Human assisted transcription factor 4 ubiquitination site for tumor diagnosis and treatment and application thereof

Abstract

The application discloses a human auxiliary transcription factor 4 ubiquitination site for tumor diagnosis and treatment and application thereof. It is found by the application that the ubiquitination level of a Lys68 site of PC4 protein is significantly increased in tumor cells or tissues, and the Lys68 site ubiquitination can be used as a diagnosis target and index; and the Lys68 site ubiquitination can enhance the RNA binding capacity of PC4, and further promote the cell cycle progression of tumor cells, promote the growth and proliferation of tumor cells, significantly promote the malignant phenotype transformation of tumors, promote the malignant progression of tumors, and a scheme for inhibiting the ubiquitination of the site can be used for developing tumor treatment drugs. Therefore, the Lys68 site of the PC4 protein can be used as a key target for developing tumor diagnosis products and targeted treatment drugs. Further, the application develops a PC4 protein Lys68 ubiquitination antigen peptide and an antibody capable of specifically recognizing the PC4 protein Lys68 ubiquitination site, and the PC4 protein Lys68 ubiquitination antigen peptide and the antibody can be applied to regulating the proliferation or growth of tumor cells, and can be used for developing clinical tumor disease diagnosis and treatment products. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a human helper transcription factor 4 (human PC4 protein) ubiquitination site for tumor diagnosis and treatment and its application. Background Technology

[0002] In medicine, cancer refers to malignant tumors originating from epithelial tissue. Clinical manifestations vary depending on the organ, location, and stage of development. However, early-stage malignant tumors often present with no obvious symptoms, and even when symptoms are present, they are often nonspecific. By the time patients develop specific symptoms, the tumor is usually already in an advanced stage. This type of disease has become one of the major threats to human life and health, with a high mortality rate and an increasingly younger age of onset. Early detection and treatment are crucial for cancer; however, currently, there is a significant lack of highly effective and specific early diagnostic indicators and treatment methods.

[0003] Multiple studies have demonstrated that the ubiquitin-proteasome pathway and deubiquitination play an important role in the development and progression of tumors, bringing the study of tumor pathogenesis to a new depth and opening up new fields and ideas for the clinical prevention and treatment of tumors. At present, the study of ubiquitination and deubiquitination has become a new research hotspot, and in-depth exploration of its mechanisms will open up a new path for the study of tumor pathogenesis and the treatment of tumors.

[0004] Positive cofactor 4 (PC4) is a potent and highly conserved nuclear protein widely distributed in biological tissues and cells. Besides participating in basal transcription mediated by RNA polymerase II, PC4 also regulates molecular biological processes such as DNA replication, chromatin remodeling, DNA repair, and RNA metabolism. Our research group was the first to report abnormally high expression of PC4 in models of malignant transformation of normal cells and in clinical specimens of various tumors, including lung cancer and osteosarcoma, and its regulation of tumor malignant progression (Shi CM, 2007, In vitro cell development; Peng Y, 2012, Cancer gene therapy; Luo P, 2019, Cell communal signal). However, the ubiquitination of PC4 protein and its relationship with tumors remain unknown. Summary of the Invention

[0005] This invention aims to study PC4 protein ubiquitination and its relationship with tumors, and to provide a new approach that can be used for tumor diagnosis and targeted therapy.

[0006] The primary objective of this invention is to provide information on the use of the Lys68 site of the PC4 protein as a tumor diagnostic target, the ubiquitination level of the Lys68 site of the PC4 protein as a tumor diagnostic indicator, and the application of reagents for detecting the ubiquitination level of the Lys68 site of the PC4 protein in the preparation of tumor diagnostic products.

[0007] The second objective of this invention is to provide the application of the Lys68 site of the PC4 protein as a target for tumor therapy or a target for regulating tumor cell proliferation, or as a target for screening, developing and / or designing products for the prevention and / or treatment of tumors, as well as the application of reagents capable of inhibiting ubiquitination of the Lys68 site of the PC4 protein in the preparation of tumor therapy products.

[0008] The third objective of this invention is to provide a method for ubiquitination of PC4 protein Lys68 (ubPC4). k68 Antigen peptide.

[0009] The fourth objective of this invention is to provide an antibody that specifically recognizes the Lys68 ubiquitination site of the PC4 protein.

[0010] The fifth objective of this invention is to provide the use of the antigenic peptide or the antibody as a product for tumor detection / diagnosis / screening, treatment or prognosis.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] Our research found that the Lys68 site in the PC4 amino acid sequence is the site of its protein ubiquitination modification; however, it is unclear whether this modification can affect tumor cell growth by influencing PC4 protein function. Our further research showed that the expression level of PC4 protein Lys68 ubiquitination in tumor cells or tissues is higher than that in normal cells or tissues; and that PC4 protein Lys68 ubiquitination significantly enhances the ability of PC4 protein to bind to RNA, thereby enhancing PC4's regulation of downstream RNA stability and promoting metabolic regulation of cell cycle-related RNAs, thus promoting cell cycle progression, tumor cell growth and proliferation, significantly promoting malignant phenotypic transformation, and promoting malignant tumor progression. Inhibition of this site's ubiquitination can be used to develop tumor therapeutic drugs. Therefore, PC4 Lys68 can serve as a key target for developing tumor diagnostic products or targeted tumor therapies. Thus, products for tumor screening, diagnosis, and monitoring of treatment efficacy, as well as targeted tumor therapies, can be developed targeting this site. Furthermore, this invention also developed a method targeting human PC4 protein Lys68 ubiquitination (ubPC4). k68 Antigen peptides, as well as antibodies that can specifically recognize the Lys68 ubiquitination site of the PC4 protein, can be applied to the diagnosis, treatment, and prognosis of diseases such as tumors.

[0013] Based on the research results of this invention, this invention claims protection for the following solutions:

[0014] Application of the Lys68 site of the PC4 protein as a target for tumor diagnosis.

[0015] Application of PC4 protein Lys68 site ubiquitination level as a tumor diagnostic indicator.

[0016] Application of reagents for detecting ubiquitination levels at the Lys68 site of PC4 protein in the preparation of tumor diagnostic products.

[0017] Application of the PC4 protein Lys68 site as a target for tumor therapy or regulation of tumor cell proliferation. This regulation aims to inhibit tumor cell proliferation, growth, and malignant progression. The regulatory mechanism involves inhibiting ubiquitination at the PC4 protein Lys68 site, which significantly weakens the PC4 protein's ability to bind to RNA, leading to cell cycle arrest and inhibiting tumor cell growth and malignant progression.

[0018] Application of the Lys68 site of the PC4 protein as a target in the screening, development and / or design of products for the prevention and / or treatment of cancer.

[0019] Application of reagents that can inhibit ubiquitination at the Lys68 site of PC4 protein in the preparation of tumor-targeted therapy products.

[0020] In addition, this invention further develops a PC4 protein Lys68 ubiquitination (ubPC4) method. k68 The antigenic peptide, with the sequence CMFQIGK(STLHLVLRLRGG)MRYVS, has lysine residues that are ubiquitinated. This antigenic peptide can not only solve the problem of effective research on ubiquitination modification of PC4 protein, but also be used for the diagnosis, treatment, and prognosis of diseases such as tumors.

[0021] Furthermore, using the antigenic peptide as an immunogen to immunize animals, a protein that specifically recognizes the Lys68 ubiquitination site of PC4 protein (ubPC4) was prepared. k68 The polyclonal antibody (named ubPC4) K68 This antibody can not only solve the problem of effective research on PC4 protein ubiquitination modification, but also be used for the diagnosis, treatment and prognosis of diseases such as tumors.

[0022] Therefore, the antigenic peptide specifically recognizes the Lys68 ubiquitination site (ubPC4) of the PC4 protein during preparation. k68 The application of polyclonal antibodies should also be within the scope of protection of this invention.

[0023] The use of the antigenic peptide or the antibody as a product for tumor detection / diagnosis / screening, targeted therapy or prognosis should also be within the scope of protection of this invention.

[0024] 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.

[0025] The tumors described in this invention include liver cancer, cervical cancer, colorectal cancer, breast cancer, and prostate cancer.

[0026] The invention includes drugs, etc.

[0027] PC4 protein, an RNA-binding protein, accelerates the G1 / S phase transition of the cell cycle while ensuring orderly mitosis by regulating the mRNA stability and protein expression levels of cell cycle-related genes in liver cancer cells, thereby promoting the proliferation, migration, and metastasis of liver cancer cells. This invention targets the ubiquitination of the PC4 protein at the Lys68 site, thereby promoting the occurrence, development, and metastasis of tumor cells. This has significant value for research on tumor development and treatment strategies.

[0028] The highly specific antibody against Lys68 ubiquitination of human PC protein prepared by this invention can be used in Western blot experiments to detect expression differences between normal cells and tumor cells. This helps to study the role of PC4 protein ubiquitination modification in the occurrence and development of tumor diseases, and provides a potential target for the diagnosis or treatment of clinical tumor diseases. It can also be used in immunological experiments such as IHC and ELISA to detect the ubiquitination level of human PC4 protein and explore its relationship with tumors and other systemic diseases. It has broad clinical application prospects in disease diagnosis, treatment and prognosis.

[0029] The present invention has the following beneficial effects:

[0030] (1) This invention reveals that the 68th lysine residue (Lys) of an RNA-binding protein PC4 is ubiquitinated, which enhances the binding of PC4 protein to RNA, leading to cell cycle arrest and growth inhibition in tumor cells. Therefore, targeted anti-tumor drugs can be developed by inhibiting ubiquitination at this site.

[0031] (2) The Lys68 site ubiquitination 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.

[0032] (3) The present invention also provides corresponding ubiquitinated 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.

[0033] (4) The antigen peptides and antibodies provided by this invention can detect the post-translational ubiquitination modification of human PC4 protein in practical applications, which facilitates the study of the correlation between ubiquitination modification of specific sites of human PC4 protein and specific biological events such as tumor cell cycle; it helps to explore the role mechanism of ubiquitination modification of human PC4 protein in the occurrence and development of tumor diseases, and can also be used to study the biological functions of kinases and hydrolases that mediate ubiquitination in the occurrence of tumor diseases, which helps to study the various biological functions of PC4 protein and has great scientific research value. Attached Figure Description

[0034] Figure 1 This shows the query results for the Lys68 site of the human PC4 protein in the PhoshoSitePlus database.

[0035] Figure 2 The mass spectrum of the Lys68 site of the human PC4 protein is shown.

[0036] Figure 3 Lys68 is highly conserved in humans, mice, chimpanzees, macaques, cattle, chickens, and African clawed frogs.

[0037] Figure 4 Western blot showed PC4 K68R PC4 WT ubiquitination expression level.

[0038] Figure 5 RIP-qPCR revealed intracellular Flag-PC4 K68R The mutation significantly reduces the ability of RNA to bind.

[0039] Figure 6 In vitro EMSA shows purified PC4 K68R The mutation significantly reduces the ability of RNA to bind.

[0040] Figure 7 PC4 is displayed. K68R Mutations reduce the stability of downstream mRNA (CCND1), thereby reducing the expression levels of CCND1 mRNA and protein.

[0041] Figure 8 CCK8 experiment shows PC4 K68R Mutations inhibit tumor cell proliferation.

[0042] Figure 9 Colony formation experiments show that PC4 K68R Mutations inhibit tumor cell growth.

[0043] Figure 10 PC4 is displayed. K68R Mutations inhibit the ability of tumor cells to invade.

[0044] Figure 11 PC4 is displayed. K68R Mutations inhibit the migration ability of tumor cells.

[0045] Figure 12 Subcutaneous tumor growth curves in mice show PC4 K68R Mutations inhibit tumor formation.

[0046] Figure 13 Mouse subcutaneous tumor size display PC4 K68R Mutations inhibit tumor formation.

[0047] Figure 14 FUCCI system cell tracking monitoring showed PC4 K68R Mutations inhibit the G1 to S phase transition in the cell cycle of tumor cells.

[0048] Figure 15 WB displays ubPC4 K68 The antibody can specifically recognize the ubiquitination modification at the PC4 Lys68 site.

[0049] Figure 16 Western blotting shows ubPC4 in paired normal cells / tissues and in liver cancer tissue. K68 Significantly decreased and increased.

[0050] Figure 17 This diagram illustrates how PC4-K68 ubiquitination promotes tumor cell growth. Detailed Implementation

[0051] The following description, in conjunction with the accompanying drawings and specific embodiments, further elaborates and illustrates the technical route for the regulation of tumor cells by ubiquitination of human PC4 protein at Lys68 site and the preparation of its specific antibody. However, the embodiments do not limit the invention in any way.

[0052] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0053] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0054] The experimental materials and methods used in the following embodiments are partially illustrated (excluding those specifically described as conventional materials and methods in the art):

[0055] 1 Experimental Cells

[0056] Human liver cancer cells Huh7 and HepG2, cervical cancer cells Hela, and normal liver cells LO2 and LX2.

[0057] 2. Experimental drugs and antibodies

[0058] 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, Actinomycin D, Matrigel Matrix, Western and IP cell lysis buffer, BCA protein concentration assay 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, crystal violet staining solution, PC4 antibody (Abcam, ab72132), CK2α antibody (CST, 2656S), PP2A antibody (BD, 610555), Flag antibody (Sigma, F1804), GAPDH antibody (Bioss, bs-2188R), β-actin antibody (CST, 3700), etc.

[0059] 3. Experimental Instruments and Consumables

[0060] 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, Transwell chamber, etc.

[0061] 3 Experimental Methods

[0062] 4.1CCK-8

[0063] 4.1.1 Cell Plating: Cells from each group (NC, shPC4, shPC4+PC4) were plated. WT and shPC4+PC4 K68RHuh7 cells were digested, counted, and prepared into a cell suspension with a density of 2×10⁴ / ml. 100 μl of the suspension was added to each well of a 96-well plate, with 6 replicates per group. The plates were then incubated in a cell culture incubator at 37°C and 5% CO₂.

[0064] 4.1.2 OD Value Detection and Growth Curve Plotting: Every 24 hours, remove a 96-well plate and prepare working solution according to the number of wells to be tested each time, using a ratio of 10 μl CCK-8 reagent to 90 μl PBS. Remove the old culture medium, add 100 μl of the newly prepared CCK-8 working solution, and incubate at 37℃ for 2 hours. Then, measure the absorbance at 450 nm using a microplate reader, and finally plot the growth curve.

[0065] 4.2 Colony formation

[0066] 4.2.1 Cell Plating: Cells from each group (NC, shPC4, shPC4+PC4) were plated. WT and shPC4+PC4 K68R Huh7 cells were digested, counted, and prepared into a cell suspension with a density of 2×10⁴ / ml. 100 μl of the suspension was added to each well of a 6-well plate, followed by 2 ml of culture medium. Three replicates were set up for each group. The plates were then incubated in a cell culture incubator at 37°C and 5% CO₂.

[0067] 4.2.2 Colony Staining and Counting: Change the culture medium every 2-3 days and observe the colony size under a microscope. The experiment can be terminated when the colonies become visible to the naked eye after 2-3 weeks. Aspirate the culture medium, wash twice with PBS, and fix with 4% paraformaldehyde for 15 minutes. Finally, stain with crystal violet for 15 minutes, dry, photograph, and count the colonies.

[0068] 4.3 Transwell assay to detect the migration and invasion abilities of breast cancer cells in each group

[0069] 4.3.1 Matrix adhesive plating: The upper chamber surface of the bottom membrane of the Transwell chamber (8 μl pore size) is coated with matrix adhesive and placed at 37°C for half an hour to allow the matrix adhesive to coagulate (step 1 is not required for migration experiments).

[0070] 4.3.2 Cell Seeding: Cells from each group (NC, shPC4, shPC4+PC4) were seeded. WT and shPC4+PC4 K68R Huh7 cells were digested to prepare a serum-free cell suspension with a density of 5 × 10⁵ / ml (cells can be pre-starved for 12 h without serum). 100 μl of the cell suspension was added to the Transwell chamber. The lower chamber contained culture medium with 20% FBS, and the upper chamber contained serum-free culture medium. It was important to ensure that there were no air bubbles between the lower chamber and the upper chamber.

[0071] 4.3.3 Staining and Statistical Analysis: After 24 hours of culture, invasive and migrating cells will cross the membrane and adhere to the lower chamber side of the membrane without falling into the lower chamber. Wash twice with PBS, then fix with 4% paraformaldehyde for 15 minutes. Finally, stain with crystal violet for 15 minutes, wipe away the unmigrated cells from the upper layer with a cotton swab, wash and dry, and observe, photograph, and count the cell number in multiple fields of view.

[0072] 4.4 Tumor-bearing nude mice

[0073] 4.4.1 The groups with good growth (NC, shPC4, shPC4+PC4) WT and shPC4+PC4 K68R Using Huh7 cells as a research model, the cells in each group were digested, counted, and prepared into a cell suspension with a density of 2.5×107 / ml, which was then placed in an ice box for later use.

[0074] 4.4.2 Five- to six-week-old male BALB / c nude mice were purchased from the Animal Center of the Third Military Medical University and divided into four groups of five mice each. 200 μl of cell suspension was injected into the back of each mouse, resulting in 5 × 10⁶ cells per mouse.

[0075] 4.4.3 Nude mice were housed in an SPF animal room. Tumor growth was observed and tumor size was measured every 3 days (tumor volume = (length × width 2) / 2). At the end of the experiment, the nude mice were sacrificed, and the tumors were removed, photographed, and weighed. Part of the tumor was frozen at -80℃, and the other part was fixed in 4% paraformaldehyde.

[0076] 4.5 qPCR detection of mRNA stability of cell cycle genes in each group

[0077] 4.5.1 Each group (NC, shPC4, shPC4+PC4) WT and shPC4+PC4 K68R After adding 5ug / ml Actinomycin D to Huh7 cells, samples were collected at 0h, 2h, 4h and 6h respectively. The culture medium in the culture flask was discarded, the cells were washed 1-2 times with PBS, and then 1ml Trizol was added. The cells in each group were scraped off with a cell scraper, collected and transferred to 1.5ml EP tubes.

[0078] 4.5.2 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 three layers; carefully transfer 450 μl of the 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 of 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;

[0079] 4.5.3 Remove genomic DNA and reverse transcribe RNA into cDNA;

[0080] 4.5.4 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 °C and 40 cycles. The specific primer sequences are shown in Table 2.

[0081] 4.5.5 Statistical analysis was performed on the mRNA expression levels of each group, and mRNA stability charts for each gene were generated.

[0082] Table 2. qPCR primer sequences for cell cycle genes

[0083]

[0084] 4.6 Western Blotting

[0085] 4.6.1 Extraction of cell proteins: Cells from each group (NC, shPC4, shPC4+PC4) were scraped directly from the culture flask. WT , and shPC4+PC4 K68R All cells were collected and transferred to centrifuge tubes; centrifuged at 1500 rpm for 5 min at room temperature; the supernatant was discarded, the bottom cells were resuspended in PBS, and transferred to 1.5 ml EP tubes; centrifuged at 1500 rpm for 5 min at room temperature; the PBS was discarded, and cell lysis buffer containing 10% cocktail protease inhibitor was added at approximately 3 times the cell volume. The cells were gently lysed by tapping with a finger, and placed in an ice bath for 30 min; centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to 1.5 ml EP tubes.

[0086] 4.6.2 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 and add 980 μL of PBS to dilute 50 times, resulting in a concentration of 0.5 mg / ml. Prepare the BCA working solution (the ratio of solution A to solution B is 50:1), preparing 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 OD562 using a microplate reader. Plot a standard curve, calculate the protein concentration, and balance the groups 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 5 min.

[0087] 4.6.3 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 layer 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).

[0088] 4.6.4 Transfer (Wet Transfer): Prepare the transfer buffer, adding methanol during the transfer preparation. Immerse the 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 it 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, from bottom to top: black clamp, filter paper, gel, PVDF membrane, filter paper, white clamp. Carefully and gently remove air bubbles with a glass rod. Clamp the clamps and place the apparatus in the electrophoresis device. Fill the inner white box with ice. Place the electrophoresis device 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.

[0089] 4.6.5 Blocking: After electroporation, rinse the PVDF membrane once in TBST solution, place it in a clean incubation box or plastic bag, add Western blotting solution for blocking, and place it on a shaker at 37°C for about 2 hours.

[0090] 4.6.6 Incubation of primary and secondary antibodies

[0091] 4.6.7 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 (p-PC4-S17) 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 three times at room temperature on a shaker, approximately 10 min each time. Dilute the secondary antibody with Western blotting 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), 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 three times at room temperature on a shaker, approximately 10 min each time.

[0092] 4.6.8 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.

[0093] 4.7 Immune tissues

[0094] 4.7.1 Sampling and Fixation: Label each sampling bottle and add an appropriate amount of 4% paraformaldehyde fixative (about 2 / 3 full). Place each tissue (liver cancer and control normal liver tissue) into the sampling bottle in sequence and fix for 24-72 hours.

[0095] 4.7.2 Tissue trimming and dehydration: Rinse the dehydration basket with boiling water to remove the wax adhering to its surface. Remove all tissue from each sampling bottle, trim each tissue as flat as possible, place them in the dehydration basket, and label them carefully. Place them in a dehydrator and dehydrate at room temperature for 16 hours.

[0096] 4.7.3 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 (more than 0.5 hours; freezing may cause the paraffin block to crack).

[0097] 4.7.4 Trimming: Remove the solidified wax block and trim it into a regular cuboid as much as possible (do not trim the cut surfaces, and trim as little as possible the top and bottom surfaces of the cut surfaces, i.e., the machine mounting surfaces; trim more on the left, right, and opposite sides of the cut surfaces). After trimming, re-stick the label.

[0098] 4.7.5 Sectioning: Half an hour before sectioning, place the wax block at -20℃. The thickness of the section is generally 5mm. Unused wax blocks can be sealed and stored at room temperature for a long time (to prevent rodents from eating them).

[0099] 4.7.6 Slide Preparation: Mark blank slides with a pencil beforehand (to prevent alcohol staining). Generally, prepare 5-6 slides for each type of tissue, with 2-3 tissue samples on each slide for use as controls. If dewaxing and staining are not performed immediately after drying, the slides can be placed in a slide box (ideally within one month for staining). Dry the slides again for 0.5 hours before dewaxing and staining.

[0100] 4.7.7 Dewaxing: Immerse the rack in xylene II-xylene I-100% ethanol-95% ethanol-80% ethanol for 10 min-10 min-5 min-5 min-5 min sequentially. After dewaxing, place the rack in a beaker and rinse with water for 3 min.

[0101] 4.7.8 Sodium citrate retrieval: 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 bath (the purpose of boiling is to expose the antigen sites). After the temperature in the beaker rises to above 93℃, start the retrieval time for 20 minutes, then remove the beaker and cool it to room temperature at around 37℃ (the sodium citrate can be recycled).

[0102] 4.7.9 Hydrogen peroxide retardation: Add 300ml of PBS solution (or double-distilled water) to a beaker, immerse the scaffold in the beaker for 10 minutes (replace the PBS once during this time) or 5 minutes x 3 times. If there are few slides, you can directly add PBS solution (or double-distilled water) to the slides, 5 minutes x 3 times. Then gently shake the slides dry (you can wipe them dry with paper), being careful not to dry them completely, keeping the slides moist. Add 3% H2O2 directly to the tissue surface of each slide, being careful not to touch the tissue, and place them at room temperature for 15 minutes (or place them in a humidified chamber). Then shake off the reagent on the slides and place them back on the scaffold.

[0103] 4.7.10 Serum Blocking: Add 300ml of PBS solution (or double-distilled water) to a beaker, immerse the scaffold in the beaker for 10 minutes (replace the PBS once during this time) or 5 minutes x 3 times. If the number of slides is small, you can directly add PBS solution (or double-distilled water) to the slides, 5 minutes x 3 times. Then gently shake the slides dry (you can wipe them dry with paper), being careful not to dry them completely, keeping the slides moist. Use a pipette to add serum to the tissue surface of each slide to block some non-specific sites. Then place the slides in a humidified chamber (if the number is small, you can directly place them in a water-filled pipette tip box), and then place the humidified chamber in a 37°C incubator for 30 minutes (if the primary antibody is prepared by diluting with serum, the 37°C incubation time can be shortened to 15 minutes).

[0104] 4.7.11 Adding Primary Antibody: Pipette the pre-prepared primary antibody (p-PC4-S17 antibody diluted 1:100) onto the tissue surface of each slide (if performing a control experiment, add PBS to the control tissue; reserve one tissue sample from the same type or slide as a control). Place the slides in a humidified chamber and then store the chamber at 4°C overnight (or at least 12 hours). Primary antibody preparation: Dilute with PBS (or serum). Generally, approximately 100 μL of antibody diluent is needed per sample. The dilution ratio can be 1:50, 1:100, or 1:200, depending on the instructions.

[0105] 4.7.12 Adding Secondary Antibody: Add 300ml of PBS solution (or double-distilled water) to a beaker, immerse the scaffold in the beaker for 10 minutes (remember to change the PBS once during this time) or 5 minutes x 3 times. If there are few slides, you can directly add PBS solution (or double-distilled water) to the slides, 5 minutes x 3 times. Then gently shake the slides dry (you can wipe them dry with paper), being careful not to dry them completely, keeping the slides moist. Add the secondary antibody directly to the tissue surface of each slide, then place the slides in a humidified chamber, and then place the humidified chamber in a 37°C incubator for 40 minutes.

[0106] 4.7.13 Adding the chromogenic reagent: Add 300ml of PBS solution (or double-distilled water) to a beaker. Immerse the support in the beaker for 10 minutes (replace the PBS once during this time) or 5 minutes x 3 times. If there are few slides, you can directly add PBS solution (or double-distilled water) to the slides, 5 minutes x 3 times. Place the support in a beaker containing PBS or double-distilled water and place it in front of a microscope for chromogenic development. Gently shake the slides dry (you can wipe them dry with paper). Use a pipette to directly add the prepared DAB chromogenic solution to the tissue surface of each slide. Develop for about 10 seconds (the exact time depends on the actual situation), then quickly wash it off in water and observe under a microscope. If the chromogenic development is insufficient, repeat the previous steps. Preparation of DAB chromogenic solution: Generally, about 100ul of DAB chromogenic solution is needed for one sample. The ratio is 1ml of reagent A + 1 drop of reagent B according to the instructions.

[0107] 4.7.14 Counterstaining: Place the developed slide back into the support (beaker) 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).

[0108] 4.7.15 Drying and dehydration: Place the rack in the oven to dry (for a few minutes, but not too dry), then place the rack in xylene II for 20 minutes and let it air dry.

[0109] 4.7.16 Mounting: Mount the slide with neutral resin, air dry, observe and photograph under a microscope.

[0110] 4.8 mRNA stability assay

[0111] 4.8.1 Each group (NC, shPC4, shPC4+PC4) WT and shPC4+PC4 K68R After adding 5ug / ml Actinomycin D to Huh7 cells, samples were collected at 0h, 2h, 4h and 6h respectively. The culture medium in the culture flask was discarded, the cells were washed 1-2 times with PBS, and then 1ml Trizol was added. The cells in each group were scraped off with a cell scraper, collected and transferred to 1.5ml EP tubes.

[0112] 4.8.2 RNA was extracted using the Trizol method;

[0113] 4.8.3 Removal of genomic DNA;

[0114] 4.8.4 Reverse transcription of cDNA;

[0115] 4.8.5 Real-time PCR

[0116] 4.9 Immunoprecipitation

[0117] 4.9.1 Protein extraction: Wash cells twice with pre-chilled PBS, and aspirate the PBS after the last wash; add pre-chilled 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.

[0118] 4.9.2 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.

[0119] 4.9.3IP: 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.

[0120] 4.9.4 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.

[0121] 4.9.5 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;

[0122] 4.9.6 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.

[0123] 4.10 Ubiquitination Modification Omics (Mass Spectrometry)

[0124] 4.10.1 Protein Extraction: Samples were removed from -80℃, and each group of samples was added with 4 times the volume of lysis buffer (8M urea, 1% protease inhibitor, 1% ubiquitinase inhibitor) and sonicated for lysis. 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 assay kit.

[0125] 4.10.2 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.

[0126] 4.10.3 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.

[0127] 4.10.4 Liquid Chromatography-Mass Spectrometry (LC-MS / MS) Analysis: 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 ionization. TMPlus 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.

[0128] 4.11 Preparation of antigenic peptides specific to the Lys68 ubiquitination site of PC4 protein

[0129] 4.11.1 Design of ubiquitinated antigenic peptide at Lys68 site of human PC4 protein: With the Lys68 site of human PC4 protein as the center, the N-terminus is MFQIG and the C-terminus is MRYVS. The designed synthetic peptide (antigen peptide) sequence is CMFQIGKMRYVS. The ubiquitinated C-terminal 12-peptide STLHLVLRLRGG is added to the Lys68 amino acid. The peptide sequence is: CMFQIGK(STLHLVLRLRGG)MRYVS.

[0130] 4.11.2 Synthesis of Antigenic Peptides: Based on the design of the hapten synthetic peptides, a ubiquitination group was added to the Lys68 site to obtain ubiquitinated antigenic peptides, which were then coupled with hemocyanin (KLH) to obtain the complete antigen for chicken immunization (ubLys68-KLH). Furthermore, the ubiquitinated synthetic peptides (antigenic peptides) were coupled with bovine serum albumin (BSA) via the maleimide method and used as packing material for affinity purification chromatography columns (ubLys68-BSA). Correspondingly, a non-ubiquitinated synthetic peptide corresponding to Lys68 was synthesized and coupled with BSA to use as packing material for affinity separation chromatography columns (Lys68-BSA), and a ubiquitin C-terminal 12-peptide STLHLVLRLRGGC was synthesized and coupled with BSA to use as packing material for affinity separation chromatography columns (Ub-BSA).

[0131] 4.12 Preparation of specific polyclonal antibodies against PC4 protein Lys68 ubiquitination

[0132] 4.12.1 Preparation of negative serum: 3 mL of blood was collected from the subwing vein of a healthy female chicken (2-3 kg, purchased from the Slack Laboratory Animal Center, Chinese Academy of Sciences) and placed in a blood collection tube. Hemostasis was achieved by applying pressure with a cotton ball. The blood was left at room temperature for approximately 1 hour until it coagulated and formed a clot. The blood was then placed at 4°C for 2 hours to allow serum precipitation. The blood was centrifuged at 2500 g for 10 minutes, and the supernatant was collected and labeled as the negative control serum. The serum was aliquoted and stored at -20°C until testing.

[0133] 4.12.2 Pre-immunoassay Screening - Western blot: Take 5 μg each of BSA standard, Lys68-BSA, ubLys68-BSA, and Ub-BSA, add appropriate 5×SDS loading buffer, boil in a water bath for 10 min to denature the proteins, and centrifuge at 10000×g for 1 min; for SDS-PAGE electrophoresis, the separating gel is 8% and the stacking gel is 5%; load samples using a pipette in the predetermined order, adding an equal volume of 1×SDS gel loading buffer to unused sample wells; electrophoresis at 80V for 20 min, then switch to 120V for approximately 50 min, until bromophenol blue reaches the bottom of the separating gel, then turn off the power. Transfer conditions: constant current 300mA, time 120 min. Block with 5% skim milk powder, shake at 37℃ for 1 h. Place the transfer membrane in a pre-immunoassay antiserum dilution buffer prepared at a ratio of 1:5000 with primary antibody dilution buffer, shake gently horizontally, and incubate overnight at 4℃. The following day, wash the membrane with 1×PBST for 10 min, repeating 4 times. Place the membrane in a 1:5000 dilution of HRP-labeled goat anti-chicken IgY secondary antibody solution diluted with 1×PBST, and incubate at 37°C for 60 min with shaking. Discard the secondary antibody solution, wash the membrane with 1×PBST for 10 min, repeating 3 times. Develop the membrane using the ECL kit according to the manufacturer's instructions, and photograph the results.

[0134] 4.12.3 Animal Immunization: Approximately 73 days after the initial immunization, the ubLys68-KLH complete antigen was dissolved in sterile 1×PBS. Using a sterile syringe, the antigen solution was drawn up, and an equal volume of Freund's complete adjuvant (CFA) was drawn up in another syringe. The two syringes were connected by a plastic tube. The mixture was repeatedly aspirated and aspirated to thoroughly mix the ubLys68-KLH complete antigen and CFA until a completely emulsified emulsion was formed that did not spread when dropped into water. The initial immunization was performed via multiple intramuscular (IM) injections at multiple points in the pectoralis major muscle and intradermal (ID) injections on both sides of the waist. The total initial immunization dose was 0.2 mg. A booster immunization was performed 21 days after the initial immunization. An antigen emulsion was prepared using Freund's incomplete adjuvant (IFA) instead of CFA and injected according to the initial immunization protocol. The total booster dose was approximately 0.1 mg. Fourteen days after the initial immunization, a second booster immunization was administered. Freund's incomplete adjuvant (IFA) was used instead of CFA as the immunizing adjuvant to prepare an antigen emulsion, which was injected following the same procedure as the initial immunization. The total amount of antigen in this booster immunization was approximately 0.1 mg. Ten days after the final booster immunization, the chickens underwent a large-scale blood collection from the abdominal aorta to collect a large amount of serum. The collected blood in centrifuge tubes was left to stand at room temperature overnight to allow the blood clots to shrink. The next day, the separated serum was aseptically centrifuged in 50 mL centrifuge tubes at 4000 g for 10 min. The supernatant was collected, aliquoted into 1 mL tubes, labeled as post-immunization antiserum (approximately 51 mL in total), and stored at -20°C.

[0135] 4.12.4 ELISA titer determination of antiserum after immunization: Ubiquitinated antigen ubLys68-BSA and non-ubiquitinated antigen Lys68-BSA, and Ub-BSA were coated separately using antigen coating buffer (CBS) and added to 96-well ELISA plates, 0.1 mL per well. The plates were sealed and incubated overnight at 4°C (at least 12 h). After coating, the liquid in the wells was discarded. Each well of the ELISA plate was thoroughly washed with 1×PBST, and the washing solution was discarded. This process was repeated three times, and residual liquid was blotted dry on filter paper after each wash. Blocking buffer (0.25% BSA / PBST), 200 μL / well, was added to each well and incubated at 37°C for 2 h. The titration plate was washed three times with 1×PBST, and residual liquid was blotted dry on filter paper after each wash. Dilute the post-immunization antiserum with 1×PBST at ratios of 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000, and 1:729000, using 1×PBS buffer as a blank control. Add the solutions to each well of a 96-well ELISA plate, seal the plate, and incubate at 37°C for 1 hour. Discard the liquid in the wells and wash the plate three times with 1×PBST. Add 100 μL / well of HRP-labeled goat anti-chicken IgY secondary antibody diluted 1:5000 with 1×PBST, and incubate at 37°C for 1 hour. Seal the plate and incubate at 37°C for 1 hour. Wash the plate five times with 1×PBST and blot dry. Add 100 μL / well of freshly prepared TMB chromogenic solution and incubate at room temperature in the dark for 30 minutes. Stop the reaction by adding 50 μL / well of 2M H₂SO₄. The OD values ​​of each well were measured at 450 nm using an ELISA reader. The ELISA values ​​of the antiserum after immunization with ubLys68 antigen at dilutions of 1:1000, 1:3000, and 1:9000 were all above 0.5.

[0136] 4.12.5 Preparation of Ubiquitinated and Non-ubiquitinated Peptide Chromatography Columns: Lys68-BSA, ubLys68-BSA, and Ub-BSA were dissolved in coupling buffer to a concentration of 1 mg / mL. 7 mg of Lys68-BSA, 6 mg of ubLys68-BSA, and 6 mg of Ub-BSA were added to 5 mL of resin, mixed well, and then added to the chromatography column. The column was mixed at room temperature for 15 minutes, and then incubated at room temperature for 30 minutes. The caps at both ends of the column were removed, and the eluent was collected and rinsed with 3 times the volume of coupling buffer. The bottom cap of the column was replaced, and 50 mM L-Cysteine·HCl was added to the coupling buffer. After mixing, an equal volume of buffer was added to the column, mixed at room temperature for 15 minutes, and then allowed to stand for 30 minutes. Remove the bottom cap, release the coupling buffer, wash the column with 6 volumes of washing buffer (1M NaCl), then rinse the column with 2 volumes of storage buffer, cap it, add 1 volume of storage buffer, and store at 4 degrees Celsius for later use.

[0137] 4.12.6 Purification of ubiquitinated antibodies: Remove the bottom cap of the chromatography column, release the storage buffer, and wash the column twice with 10 mL of binding buffer. Dilute the ubLys68 antiserum with an equal volume of PBS, add it to the corresponding ubiquitinated chromatography column, collect the eluent, repeat twice to obtain the ubLys68 eluent, then wash the chromatography column three times with 10 mL of washing buffer, and finally elute the chromatography column with elution buffer to obtain the corresponding crude ubLys68 ubiquitinated antibody, dissolved in 1×PBS. Add the collected crude ubLys68 ubiquitinated antibody to the corresponding rinsed non-ubiquitinated column, collect the eluent, repeat twice; add the eluent to the rinsed Ub-BSA column, collect the eluent, repeat twice, retain the final eluent at the bottom to obtain the corresponding Lys68 ubiquitinated antibody, dissolve the antibody in 1×PBS, and add 0.1% NaN3 for later use. The titer of purified ubiquitinated antibodies and their recognition of ubiquitinated target antigen peptides, non-ubiquitinated synthetic peptides, and Ub peptides were detected by ELISA. First, synthetic ubiquitinated peptide antigen-BSA, non-ubiquitinated synthetic peptide antigen-BSA, and Ub-BSA were coated separately. Different dilutions of pre-purification serum and purified antiserum were added for incubation. After washing, HRP-labeled goat anti-chicken IgY dilution was added, followed by incubation, washing, TMB color development, and OD450 measurement after reaction termination. Results showed that when the Lys68 ubiquitinated antibody dilution was 1:9000, the OD450 value against the ubiquitinated antigen was greater than 0.75, and the OD450 value against the non-ubiquitinated antigen was less than 0.25 with a P / N value greater than 3. Therefore, the titer of the purified Lys68 ubiquitinated antibody was at least 1:9000.

[0138] The following examples illustrate the experimental results and analysis:

[0139] Example 1: Identification of ubiquitination sites in human PC4 protein.

[0140] First, the protein ubiquitination site database PhoshohoSitePlus ( Figure 1 The search for potential ubiquitination sites in the human PC4 protein was conducted, followed by ubiquitination-based mass spectrometry analysis. Figure 2 This confirms that Lys68 is a ubiquitination site in the human PC4 amino acid sequence. Sequence alignment shows that Lys68 is highly conserved in humans, mice, chimpanzees, macaques, cattle, chickens, and African clawed frogs. Figure 3 Next, construct a PC4 that is not ubiquitinated. K68R The mutant was transfected into Huh7 cells with wild-type and mutant PC4, and the results showed that PC4 K68R Mutant, not wild-type PC4 (PC4) WT ), eliminated PC4 ubiquitination ( Figure 4 These results ultimately identified Lys68 as the specific ubiquitination site for the human PC4 protein.

[0141] Example 2: Lys68 ubiquitination of PC4 enhances its RNA binding ability and promotes metabolic regulation of cell cycle-related RNAs.

[0142] Previous data from our research group indicate 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 plays a crucial role in the coordinated control of cell cycle progression. To investigate whether Lys68 ubiquitination of PC4 affects its RNA-binding ability, this invention first constructs different flag-tagged PC4 mutant vectors (Flag-PC4). WT and Flag-PC4 K68R Then, in vivo RIP-qPCR and in vitro EMSA experiments were performed, and the results showed that it was similar to Flag-PC4. WT Compared to Flag-PC4 K68R The mutation significantly reduced the RNA binding ability (the deletion of the CTD region of the PC4 protein significantly reduced its RNA binding ability, serving as a positive control for this experiment). Figure 5-6 This suggests that Lys68 ubiquitination of PC4 promotes the binding of PC4 protein to RNA. Subsequently, endogenous PC4 was knocked down in Huh7 cells while exogenous PC4 was stably expressed. WT and PC4 K68R By detecting cell cycle-related mRNA stability, it was demonstrated that it is related to PC4. WT Compared to PC4 K68R Mutations significantly affect the stability of downstream gene mRNA, as well as mRNA and protein expression levels. Figure 7 These results confirm that Lys68 ubiquitination is crucial for PC4-mediated regulation of downstream target gene mRNA stability.

[0143] Example 3: Lys68 ubiquitinated PC4 can promote cell cycle operation, cell growth and malignant progression of tumor cells.

[0144] This invention evaluates the role of PC4 Lys68 ubiquitination in tumor malignancy. First, CCK8 and colony formation assays were used to examine the effect of endogenous PC4 knockdown on stable PC4 progression. WT and PC4 K68R Regarding the phenotype of Huh7 cell proliferation and metastasis, experimental results showed that PC4 K68R Mutant, not wild-type PC4 WT It significantly inhibited the growth, invasion, and metastasis of Huh7 cells. Figure 8-11 ).

[0145] Next, to further determine the function of Lys68 ubiquitination-mediated PC4, this invention constructed a Huh7 cell tumor-bearing mouse model. The results showed that compared with wild-type PC4... WT , expressing PC4 K68R The mutant tumors grew more slowly, but the mice had a better prognosis. Figure 12-13 ).

[0146] Next, this invention investigates whether PC4 Lys68 ubiquitination affects the malignant progression of tumor cells by regulating cell cycle progression. Based on previous experimental data from our research group, overexpression of PC4 accelerates the G1 to S phase transition of tumor cells. Therefore, this invention uses the FUCCI system to track the G1 to S phase transition process, and the results show that compared to wild-type PC4… WT PC4 K68R The mutant prolongs the G1 interphase, resulting in significant G1 phase arrest in cells. Figure 14 In this situation, the inactivation of the PC4 protein's RNA binding ability hinders cell cycle progression, thereby inhibiting tumor cell growth.

[0147] The above experimental results confirm that Lys68 ubiquitination modification of PC4 can significantly promote the malignant phenotypic transformation of tumors, suggesting that Lys68 of PC4 can be used as a key target for the development of tumor-targeted drugs.

[0148] Example 4: Preparation of a specific synthetic peptide (antigen peptide) targeting the Lys68 ubiquitination site of PC4.

[0149] To further investigate Lys68 ubiquitination of PC4 (hereinafter simplified as ubPC4) k68 To regulate tumor cell growth, this invention prepares a specific ubPC4... k68 The antigenic peptide was first analyzed for its antigenicity and homology. A corresponding antigenic peptide sequence was designed, centered on the Lys68 site of the human PC4 protein, with the N-terminus MFQIG and the C-terminus MRYVS. The designed synthetic peptide (antigenic peptide) sequence was CMFQIGKMRYVS. A ubiquitinated C-terminal 12-peptide STLHLVLRLRGG was added to the Lys68 amino acid, resulting in the peptide sequence: CMFQIGK(STLHLVLRLRGG)MRYVS.

[0150] The designed ubPC4 was synthesized using peptide synthesis technology. K68An antigenic peptide was synthesized and coupled with the carrier protein hemocyanin (KLH) to prepare ubiquitinated antibodies as an immunogen; it was also coupled with the carrier protein bovine serum albumin (BSA) to be used as a packing material for affinity purification (Ub-Lys68-BSA); correspondingly, a non-ubiquitinated synthetic peptide corresponding to Lys68 was synthesized and coupled with bovine serum albumin (BSA) to be used as a packing material for affinity separation chromatography (Lys68-BSA), and a ubiquitin C-terminal 12-peptide STLHLVLRLRGGC was synthesized and coupled with bovine serum albumin (BSA) to be used as a packing material for affinity separation chromatography (Ub-BSA).

[0151] Example 5: Preparation of polyclonal antibodies that specifically recognize the Lys68 ubiquitination site of PC4

[0152] To further study ubPC4 K68 Regarding the function of regulating tumor cell growth, this invention prepares a method that specifically recognizes ubPC4. K68 Polyclonal antibodies. First, ubPC4 was used. K68 SPF-grade chickens were immunized with a combination of antigen peptide-KLH conjugate whole antigen and adjuvant via intramuscular injection into the pectoralis major muscle, intradermal injection into both sides of the waist, and multiple injections of antigen emulsion. The immunization regimen included one priming injection, 3-4 booster injections, and a final direct antigen injection. The titer of the antiserum against the target synthetic peptide was determined by ELISA. Subsequently, a cyclic purification technique using bovine serum albumin (BSA) affinity chromatography was employed to purify the PC4-K68 site-specific ubiquitinated antibody, named ubPC4. K68 The antibodies were then tested again by ELISA to determine their titer and recognition of ubiquitinated target antigen peptides and non-ubiquitinated synthetic peptides. Finally, the antibodies were identified by dot blot assay.

[0153] Example 6: Application of PC4 protein Lys68 site ubiquitination antibody in tumors

[0154] To further verify the effects of ubPC4 in cells... K68 The antibody can specifically recognize the Lys68 ubiquitination modification site of the PC4 protein. In this invention, the recombinant vector Flag-PC4 was overexpressed in Huh7 cells. WT Or Flag-PC4 K68R Cellular proteins were extracted 24 hours later, and ubPC4 was found in the immunoprecipitation Flag product. K68 The antibody can recognize PC4 protein in the ubiquitinated state at the Lys68 site, but cannot recognize PC4 protein in the non-ubiquitinated state at the Lys68 site. Figure 15 This demonstrates that the ubiquitinated antibody has good specificity.

[0155] Further utilize ubPC4 K68 Antibody, ubPC4 in liver cancer cells / tissues detected by immunohistochemistry and Western blot. K68 The level of ubPC4 in paired normal cells / tissues and tumor cells / tissues. K68 Significantly decreases and increases ( Figure 16 This indicates that Lys68 ubiquitination of PC4 plays an important role in tumorigenesis and development.

[0156] In summary, PC4-K68 ubiquitination promotes tumor cell growth. Figure 17 Preparation of materials specifically recognizing ubPC4 K68 In practical applications, the antibodies can be used in immunological experiments such as WB, ELISA, and immunohistochemistry to detect the posttranscriptional ubiquitination modification of human PC4 protein, thereby exploring the significance of human PC4 protein ubiquitination modification in tumor-related diseases.

[0157] PC4 is an RNA-binding protein (RBP) that plays a crucial role in early embryonic development, stem cell differentiation / reprogramming, tumorigenesis, and metabolism in mouse embryos. Previous evidence from our research group showed that in tumor cells, PC4 can bind specific cell cycle-related RNAs at different cell cycle phases. By influencing RNA stability or degradation, it accelerates the transition from G1 to S phase while ensuring orderly M phase cell division, thereby promoting rapid tumor cell proliferation and division, and driving the malignant progression of tumors. However, whether specific ubiquitination modifications on the PC4 protein affect PC4's role as an RBP in cell cycle regulation remains unclear. This invention is the first to demonstrate that ubiquitination at the PC4-Lys68 site enhances the ability of PC4 to bind to RNA, thereby strengthening PC4's regulation of downstream RNA stability and ultimately promoting tumor cell growth. This invention also discovered that PC4... K68R Following mutation, K68 ubiquitination ceases, leading to a weakened binding affinity of the PC4 protein to downstream target genes. This results in cell cycle arrest in tumor cells, inhibiting their growth, invasion, and migration. More importantly, ubPC4 is involved in various human malignancies. K68The ubiquitination level is significantly increased, and this increase is positively correlated with the malignant phenotype of tumors. In summary, this invention provides that the ubiquitination site at the Lys68 position of the RNA-binding protein PC4 can promote the malignant progression of tumors. Specifically, this invention prepares polyclonal antibodies targeting this ubiquitination site. On the one hand, this helps to explore the effects of Lys68 ubiquitination modification of the PC4 protein on specific biological events such as cell proliferation and its role in the development and progression of diseases such as tumors; on the other hand, it allows for the linkage of antibodies with drugs to conduct tumor-targeted therapy, and the linkage of antibodies with radiolabels for radioimmunoassay to assist in tumor diagnosis.

[0158] 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 reagents for detecting ubiquitination levels at the Lys68 site of PC4 protein in the preparation of tumor diagnostic products.

2. The application of a reagent capable of inhibiting ubiquitination at the Lys68 site of PC4 protein in the preparation of tumor therapeutic products, wherein the reagent comprises a PC4 protein Lys68 ubiquitinated antigenic peptide, the sequence of which is CMFQIGKMRYVS, and the side chain of the Lys68 amino acid is modified with STLHLVLRLRGG.

3. A PC4 protein Lys68 ubiquitinated antigenic peptide, characterized in that, Its sequence is CMFQIGKMRYVS, and the side chain of amino acid Lys68 is modified with STLHLVLRLRGG.

4. The use of the antigenic peptide of claim 3 in the preparation of an antibody that specifically recognizes the Lys68 ubiquitination site of the PC4 protein.

5. An antibody that specifically recognizes the Lys68 ubiquitination site of the PC4 protein, characterized in that, It is prepared from the antigenic peptide described in claim 3 as an immunogen.

6. The use of the antigenic peptide of claim 3 or the antibody of claim 5 in the preparation or use of tumor detection / diagnosis / screening or treatment products.

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