Application of ZNF473 / TRIM28 / EMT as target in screening of drugs for treating AEJ

Abstract

The invention belongs to the technical field of biological medicine, and provides application of ZNF473 / TRIM28 / EMT as a target in screening of drugs for treating AEJ. The medicine for treating AEJ is an inhibitor for inhibiting or reducing ZNF473 expression, or an inhibitor or a blocking agent for inhibiting or blocking combination of ZNF473 and TRIM28, or an inhibitor for inhibiting EMT expression. Through molecular docking, it is preliminarily proved that ZNF473 is combined with TRIM28. Cell experiments also prove that ZNF473 is combined with TRIM28, and then malignant phenotypes of AEJ are promoted through EMT. The transcription factor plays a key regulation role in cells, and the interaction of the transcription factor plays an important role in the development of tumors and the formation of malignant phenotypes.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of ZNF473 / TRIM28 / EMT as a target in screening drugs for treating AEJ. Background Art

[0002] Adenocarcinoma of the esophagogastric junction (AEJ) is a type of adenocarcinoma that arises in the region connecting the distal esophagus and proximal stomach. Despite a continued downward trend in the incidence of gastric cancer, the incidence and mortality of AEJ cancer have not significantly decreased. AEG has become a major health threat to the Chinese population and has also imposed a significant health and economic burden.

[0003] Zinc finger protein 473 (ZNF473), also known as KIAA1141, ZincFinger Protein 100 Homolog, DKFZP434N043, HZFP100, ZFP100, Zfp-100, and ZN473, encodes a C2H2-type zinc finger protein. The human genome reference sequence is GRCh38 (Genome Reference Consorium human genome (build 38)), also known as GRCh38 or hg38. This version was released by the Genome Reference Consorium in December 2013.

[0004] ZNF473 is mainly located in the nucleus, with a small amount possibly located in the cytoplasm, suggesting that ZNF473 may function as a transcription factor.

[0005] The TRIM (Tripartite Motif) family is a group of proteins that play important roles in diverse biological processes, including cell proliferation, differentiation, and immune responses. Recent research on TRIM family members has revealed that TRIMs not only participate in normal physiological functions but are also closely associated with the development and progression of various diseases and cancers. While studies have revealed the important roles of the TRIM family in cell signaling, immune regulation, and tumor biology, the specific mechanisms underlying this role remain to be explored.

[0006] The TRIM family is a family of proteins with specific structural features, primarily composed of a RING finger structure, two B-box structures, and a Coiled-coil structure. The combination of these structures enables TRIM family members to perform multiple functions within cells, participating in the regulation of important biological processes such as cell proliferation, differentiation, apoptosis, and immune responses. Based on their structure and function, TRIM family members are divided into multiple subfamilies. Currently, there are more than 70 known TRIM proteins, which are widely present in a variety of organisms, including humans, mice, and plants. The diversity of TRIM family members enables them to play an important role in cell biology. For example, TRIM21, as an E3 ubiquitin ligase, can mediate the ubiquitination of target proteins, thereby affecting protein degradation and cell signal transduction. In addition, TRIM family members are also involved in processes such as antiviral immunity, cell cycle regulation, and tumorigenesis.

[0007] The TRIM protein family includes a class of E3 ubiquitin ligases with conserved domains that are widely involved in various biological processes within the cell. The basic domains of TRIM proteins typically include a RING (Really Interesting New Gene) finger domain, two or more B-box domains, and a CC (Coiled-Coil) domain. These domains play a crucial role in the function of TRIM proteins. The RING finger domain is responsible for binding to ubiquitin and mediating the ubiquitination process, while the B-box domain may be involved in protein interactions and stability. The CC domain is often believed to play a key role in the self-aggregation of TRIM family members and their interactions with other proteins. This feature enables TRIM proteins to play an important role in regulating cell signaling and immune responses. Summary of the Invention

[0008] The present invention provides the use of ZNF473 / TRIM28 / EMT as a target in screening drugs for treating AEJ.

[0009] The present invention is achieved by the following technical solution: the use of ZNF473 / TRIM28 / EMT as a target in screening drugs for treating AEJ, wherein the drugs for treating AEJ are inhibitors that inhibit or reduce ZNF473 expression, or inhibitors or blockers that inhibit or hinder the binding of ZNF473 to TRIM28, or inhibitors that inhibit EMT expression.

[0010] Furthermore, the inhibition or reduction of ZNF473 expression is ZNF473 mutation, copy number deletion or low expression.

[0011] This study preliminarily demonstrated the binding of ZNF473 to TRIM28 through molecular docking. Cellular experiments also demonstrated that ZNF473 and TRIM28 bind to each other, thereby promoting the malignant phenotype of AEJ through EMT. Transcription factors play a key regulatory role within cells, and their interactions play a crucial role in tumor development and the formation of malignant phenotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is an experiment to knock down ZNF473 function in AGS cells. In the figure: A is the proliferation curve; B is the colony formation assay; C is the migration assay; D is the changes in EMT indicator proteins. Figure 2 This is an experiment to knock down TRIM28 function in AGS cells; in the figure: A is the proliferation curve; B is the colony formation assay; C is the migration assay; D is the changes in EMT indicator proteins; Figure 3 3D interaction diagram of ZNF473 and TRIM28; Figure 4 Figure 1 is a diagram of ZNF473 and TRIM28 binding proteins; Figure 5 ZNF473 depends on TRIM28 to function; Figure 6 The standard curve for protein concentration detection by BCA method. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0015] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0016] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0017] 1. Experimental methods: 1. Cell Culture: The cell lines and corresponding culture medium compositions used in experimental culture are shown in Table 1. AGS and HGC cell lines were purchased from the National Model and Specialty Laboratory Cell Resource Bank / Cell Bank of the Committee for the Collection of Typical Cultures of the Chinese Academy of Sciences, and GES-1 was purchased from our company. All purchased cells were recovered cells (culture flasks). Upon receipt of the cells, verify that the labeling on the culture flasks is consistent with the order information and that there are no abnormalities such as damage or leakage. Spray the culture flasks with alcohol and place them in a 37°C, 5% CO2 incubator for 2 hours. Observe and photograph under a microscope, and then immediately passage.

[0018] Table 1: Culture systems of various cell lines 2. Cell Passaging: The first passaging should be at a 1:2 ratio, using fresh complete medium and a new culture flask; do not reuse the culture medium from the original culture flask. If some adherent cells lose their adhesion or detach during transport, transfer all the culture medium from the culture flask to a centrifuge tube. Centrifuge at 100 rpm for 5 minutes, discard the supernatant, add 1-2 mL of trypsin, gently pipette, and resuspend. After 1-2 minutes, add 3-6 mL of complete medium to terminate the reaction. Centrifuge again, discard the supernatant, and resuspend in 1-3 mL of complete medium. Transfer the cells to a new T25 culture flask (passaging at a 1:2 ratio), add 8-10 mL of new complete medium per flask, and culture as usual.

[0019] Processing steps for cells in the receiving flask: Observe cell morphology under a microscope and photograph the flask. (If the culture medium has turned yellow, this indicates a high cell population and high nutrient consumption, requiring prompt processing.) Allow to stand in an incubator for 3 hours. Discard the liquid from the original flask, add 2 mL of trypsin, incubate for 2 minutes, gently tap, and terminate with 2 mL of complete culture medium. Aspirate the culture medium and gently pipette the cells away from the flask, then transfer the cells to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. After completion, discard the supernatant, add 2 mL of complete culture medium, and gently pipette to completely dissolve the cells. Divide the cells into two new flasks and fill the flask to 6 mL with complete culture medium. Use the cross or figure-of-eight technique to evenly distribute the cells between the flasks. Tighten the caps, wipe the flasks with alcohol to disinfect, and label with a marker the cell type, passage number, and treatment time. Transfer to an incubator and observe after 24 hours.

[0020] 3. Gene knockdown: Culture cells in six-well plates. When the cell confluence reaches 50-70%, replace the complete medium with serum-free medium overnight to ensure consistent cell status.

[0021] (1) Mix 5 μL of lipo2000 transfection reagent with 500 μL of Opti-MEM serum-free medium and incubate for 5 minutes; (2) 5 μL of target gene siRNA was mixed with 500 μL of Opti-MEM serum-free medium and then mixed with the mixture in step (1) and incubated for 25 minutes; (3) Aspirate the culture medium in the well plate and add Opti-MEM serum-free medium mixed with Lipo2000 and target gene siRNA. After incubation in the incubator for 6 hours, replace with complete medium. After 48-72 hours, collect cells and extract RNA or protein.

[0022] 4. RNA Extraction: Add 500 μL of Trizol to each well, pipette evenly and repeatedly, then collect into a 1.5 mL enzyme-free centrifuge tube. Add 200 μL of chloroform per 1 mL of Trizol (for a single well or more than two wells, reduce the amount of chloroform or increase the number of centrifuge tubes proportionally), vortex to mix, and incubate at room temperature for 5 minutes. Centrifuge at 14,000 × g for 15 minutes. The tube will now have three layers: the first layer is light red, the original color of the Trizol reagent; the second layer is white, with irregular membranes; and the third layer is a transparent, clear liquid.

[0023] Carefully aspirate the upper clear liquid and transfer it to a new enzyme-free centrifuge tube (normally about 500uL), add an equal volume of isopropanol, and centrifuge at 14000×G for 15 minutes. 6 A white precipitate may appear.

[0024] Decant the liquid and add 1 mL of pre-chilled 70% alcohol (prepared from anhydrous ethanol). Pipet the precipitate to float. If extracting a large amount, there is no need to disrupt the precipitate. Centrifuge at 7500 rpm for 10 minutes. Pour off the 70% alcohol and, using a yellow pipette and then a white pipette, remove as much of the remaining liquid as possible and discard. Allow to stand at room temperature for approximately 5 to 10 minutes (if a precipitate forms), until the edges of the precipitate become slightly transparent. Add 40 μL of DEPC water and pipette until the precipitate dissolves. At this point, the extracted RNA concentration can be directly measured. (It is recommended to maintain the same orientation of the centrifuge tubes throughout the centrifugation process. If extracting a small amount of RNA and no precipitate is present, gently pipette and perfuse according to the tube position.)

[0025] 5. Protein extraction and concentration determination: Trypsinize or scrape cells. After centrifugation, discard the supernatant as completely as possible. Add the prepared RIPA solution (add PMSF and protease inhibitors depending on the sample size). Incubate on ice for 10 minutes, sonicate for 15 seconds three times, and then centrifuge at 16,000 rpm for 20 minutes. The supernatant is the protein. Determine cell concentration using the BCA microplate assay. Prepare the BSA standard according to the manufacturer's instructions. Use 1x PBS as the diluent.

[0026] 6. Extract cell proteins using the CO-IP kit, remove non-specific binding, elute after immunoprecipitation, and detect the expression of target protein by Western Blot.

[0027] Table 2: Preparation of protein concentration standards using the BSA method To prepare BSA standards of various concentrations, prepare them in labeled 1.5 mL centrifuge tubes as shown in Table 2. (A and I do not need to be prepared in advance, as only one liquid is added). The prepared BSA standards can be stored at room temperature until further testing. To prepare samples, dilute the cell protein 10-fold with 1x PBS and keep on ice.

[0028] Prepare BCA working solution: Calculate BCA working solution, add 100uL or 200uL working solution to each sample in the microplate. Take 10uL of standard and sample and add them to the microplate, add 100uBCA working solution to each well (sample: BCA working solution = 1:10), mix thoroughly to avoid bubbles, cover the microplate, and incubate at 37°C for 30min. Set the enzyme reader to 562nm wavelength to detect absorbance. Draw a standard curve based on the absorbance of the BSA standard (minus the OD value of the blank well in the standard). Calculate the sample protein concentration based on the standard curve and the dilution multiple of the sample. The standard curve is as follows: Figure 6 shown.

[0029] 6. Western blot detection of protein expression: SDS-PAGE electrophoresis gel was configured according to Table 3.

[0030] Table 3: SDS-PAGE gel configuration Load 30 μg of total protein in a 20 μL volume. Calculate the required protein volume for each group based on protein concentration and pipette each group of protein into an EP tube. Add 5 μL of loading buffer to each tube and bring the volume up to 20 μL with protein lysis buffer. Mix thoroughly and boil in boiling water for 10 minutes to completely denature the proteins. Bring to room temperature and store at -80°C. Place the prepared gel in an electrophoresis tank (vertically). Slowly add running buffer to the tank until the liquid level reaches the outer surface of the gel. Wet transfer: Transfer the proteins to a PVDF membrane by wet transfer. Blocking: Block the PVDF membrane with 5% nonfat dry milk for 1 hour. Primary antibody incubation: Dilute the primary antibody appropriately in blocking buffer and incubate the PVDF membrane in the primary antibody overnight at 4°C on a shaker. Membrane washing: The next day, remove the antibody and wash the membrane three times with TBST for 10 minutes. Secondary antibody incubation: Incubate the PVDF membrane in the secondary antibody for 1 hour at room temperature on a shaker. Wash the membrane with TBST (10 min x 3). For ECL development, add ECL solution (solution A and solution B in a 1:1 ratio) onto the membrane and photograph using a gel imaging system. Analyze bands using ImageJ software, and plot using GraphPad Prism 9 software.

[0031] 7. Cell Function Assays: Flag ZNF473 (oe-ZNF473) and empty vector (oe-NC) were purchased from Hanbio Biotech (Shanghai, China). AGS cells were used to establish a stable ZNF473 knockdown model, and HGC cells were used to establish a stable ZNF473 overexpression model. Cells were cultured and transfected with the indicated lentiviruses according to the manufacturer's instructions. Cells infected with the indicated lentiviruses were selected with puromycin (Hanbio, Shanghai, China) one week after infection. RT-qPCR analysis was used to determine transfection efficiency and green fluorescent protein expression under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The sequence of sh-ZNF473 is GTAAGATTCTCTTCAGACGAA.

[0032] A total of 10 3 Cells were seeded into 96-well plates, with five replicate wells per well, and then incubated at 37°C for 1 hour using a Cell Counting Kit-8 (CCK-8, MEILUN, China). The absorbance of the cells at 450 nm was observed at the same time for 5 consecutive days.

[0033] In the colony formation assay, 1×10 3Treated cells were seeded onto 6-well plates. After incubation for at least two weeks, the plates were gently rinsed twice with phosphate-buffered saline (PBS) (do not rinse directly on the cell surface), then treated with formaldehyde for 15 minutes and finally stained with 0.1% crystal violet solution within one hour. Colony counts were determined and evaluated using ImageJ.

[0034] Migration experiments were performed with 2 × 10 5 Treated cells (volume 200uL) were inoculated into the transwell chamber, and 500uL complete culture medium was added to the lower chamber to remove bubbles. After 48-72 hours, the cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet. After washing off the staining solution, the cells were photographed under a microscope.

[0035] Invasion assay, 6 hours before the experiment, Matrigel was mixed with Opti-MEM serum-free medium at a ratio of 1:9 on ice and then added to the transwell chamber, which was placed in a 37°C incubator for 2 hours. 6 Treated cells (volume 200uL) were inoculated into the transwell chamber, and 500uL complete culture medium was added to the lower chamber to remove bubbles. After 72 hours, the cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet. After washing off the staining solution, the cells were photographed under a microscope.

[0036] 8. Amino acid analysis A. Obtain amino acid sequence: Search for the target protein gene on the Uniprot website (https: / / www.uniprot.org / ), select "HUMAN", and then select "Aminoacids" and "(gotosequence)" in the "Aminoacids" column. Select "Download" under the Sequence module on the new page to obtain the unnumbered amino acid sequence.

[0037] B. Protein structure and function prediction: Select the "ProtParam" module in the ExPASy database (https: / / www.expasy.org / ), enter the amino acid sequence of the target protein on the "ProtParam" module page (https: / / www.expasy.org / resources / protparam), and click "Compute parameters" to obtain the results.

[0038] 4. Molecular Docking: Select the full-length AlphaFold predicted structures of ZN473_HUMAN (Uniprot ID: Q8WTR7) and TIF1B_HUMAN (Uniprot ID: Q13263) as the receptor and ligand proteins. Set docking parameters and perform protein-protein docking. Comprehensively describe the binding interface of the protein-protein complex and conduct a systematic analysis. Use PyMOL to supplement the interaction details.

[0039] 2. Experimental results: 1. After knocking down and silencing ZNF473 in AGS cells, the results are as follows Figure 1 As shown in A, the proliferation ability of AGS cells decreased significantly, and the statistical analysis t=29.92 at 96h. P <0.0001; Figure 1 B. Colony formation assay showed that the cell colonies were significantly reduced after knockdown of ZNF473; Figure 1 C Migration experiment proved that the number of AGS cells migrating after ZNF473 knockdown was reduced compared with the non-knockdown group; invasion experiment also proved that the number of AGS cells after ZNF473 knockdown was reduced compared with the non-knockdown group. Figure 1 D Western Blot experimental results showed that after knocking down ZNF473, E-acd expression increased and N-cad decreased, suggesting that ZNF473 may affect the biological process of AGS through EMT.

[0040] 2. After knocking down and silencing TRIM28 in AGS cells, the results are as follows Figure 2 As shown in A, the proliferation ability of AGS cells decreased significantly, and the statistical analysis t=12.57 at 96h. P <0.0001; Figure 2 B. Colony formation experiment showed that the cell colonies were significantly reduced after knocking down TRIM28; Figure 2 C Migration experiment proved that the number of AGS cells migrating after knocking down TRIM28 was reduced compared with that in the NC group; invasion experiment also proved that the number of AGS cells was reduced after knocking down TRIM28 compared with that in the NC group. Figure 2 D Western Blot experimental results showed that after knocking down TRIM28, the expression of E-acd increased and N-cad decreased, while there was no significant difference in the expression of ZNF473, suggesting that ZNF473 may affect the biological process of AGS by acting on EMT through TRIM28.

[0041] TRIM28 is highly expressed in AGS and HGC cells. The importance of the TRIM family in cell biology cannot be underestimated. As a multifunctional class of E3 ubiquitin ligases, TRIM family members play a central role in regulating cell proliferation, differentiation, apoptosis, and immune responses. Their unique structure and function enable them to serve as hubs for signal transduction, influencing diverse cellular processes. Therefore, further exploration of the role of the TRIM family in cell biology will not only help us understand fundamental biological mechanisms but also provide new insights into the pathogenesis of disease.

[0042] 3. ZNF473 binds to TRIM28 A. Amino acid composition analysis ZNF473 amino acid analysis: The Uniprot database name for ZNF473 is Q8WTR7·ZN473. ZNF473 has 871 amino acids, a molecular weight of 100182.33, approximately 100 kDa, and an isoelectric point (PI) of 8.63, suggesting that ZNF473 is a basic protein. There are 101 aspartic acid (Asp) and glutamic acid (Glu) residues, and 121 lysine (Lys) and arginine (Arg) residues. The molecular formula is: C 4336 H 6694 N 1312 O 1320 S 58 The total number of atoms is 13,720. The instability coefficient is 45.29, which is greater than 40, indicating that the ZNF473 protein is unstable. Care should be taken to minimize protein degradation during experiments. The specific amino acid composition is shown in Table 4. The atomic composition of ZNF473 is shown in Table 5. SOPMA analysis is shown in Table 6.

[0043] Table 4: ZNF473 amino acid composition Table 5: Atomic composition of ZNF473 Table 6: SOPMA analysis of ZNF473 B. TRIM28 amino acid analysis The Uniprot database name for TRIM28 is Q13263·TIF1B_HUMAN. TRIM28 consists of 835 amino acids, has a molecular weight of 88549.66, and an isoelectric point (PI) of 5.52, suggesting that TRIM28 is an acidic protein. It contains 105 aspartic acid (Asp) and glutamic acid (Glu) residues, and 84 lysine (Lys) and arginine (Arg) residues.

[0044] The molecular formula is: C 3807 H 608 2N 1106 O 1231 S 47 The total number of atoms is 12,273. The instability coefficient is 46.43, which is greater than 40, indicating that the TRIM28 protein is unstable. Care should be taken to minimize protein degradation during experiments. The amino acid composition of TRIM28 is shown in Table 7, the atomic composition of TRIM28 is shown in Table 8, and the SOPMA analysis is shown in Table 9.

[0045] Table 7: Amino acid composition of TRIM28 Table 8: Atomic composition of TRIM28 Table 9: SOPMA analysis of TRIM28 C. Molecular docking: Figure 3 Shown is the interaction between ZN473 and TRIM28, with ZN473 used as the reference chain for interaction analysis.

[0046] There are 34 pairs of hydrophobic interactions and 37 pairs of hydrogen bonds between the two pairs of proteins. The hotspot residues involved in the interaction are mainly ALA-35, LEU-36, ASP-37, ASN-38, CYS-39, LEU-42, PHE-43, GLU-60, ARG-233, GLU-243, GLN-244, GLY-245, PHE-246, ASP-247, ARG-615, TYR-619, GLU-623, GLN-624, LYS-635, PHE-639, LYS-65 3, HIS-657, LYS-663, ILE-667, GLU-671, LYS-675, LYS-678, ASP-680, ARG-681, VAL-682, PHE-683, THR-684, GLN-685, ARG-686, TYR-688, GLN-691, HIS-692, THR-695, GLN-713, LYS-719, TYR-730, VAL-731, and HIS-854. The interaction forces, amino acid names, and positions are shown in Tables 10 and 11.

[0047] Table 10: Hydrophobic interactions Table 11: Hydrogen Bonding Hydrophobic interactions: The aggregation of hydrophobic molecules or chemical groups in aqueous solution reduces their surface area in contact with water, leading to the release of water molecules into the body. With the increase in entropy, the coordination of hydrophobic amino acids with the corresponding ligand groups plays a dominant role in protein-ligand binding.

[0048] Hydrogen bonding: Hydrogen bonding is considered the most important of all directional covalent interactions, and it is hypothesized that each additional hydrogen bond increases the binding affinity of a ligand by an order of magnitude. Polar covalent bonds form between a donor group (DH), which provides the positive end in the form of a hydrogen atom, and an acceptor group with high electron density. Typical values ​​of hydrogen bond energy range from 10 to 40 kJ / mol.

[0049] Docking score. A smaller docking score (negative value) indicates a more likely binding model. When the confidence score is higher than 0.7, the probability of binding between the two molecules is high. The molecular docking models are shown in Table 12.

[0050] Table 12: Molecular docking model D. ZNF473 and TRIM28 binding protein diagram like Figure 4 Proteins were extracted from AGS and HGC cells and treated with anti-ZNF473 and anti-TRIM28 antibodies, respectively. Western blot analysis was performed to detect ZNF473 and TRIM28 expression. No protein binding was observed in the IgG lanes; however, ZNF473 and TRIM28 were detected in both AGS and HGC cells under various treatments.

[0051] 4. ZNF473 promotes AEJ malignant phenotype through TRIM28 Molecular docking results have demonstrated that ZNF473 binds to TRIM28; cellular experiments have also demonstrated that ZNF473 and TRIM28 bind to each other, thereby promoting the malignant phenotype of AEJ through EMT. Transcription factors play a key regulatory role within cells, and their interactions play a crucial role in tumor development and the formation of malignant phenotypes.

[0052] like Figure 5 A, Using the empty vector as the control, overexpression of ZNF473 significantly increased the proliferation capacity, while overexpression of ZNF473 and knockdown of TRIM28 decreased the proliferation capacity, which was between that of overexpression and the empty vector.

[0053] Figure 5 B, Using the empty vector as the control, the colony number increased significantly after overexpression of ZNF473, while the colony number decreased after overexpression of ZNF473 and knockdown of TRIM28, and was between the overexpression and empty vector. Figure 5 C, Overexpression of ZNF473 and knockdown of TRIM28 in HGC cells were used to examine changes in EMT-related pathways. The results showed that, compared with the empty vector used as a control, E-cad was decreased after ZNF473 overexpression; however, E-cad was increased after ZNF473 overexpression and TRIM28 knockdown. Regarding N-cad, compared with the empty vector used as a control, N-cad was increased after ZNF473 overexpression; however, N-cad was decreased after ZNF473 overexpression and TRIM28 knockdown. Changes in TRIM28 were consistent with those in N-cad.

[0054] Figure 5 D, After overexpressing ZNF473 and knocking down TRIM28 in HGC cells, migration and invasion experiments were performed. Using the empty vector (Vector) as a control, the number of penetrating cells in the field of view increased after overexpressing ZNF473, while the number of penetrating cells decreased after overexpressing ZNF473 and knocking down TRIM28, and was between the empty vector and overexpression.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of ZNF473 / TRIM28 / EMT as a target in screening drugs for treating AEJ, characterized by: The drug for treating AEJ is an inhibitor that inhibits or reduces the expression of ZNF473, or an inhibitor or blocker that inhibits or blocks the binding of ZNF473 to TRIM28, or an inhibitor that inhibits the expression of EMT.

2. The use according to claim 1, characterized in that: The inhibition or reduction of ZNF473 expression is ZNF473 mutation, copy number deletion or low expression.

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