse with water for 3 minutes. Observe under the microscope to see if it is re-stained (if not, repeat this step; if the re-staining is heavy, place in hydrochloric acid for 6 seconds and rinse with water for 3 minutes).

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

[0161] 16) Sealing: Seal the slides with drops of neutral gum, let them dry, observe under a microscope and take photos.

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

[0163] 1) Protein extraction: Wash the cells twice with pre-cooled PBS, and aspirate the PBS for the last time; add pre-cooled Lysis buffer; use a pipette to disperse the cells, transfer the suspension to a 1.5 mL EP tube, and shake slowly at 4°C for 15 min; centrifuge at 4°C, 14,000 rpm for 15 min, and immediately transfer the supernatant to a new centrifuge tube.

[0164] 2) Remove large molecular proteins: Prepare agarose beads, wash the beads twice with Lysis buffer, and then prepare them to 50% concentration with Lysis buffer; add 100 μL of agarose beads to every 1 mL of total protein, and shake at 4°C for 10 minutes. Centrifuge at 4°C, 14,000 rpm for 15 minutes, transfer the supernatant to a new centrifuge tube, and remove the agarose beads. Make a protein curve and determine the protein concentration. Dilute the total protein to about 1 ug / μL with Lysis buffer.

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

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

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

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

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

[0170] 1) Protein extraction: The samples were taken out from -80°C, and 4 volumes of lysis buffer (8 M urea, 1% protease inhibitor, 1% ubiquitinase inhibitor) were added to each group of samples, and ultrasonic lysis was performed. The samples were centrifuged at 4°C, 12000g for 10 min to remove cell debris, and the supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

[0171] 2) Trypsin hydrolysis: Take an equal amount of each sample protein for enzymatic hydrolysis, and adjust the volume to the same with lysis buffer. Slowly add TCA at a final concentration of 20%, vortex to mix, and precipitate at 4°C for 2h. Centrifuge at 4500g for 5min, discard the supernatant, and wash the precipitate with pre-cooled acetone 2-3 times. After drying the precipitate, add TEAB at a final concentration of 200mM, ultrasonically break up the precipitate, add trypsin at a ratio of 1:50 (protease: protein, m / m), and hydrolyze overnight. Add dithiothreitol (DTT) to a final concentration of 5mM and reduce at 56°C for 30min. Then add iodoacetamide (IAA) to a final concentration of 11mM and incubate at room temperature in the dark for 15min.

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

[0173] 4) Liquid chromatography-mass spectrometry analysis: The peptides were dissolved in liquid chromatography mobile phase A and separated using the EASY-nLC1000 ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient settings: 0-86min, 3%~17% B; 86-110min, 17%~28% B; 110-115min, 28%~80% B; 115-120min, 80% B, the flow rate was maintained at 500nL / min. After the peptides were separated by the ultra-high performance liquid phase system, they were injected into the NSI ion source for ionization and then into the Q Exactive TM Plus mass spectrometry was used for analysis. The ion source voltage was set to 2.2 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scanning range was set to 350-1800 m / z, and the scanning resolution was set to 70,000; the secondary mass spectrometry scanning range had a fixed starting point of 100 m / z, and the secondary scanning resolution was set to 35,000. The data acquisition mode used a data-dependent scanning (DDA) program, that is, after the primary scan, the top 20 peptide parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometry, the 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 of the tandem mass spectrometry scan was set to 15 seconds to avoid repeated scanning of the parent ion.

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

[0175] (1) Rabbit immunization: ① Prepare immunization materials: dilute the immunogen with physiological saline, and then mix it with the corresponding adjuvant in a 1:1 ratio. The antigen and adjuvant are completely mixed to form a stable emulsion. The antigen mixture is extracted with a syringe and injected into two points under the skin of the shoulders and two points in the muscles of the hind legs of SPF experimental New Zealand white rabbits. Approximately 1 / 4 volume of the immunogen is injected into each area. In this way, the immunogen can persist and thus enhance the immune response. ② Immunization: Each rabbit is immunized 6 times in total, on the 1st day, 7th day, 14th day, 21st day, 28th day, and 40th day. ③Blood collection: The first blood collection is on the 28th day, 2 mL of blood is collected and centrifuged, the supernatant is collected after centrifugation and sent to the laboratory for serum screening test, including ELISA or Dot blot; the 2nd / 3rd / 4th blood collection is on the 35th day, 47th day, and 52nd day, 30 mL of blood is collected each time, the supernatant is collected after centrifugation and sent to the laboratory for serum screening test, including ELISA or Dot blot; the serum with positive test results is purified.

[0176] (2) Purification of PC4 protein Lys35 methylated antibody: ①Prepare protein A affinity column: Usually select 5mL or 10mL protein A filler, mix and stir equal volumes of filler and PBS buffer solution, and remove bubbles in the filler by vacuuming. Slowly add protein A filler into the glass column to prepare the chromatography column. Avoid column drying during this process. After filling, equilibrate the column with 10 times the volume of pre-cooled PBS buffer solution. ②Protein A affinity chromatography: After filtering the serum with a filter, load it onto the equilibrated protein A chromatography column. In order to detect the binding efficiency of the antiserum with the filler, the load effluent needs to be retained. Wash the column with PBS buffer solution and then elute with 150mM glycine buffer. Collect the eluate and add neutralization buffer solution to adjust the pH to 7. ③Enrich the target antibody: Load the crude pure IgG obtained after protein A purification onto the equilibrated antigen polypeptide 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 a non-modified affinity chromatography column and directly collect the effluent (FT3) to remove non-specific antibody components. ⑤ Antibody preservation: Determine the protein content. Add 10% glycerol to preserve the antibody, and store the purified antibody at -20°C after aliquoting.

[0177] (3) Quality control of PC4 protein Lys35 methylation antibody: PC4-K35me2 antibody was purified by affinity chromatography using antibody affinity separation-affinity purification cycle purification technology. The antibody titer and recognition of methylated target antigen peptide and non-methylated synthetic peptide were detected by ELISA after purification. The antibody was then identified by protein dot blot and Western blot experiments. The details are as follows:

[0178] 1) ELISA detection: ① Antigen coating: dilute the antigen with coating solution, add 50ug / well to the ELISA plate in turn, and incubate in a refrigerator at 4℃ overnight or in a 37℃ oven for 2h. ② Washing: Take out the ELISA plate coated the day before, add 1×TBST and wash 3 times. ③ Blocking: Add 1% BSA blocking solution to the cleaned ELISA plate, incubate at 37℃ for 1h, and then wash 1-3 times. ④ Primary antibody incubation: Dilute the antibody 3 times starting from 1:1K. Adjust the dilution volume according to the actual situation. Add to the ELISA plate in turn, incubate at 37℃ for 1.5h, and then wash 1-3 times. ⑤ Secondary antibody incubation: dilute the secondary antibody to 1:10K with 1% BSA blocking solution, incubate at room temperature or 37℃ for 45min, and then wash 1-3 times. ⑥ Color development: Add TMB color development solution for 5-10min, then 1M sulfuric acid to terminate the display reaction, and read the data with an ELISA reader.

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

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

[0181] The following examples are experimental results and analysis descriptions:

[0182] Example 1 Determination of human PC4 protein methylation sites

[0183] First, through mass spectrometry analysis of methylome ( Figure 1 ), found that Lys35 is a methylation site in the amino acid sequence of human PC4. Protein sequence comparison showed that Lys35 is highly conserved in the genomes of humans, rats, mice, gorillas, macaques, cattle, and chickens ( Figure 2 ).

[0184] The results of the CO-IP experiment (using PC4 antibody to capture the enriched protein) confirmed the methylation modification of PC4 protein in Huh7 cells ( Figure 3 Next, PC4 without methylation was constructed. K35R Mutant vectors (specific sequence experimental grouping).

[0185] Transfection of Flag-tagged overexpression recombinant vector Flag-PC4 into Huh7 cells WT and Flag-PC4 K35R The results of the CO-IP experiment (using Flag antibody to capture enriched proteins) are as follows Figure 3 As shown in Figure B, the results show that with PC4 WT Group comparison, PC4 K35R PC4 methylation disappeared in the mutant group. These results finally identified Lys35 as the specific methylation site of human PC4 protein.

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

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

[0188] In order to study the modifying enzyme of Lys35 methylation of PC4, the lysine methyltransferase SETDB1 was discovered by analyzing the protein spectrum of PC4 interaction. Huh7 cells in different cell cycles were collected after cell cycle synchronization, and IP experiments (using PC4 antibody to capture enriched proteins) confirmed that SETDB1 has a strong binding ability with PC4 protein in the G2 phase ( Figure 4 Figure A).

[0189] Subsequently, the present invention constructed a SETDB1 plasmid with an HA tag for transfection, and IP experiments (using PC4 antibody to capture enriched proteins) confirmed that the wild type of SETDB1 can increase the methylation of Lys35 of PC4 protein ( Figure 4 Figure B).

[0190] At the same time, the SETDB1 knockdown experiment was carried out by transfecting siRNA with Lipo3000, that is, NC group siRNA (sequence: UUCUCCGAACGUGUCACGUTT), SETDB1-1 group siRNA (sequence: GGCCAAAUCAACCCUAACUTT) and SETDB1-2 group siRNA (sequence: CCGAGGCUUUGCUCUUAAATT) were transfected into Huh7 cells respectively. The results of CO-IP experiment (using PC4 antibody to capture enriched protein) showed that compared with the NC group, inhibition of SETDB1 expression could reduce the Lys35 methylation of PC4 protein ( Figure 4 Figure C).

[0191] The above results confirmed that the methyltransferase SETDB1 regulated the methylation of Lys35 of PC4.

[0192] Next, the present invention constructs a PC4 mutant vector with a Flag tag (overexpression recombinant vector Flag-PC4 WT and overexpression recombinant vector Flag-PC4 K35R ), cells were transfected with different PC4 mutant vectors or treated with 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 with different treatments was detected. The results showed that inhibition of methylation modification of Lys35 site of PC4 protein (PC4 K35R Mutation or SETDB1-IN) increases the stability of downstream CENPF mRNA ( Figure 5 Figure A), causing abnormal increase in CENPF protein expression level ( Figure 5 Figure B), leading to imbalanced expression of centromere proteins and spindle assembly disorders, ultimately causing mitotic catastrophe ( Figure 6 These results confirm that Lys35 methylation is essential for PC4 to regulate the mRNA stability of downstream centromere-related genes and ensure correct spindle assembly and mitosis.

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

[0195] The present invention evaluates the role of Lys35 methylation of PC4 protein in tumor malignant progression and tumor treatment.

[0196] First, flow cytometry was used to detect the endogenous knockdown of PC4 followed by stable PC4 WT and PC4 K35R The results showed that compared with shPC4+PC4 WT Treatment group, shPC4+PC4 K35R The treatment group significantly increased the apoptosis level of Huh7 cells ( Figure 7 ).

[0197] Secondly, as a classic tumor chemotherapy drug, paclitaxel destroys microtubule polymerization by isolating tubulin monomers and blocking mitosis, but the common drug resistance phenomenon limits its clinical efficacy. Given that blocking PC4-K35me2 can lead to abnormal spindle assembly, the present invention further confirms its effect on the sensitivity of paclitaxel chemotherapy. The results of the effect of paclitaxel on the survival rate of Huh7 cells under different treatments are shown in the figure. Figure 8 As shown in Figure A, the effects of docetaxel on the survival rate of Huh7 cells under different treatments are shown in Figure 8 As shown in Figure B, the results of in vitro cell experiments showed that inhibition of PC4 protein Lys35 site methylation modification (PC4 K35R Mutations or SETDB1 inhibitors) can increase the sensitivity of tumor cells to the treatment of taxanes (paclitaxel and docetaxel).

[0198] The results of nude mouse tumor-bearing experiments also confirmed that inhibition of PC4 protein Lys35 site methylation modification (PC4 K35Rmutations or SETDB1 inhibitors) can inhibit tumor growth and increase tumor sensitivity to paclitaxel ( Fig. 9 ).

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

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

[0201] In order to further study the function of PC4 Lys35 methylation (hereinafter simplified as PC4-K35me2) in regulating tumor cell growth, the present invention prepared a specific PC4-K35me2 antigen peptide. First, the antigenicity and homology of the site were analyzed, and the corresponding antigen peptide sequence was designed. The Lys35 site of human PC4 protein was centered, with QVAPE at the N-terminus and PVKKQ at the C-terminus. A methylated C-terminus was added to the amino acid Lys35. The designed synthetic peptide (antigenic peptide) core sequence was: QVAPE-(Dimethyl)K-PVKKQ (SEQ ID NO.2). Finally, two modified antigen polypeptides A / B and two modified control polypeptides C / D were designed and synthesized using peptide synthesis technology, and coupled with the carrier protein hemocyanin (KLH) for animal immunization, purification and detection; at the same time, a non-modified control polypeptide was designed and synthesized, and coupled with the carrier protein hemocyanin (KLH) for purification and detection. The results of the peptide mass spectrometry detection were all qualified, and the specific peptide information is shown in Table 2:

[0202] Table 2 Peptide information

[0203]

[0204]

[0205] Example 5 Preparation of a polyclonal antibody that specifically recognizes the Lys35 methylation site of PC4

[0206] In order to further study the function of PC4-K35me2 in regulating tumor cell growth, the present invention prepares a polyclonal antibody that specifically recognizes PC4-K35me2.

[0207] First, all PC4-K35me2 antigen peptides in Example 4 were coupled with KLH carrier protein to prepare complete antigens, which were then combined with adjuvants to immunize SPF rabbits, and multiple injections of antigen emulsions were intradermally injected. The number of immunizations included one priming injection, 3 to 4 booster injections, and the last antigen direct injection. The titer of the antiserum against the target synthetic peptide was determined by ELISA; then affinity chromatography was used to perform antibody affinity separation-affinity purification cycles to purify PC4-K35 site-specific methylated antibodies, named PC4-K35me2 antibodies, and the titer of the purified antibodies and the recognition of the methylated target antigen peptide and the non-methylated synthetic peptide were detected by ELISA again. Finally, protein dot hybridization (Dot blot) and Western blot experiments were used to identify the antibodies.

[0208] ELISA test results Fig.10 As shown, the results indicate that the recognition ability of Ab1 and Ab2 antibodies for the modified peptide is not less than 1:50K dilution, and is more than 10 times stronger than the ability to recognize the non-modified negative peptide.

[0209] Dot blot test results are as follows Fig.11 As shown, the results indicate that the lower limit of detection of the modified antigenic peptide by the antibody reaches 1 ng, and the hybridization signal of the antibody recognizing the modified peptide is 10 times stronger than the hybridization signal of the antibody recognizing the non-modified peptide.

[0210] Western blot test results Fig.12 As shown in the figure, the results show that an obvious band of about 15kDa can be detected in Jurkat lysate, and MCF cells also have a weaker band at this position. This band is 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] In order to further verify that the PC4-K35me2 antibody can specifically recognize the PC4 protein Lys35 methylation modification site in cells, the present invention transfected the overexpression recombinant vector Flag-PC4 in Huh7 cells. WT Or overexpression recombinant vector Flag-PC4 K35R After 24 hours, the cell proteins were extracted. In the immunoprecipitated Flag product, the PC4-K35me2 antibody could recognize the PC4 protein in the methylated state at Lys35, but could not recognize the PC4 protein in the unmethylated state at Lys35 ( Fig.13 ), proving that the methylation antibody has good specificity.

[0213] PC4-K35me2 antibody was further used to detect the level of PC4-K35me2 in liver cancer cells / tissues by immunohistochemistry and Western blot. Compared with the paired normal cells / tissues, PC4-K35me2 in tumor cells / tissues was significantly increased ( Fig.14 ), indicating that Lys35 methylation of PC4 plays an important role in the occurrence and development of tumors and has potential diagnostic and therapeutic value.

[0214] In summary, PC4-K35 methylation modification promotes tumor cell growth by regulating mitosis / cell cycle ( Fig.15 ); The preparation of antibodies that specifically recognize PC4-K35me2 can be used 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 important roles in early embryonic development, stem cell differentiation / reprogramming, tumorigenesis, and metabolism in mouse embryos.

[0216] The present invention is the first to study and conclude that methylation of the PC4-Lys35 site will directly bind to and regulate the mRNA stability and expression level of the centromere-related gene CENPF, maintain correct spindle assembly and mitosis, and thereby promote cell cycle progression, tumor cell growth and malignant progression of tumor cells.

[0217] The present invention also found that PC4 K35R After the mutation, the methylation of K35 disappears, which in turn increases the stability of downstream CENPF mRNA and abnormally increases the protein expression level, leading to imbalanced centromere protein expression and spindle assembly disorders, 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, the present invention provides a methylation site at Lys35 of RNA binding protein PC4 that can promote the malignant progression of tumors. In particular, the present invention prepares a polyclonal antibody against methylation at the site, which, on the one hand, helps to explore the effects of methylation modification at Lys35 of PC4 protein on specific biological events such as mitosis / cell proliferation and its role in the occurrence and development of diseases such as tumors; on the other hand, the antibody is linked to a drug to carry out tumor targeted therapy, and the antibody is linked to a radioactive marker for radioimmunoassay to assist in the diagnosis of tumors.

[0220] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

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

2. Application of reagents for detecting the methylation level of Lys35 site of PC4 protein in the preparation of tumor diagnostic products.

3. Use of the Lys35 site of PC4 protein as a target in the preparation of drugs for preventing and / or treating tumors, or in the screening of drugs for preventing and / or treating tumors.

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

5. Application of reagents that inhibit methylation at the Lys35 site of PC4 protein in the preparation of tumor treatment products.

6. Use of SETDB1 inhibitors as or in the preparation of products for inhibiting methylation modification of Lys35 site of PC4 protein, or in the preparation of products for treating tumors.

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

2.

8. An antibody that specifically recognizes the Lys35 methylation site of PC4 protein, characterized in that: The antigen peptide according to claim 7 is used as an immunogen to prepare the immunogen.

9. Use of the antigen peptide according to claim 7 or the antibody according to claim 8 as or in the preparation of a product for tumor detection / diagnosis / screening, targeted therapy or prognosis determination.

10. A drug for treating tumors, characterized in that: Includes agents that inhibit methylation of Lys35 site of PC4 protein.

Images