Application of ZNF131-354aa in preparation of antitumor drugs

Abstract

The invention provides application of ZNF131-354aa in preparation of an anti-tumor drug, and relates to the technical field of biological medicines. The invention relates to an application of ZNF131-354aa in preparation of an anti-tumor drug, and the ZNF131-354aa is used as an action target of the anti-tumor drug. ZNF131-354aa is used as a target spot of the anti-tumor drug, and a method and thought are provided for developing a novel anti-tumor drug; meanwhile, the anti-tumor drug directly acts on a target spot, the specificity is good, the influence on normal cells is reduced, and then side effects are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to use of ZNF131-354aa in preparing anti-tumor drugs. Background Art

[0002] The effectiveness and specificity of tumor treatment have always been the core issues of cancer research and clinical treatment. Existing anti-tumor drugs have significant limitations in terms of therapeutic effects and side effects, especially poor specificity, which easily affects normal cells and leads to side effects. Summary of the invention

[0003] The problem solved by the present invention is how to effectively solve the problem of poor specificity of anti-tumor drugs.

[0004] In order to solve the above problems, the present invention provides the use of ZNF131-354aa in preparing anti-tumor drugs.

[0005] The present invention provides use of ZNF131-354aa in preparing an anti-tumor drug, wherein ZNF131-354aa serves as a target of the anti-tumor drug.

[0006] Optionally, the sequence of ZNF131-354aa is:

[0007] MYVSTVRNSLTILDILKNIFENIQSSPTAMEAEETMECLQEFPEHHKMILDR

[0008] LNEQREQDRFTDITLIVDGHHFKAHKAVLAACSKFFYKFFQEFTQEPLVEIE

[0009] GVSKMAFRHLIEFTYTAKLMIQGEEEANDVWKAAEFLQMLEAIKALEVRN

[0010] KENSAPLEENTTGKNEAKKRKIAETSNVITESLPSAESEPVEIEVEIAEGTIE

[0011] VEDEGIETLEEVASAKQSVKYIQSTGSSDDSALALLADITSKYRQGDRKGQI

[0012] KEDGCPSDPTSKQEHMKSHSTESFKCEICNKRYLRESAWKQHLNCYHLEEGGVSKKQRTGKKIHVCQYCEKQFDHFGHFKEHLRKHTEQPDGHGG (SEQ ID NO: 1).

[0013] Alternatively, ZNF131-354aa is encoded by the active IRES sequence of circZNF131.

[0014]

[0015] Alternatively, the active IRES sequence is 28 nucleotides to 51 nucleotides, 52 nucleotides to 166 nucleotides, and 167 nucleotides to 224 nucleotides of circZNF131.

[0016] Alternatively, ZNF131-354aa can inhibit the transcription of carboxy-terminal binding protein 2 and thereby regulate the expression of E-cadherin and PTEN tumor suppressor genes.

[0017] Optionally, the tumor comprises gastric cancer.

[0018] Alternatively, anti-tumor drugs acting on the ZNF131-354aa target can affect the proliferation, migration and invasion of the gastric cancer cell line AGS.

[0019] Alternatively, anti-tumor drugs acting on the ZNF131-354aa target can affect the proliferation, migration and invasion of the gastric cancer cell line HGC-27.

[0020] Optionally, ZNF131-354aa includes a specific peptide sequence: YVSTVRNSLTILDILK (SEQ ID NO: 3).

[0021] The beneficial effects of the use of ZNF131-354aa in the preparation of anti-tumor drugs of the present invention are: using ZNF131-354aa as a target of anti-tumor drugs provides a method and idea for developing new anti-tumor drugs; at the same time, the anti-tumor drugs directly act on the target with good specificity, reduce the impact on normal cells, and thus reduce the generation of side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the verification that circZNF131 can encode ZNF131-354aa protein in this example;

[0023] Figure 2 This is a schematic diagram of the ability verification of circZNF131 encoding ZNF131-354aa protein in this example;

[0024] Figure 3 Schematic diagram of the effect of ZNF131-354aa on the proliferation ability of gastric cancer cell lines (AGS and HGC-27) in this example;

[0025] Figure 4 Schematic diagram of the effect of ZNF131-354aa on the migration and invasion ability of gastric cancer cell lines (AGS and HGC-27) in this example;

[0026] Figure 5Schematic diagram of Western blotting detection of protein expression regulated by ZNF131-354aa in gastric cancer cell lines (AGS and HGC-27) in this example. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be interpreted as being limited to the embodiments described herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are only for exemplary purposes and are not intended to limit the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific implementation methods and are not intended to limit this application.

[0029] The term "including" and its variations used in this article are open inclusions, that is, "including but not limited to"; the term "based on" is "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in the present invention are used to distinguish different objects, rather than to describe a specific order or a primary and secondary relationship. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, the meaning of "multiple" is two or more.

[0030] In the related art, gastric cancer is a global malignant tumor and one of the common causes of cancer death. The age-standardized morbidity and mortality of gastric cancer in my country are both high. Since most gastric cancer patients are often in the advanced stage when diagnosed, and the effects of surgery, chemotherapy and other treatments are not ideal, it has become necessary to explore the early diagnosis of gastric cancer. The unclear mechanism of gastric cancer occurrence is a key issue that affects the difficulty of early diagnosis of gastric cancer. Therefore, studying the molecular mechanism of gastric cancer occurrence has important clinical and scientific significance. Circular RNA (circRNA) is a single-stranded covalently closed circular RNA molecule formed by reverse splicing of linear RNA. CircRNA exerts biological functions through various pathways, including competitive adsorption of microRNA (miRNA) as a molecular sponge, competitive binding to proteins to regulate cellular molecular networks, and guiding protein synthesis through internal ribosome entry sites (IRES) as translation templates. The proteins translated into these circRNAs also have important biological functions, thus playing an important role in cell function regulation and tumorigenesis.

[0031] Existing anti-tumor drugs have limitations in terms of therapeutic effects and side effects, and new and more effective targets are urgently needed to develop anti-tumor drugs. ZNF131-354aa (the protein encoded by circZNF131) is an emerging tumor treatment target, and its potential mechanism of action has not been fully studied. The present invention provides a scientific basis for the research and development of anti-tumor drugs by deeply studying the mechanism of action of ZNF131-354aa in the occurrence and development of gastric cancer, and can develop new anti-tumor drugs with new mechanisms, in order to improve the therapeutic effect and overcome the shortcomings of existing treatment methods.

[0032] In recent years, circRNA has attracted extensive attention in cancer research due to its unique circular structure and stability. Existing technologies have found that certain circRNAs play important regulatory roles in different types of cancer.

[0033] Some studies focus on specific circRNAs, such as circHIPK3 and circ_100290, and explore their roles in tumor occurrence and development by analyzing their expression levels in cancer cells and their relationship with cancer-related signaling pathways.

[0034] Study plan:

[0035] ① Expression regulation: Overexpression plasmid and short hairpin RNA (shRNA) technology are used to upregulate and downregulate the expression level of target circRNA, respectively, and evaluate their effects on cancer cell proliferation, migration and invasion.

[0036] ② Functional detection: CCK-8, EdU, Transwell and other experiments were used to detect the effects of circRNA on cancer cell proliferation, migration and invasion; in addition, flow cytometry was used to analyze cell cycle changes.

[0037] ③Molecular mechanism research: RNA pulldown and RNA immunoprecipitation (RIP) experiments are used to verify the binding of circRNA to protein; chromatin immunoprecipitation (ChIP) experiments are used to verify the binding of DNA to protein; protein blotting is used to detect changes in protein levels.

[0038] Some studies focus on the function and mechanism of circRNA in cancer.

[0039] Several studies have shown that the expression of circRNA in cancer is closely related to tumor growth, metastasis and invasion. Some circRNAs can regulate cell signaling pathways and affect the behavior of cancer cells by acting as molecular sponges to absorb miRNA or bind to proteins.

[0040] In gastric cancer, circ_100269 was found to be associated with tumor suppression by adsorbing miR-223 and reducing the inhibition of miR-223 on fibroblast growth factor receptor-1 (FGFR1), thereby promoting the proliferation and migration of tumor cells.

[0041] Study plan:

[0042] ① Expression regulation: Use circRNA overexpression plasmids and shRNA technology to study the function of target circRNA. Through transfection experiments, analyze its effects on cell biological behavior.

[0043] ② Functional detection: Cell proliferation, migration and invasion experiments were used to detect the effects of circRNA on cancer cell behavior; flow cytometry was used to analyze cell cycle changes.

[0044] ③Molecular mechanism research: RNA pulldown, RIP and ChIP experiments are used to study the interaction between circRNA and proteins and DNA; protein blotting is used to detect the expression levels of key proteins.

[0045] In view of the problems existing in the above-mentioned related technologies, this embodiment provides the use of ZNF131-354aa in the preparation of anti-tumor drugs.

[0046] The embodiment of the present invention provides a use of ZNF131-354aa in the preparation of an anti-tumor drug, wherein ZNF131-354aa serves as a target of the anti-tumor drug.

[0047] In this embodiment, ZNF131-354aa is used as the target of anti-tumor drugs, which provides methods and ideas for developing new anti-tumor drugs; at the same time, the anti-tumor drugs act directly on the target with good specificity, reduce the impact on normal cells, and thus reduce the occurrence of side effects.

[0048] Optionally, the sequence of ZNF131-354aa is:

[0049] MYVSTVRNSLTILDILKNIFENIQSSPTAMEAEETMECLQEFPEHHKMILDR

[0050] LNEQREQDRFTDITLIVDGHHFKAHKAVLAACSKFFYKFFQEFTQEPLVEIE

[0051] GVSKMAFRHLIEFTYTAKLMIQGEEEANDVWKAAEFLQMLEAIKALEVRN

[0052] KENSAPLEENTTGKNEAKKRKIAETSNVITESLPSAESEPVEIEVEIAEGTIE

[0053] VEDEGIETLEEVASAKQSVKYIQSTGSSDDSALALLADITSKYRQGDRKGQI

[0054] KEDGCPSDPTSKQEHMKSHSTESFKCEICNKRYLRESAWKQHLNCYHLEEGGVSKKQRTGKKIHVCQYCEKQFDHFGHFKEHLRKHTEQPDGHGG (SEQ ID NO: 1).

[0055] Specifically, the target protein was pulled down by immunoprecipitation and then subjected to mass spectrometry analysis, and the sequence of ZNF131-354aa was identified.

[0056] Alternatively, ZNF131-354aa is encoded by the active IRES sequence of circZNF131.

[0057] In this optional embodiment, the database predicts that the three IRES sequences with the highest scores in circZNF131 are active IRES sequences.

[0058]

[0059] Specifically, the circulated circZNF131 is shown in Figure 1 Middle A.

[0060] Alternatively, the active IRES sequence is 28 nucleotides to 51 nucleotides, 52 nucleotides to 166 nucleotides, and 167 nucleotides to 224 nucleotides of circZNF131.

[0061] Specifically, the chromosomal region of the circZNF131 gene and the back-splicing site were verified by Sanger sequencing to obtain an active IRES sequence.

[0062] Alternatively, ZNF131-354aa can inhibit the transcription of c-terminal binding protein 2 (CTBP2) and thus regulate the expression of E-cadherin and PTEN tumor suppressor genes.

[0063] Specifically, ZNF131-354aa regulates the expression of E-cadherin and PTEN tumor suppressor genes by inhibiting the transcription of carboxyl-terminal binding protein 2, thereby serving as a target for anti-tumor drugs.

[0064] Optionally, the tumor comprises gastric cancer.

[0065] Optionally, anti-tumor drugs act on the ZNF131-354aa target and can affect the proliferation, migration and invasion of the gastric cancer cell line AGS or the gastric cancer cell line HGC-27.

[0066] Specifically, anti-tumor drugs can affect the proliferation, migration and invasion of gastric cancer cell line AGS or gastric cancer cell line HGC-27 by regulating the expression of ZNF131-354aa target.

[0067] Optionally, ZNF131-354aa includes a specific peptide sequence: YVSTVRNSLTILDILK (SEQ ID NO: 3).

[0068] Specifically, the specific peptide "YVSTVRNSLTILDILK" (SEQ ID NO: 3) was able to prove that circZNF131 encodes the protein ZNF131-354aa.

[0069] The present invention is further illustrated and verified below in conjunction with specific embodiments.

[0070] Example 1

[0071] 1. Overexpression of circRNA: Use circRNA overexpression plasmid to upregulate the expression of circZNF131.

[0072]

[0073] Add 20 μL to 30 μL of E. coli DH5α containing circRNA overexpression plasmid and 2.5 μL of ampicillin to a 15 mL centrifuge tube containing 5 mL of LB medium, centrifuge at 200 rpm, and incubate at 37°C for 12 h to 16 h. Take out the centrifuge tube and observe the turbidity of the medium to determine the expansion of E. coli.

[0074] The plasmid was extracted using the EZNA Fastfilter plasmid DNA extraction kit produced by Omega. The specific steps are as follows:

[0075] 1) Centrifuge at 10,000 rpm for 1 min, discard the supernatant, add 180 μL of Solution I / RNase A, pipette to mix, and then aspirate into a new 1.5 mL EP tube.

[0076] 2) Add 180 μL of solution II, invert and mix several times, and let stand for 2 to 3 minutes. Be careful not to shake violently. Solution I and solution II in the EZNA Fastfilter Plasmid DNA Extraction Kit are ready-made solutions and do not need to be prepared.

[0077] 3) Add 90 μL of N3 buffer and mix by inversion until a white precipitate appears. The components are 3M potassium acetate and 5M acetic acid.

[0078] 4) Add 160 μL of GBT buffer provided in the kit, immediately invert and mix 5 times, and centrifuge at 13,000 g for 10 min.

[0079] 5) Insert the HiBind column into a 1.5 mL EP tube, insert the FastFilter column into the HiBind column, and transfer the supernatant into the FastFilter column.

[0080] 6) Centrifuge at 13000g for 1 min, discard the FastFilter column, and discard the filtrate to recover the EP tube.

[0081] 7) Add 200 μL of HBC buffer provided in the kit and add isopropanol to mix before use. Centrifuge at 13000 g for 20 seconds and discard the filtrate.

[0082] 8) Add 400 μL of DNA cleaning buffer provided in the kit and add isopropanol to mix before use. Centrifuge at 13000 g for 2 min and discard the filtrate.

[0083] 9) Place the Hibind column in a clean 1.5 mL EP tube, add 30 μL elution buffer, and let stand for 5 min. Centrifuge at 13000 g for 1 min, discard the Hibind column, and store the plasmid solution at -20°C.

[0084] 2. Plasmid transfection: the plasmid transfection concentration is 2 μg / mL.

[0085] Before transfection, the gastric cancer cell (AGS and HGC-27) suspension was plated in a 6-well plate, and the density of gastric cancer cells during transfection was 70% to 80%. The specific steps are:

[0086] 1) Aspirate the culture medium in the 6-well plate, wash twice with PBS, add 1 mL of culture medium without double antibody to each well, and place in the incubator for later use.

[0087] 2) Take out the 1.5mL EP tubes that have been sterilized by high temperature, and place the upper and lower rows of EP tubes on the foam rack according to the wells to be transfected. Add 50μL / μg of serum-free and dual-antibody high-glucose DMEM culture medium to each EP tube.

[0088] 3) Add plasmid to the upper row of EP tubes, and add EZ Trans cell transfection reagent (produced by Shanghai Li Ji Biological Company) to the lower row of EP tubes at a ratio of 1:3-1:4. Mix them well. Immediately add the culture medium with EZ Trans cell transfection reagent to the upper row of EP tubes. This mixing order cannot be reversed. Mix by gently pipetting and let stand at room temperature for 10-15 minutes to form EZ Trans-DNA complex.

[0089] 4) After gently blowing the plasmid-liposome complex solution in the EP tube, add it to the 6-well plate, shake it gently to mix, and then place it in the incubator for 6h to 18h.

[0090] 5) The next day, replace the cell culture medium and culture overnight. After 48 hours of transfection, the transfection is complete. The transfection efficiency of the plasmid carrying green fluorescent protein (GFP) can be observed using a fluorescence microscope.

[0091] 2. Functional testing of gastric cancer cells.

[0092] Cell proliferation was detected by Enhanced Cell Counting Kit-8 (CCK-8). The cell concentration was 2000 / 100 μL, and the OD value (450 nm) on the 0th, 1st, 2nd, 3rd and 4th day was detected by microplate reader, and the records were kept to draw the cell proliferation curve;

[0093] Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay. After 48 hours of cell transfection in the 6-well plate, 1 mL of culture medium was aspirated, 2 μL / well of EdU working solution was diluted to 1:1000 with cell culture medium at 1:500, and the culture medium containing EdU was gently mixed, and 1 mL was added to each well of the 6-well plate. The 6-well plate was gently shaken to mix, and the incubation time was 2 hours in the cell culture incubator. The culture medium was removed, 1 mL of 4% paraformaldehyde was added to fix the cells, and the cells were placed at room temperature for 15 minutes. The fixative was aspirated, and 1 mL of immunostaining blocking solution was added to each well to wash the cells, and the cells were washed 3 times. The washing solution was aspirated, and 1 mL of permeabilization solution [3% Triton X-100 in phosphate buffered saline (PBS)] was added to each well, and the cells were incubated at room temperature for 15 minutes. Aspirate the permeabilization solution, add 1 mL of blocking solution to each well to wash the cells, wash the cells 2 to 3 times. Aspirate the washing solution, add 500 μL / well of Click reaction solution, and incubate in the dark for 30 minutes. After aspirating the reaction solution, wash the cells 2 to 3 times with washing solution. Add 1 mL / well of Hoechst fluorescent dye 33342 (Hoechst 33342) reaction solution and incubate in the dark for 10 minutes. Wash 3 times with immunostaining blocking solution. Use a fluorescent microscope to detect the staining effect.

[0094] Cell migration and invasion were detected by migration and invasion assay (Transwell). The cell concentration was 20000 / 200μL, migration culture was 24h, and invasion culture was 48h. After the incubation time, the liquid in the upper and lower chambers of Transwell was aspirated, and 1mL / well of PBS was slowly added along the wall of the upper chamber to wash twice, and PBS was aspirated. 1mL of 4% paraformaldehyde was added to each well and incubated for 30min to fix the cells. The 4% paraformaldehyde was aspirated, and the cells were washed twice again with PBS, and then 1mL of 0.1% crystal violet was added to each well, and the cells were stained for 30min on a horizontal shaker at 80rpm~100rpm. The crystal violet was aspirated, and the chamber was gently washed with ultrapure water and placed in an oven to dry. Then, an inverted microscope was used to take pictures and count.

[0095] 3. Construction of circular overexpression vector circZNF131-mut with ATG mutation in the start codon of the open reading frame of circZNF131 protein and linear open reading frame overexpression vector ZNF131-354aa-GFP with GFP (both purchased from Guangzhou Jisai Biotechnology Co., Ltd.). According to the circRNADb (http: / / reprod.njmu.edu.cn / cgi-bin / circrnadb / circRNADb.php) and circBank (http: / / www.circbank.cn / ) databases, the highest-scoring IRES sequence of circZNF131 was predicted, and dual-luciferase reporter plasmids with three sequences of 28 nucleotides (nt) to 51nt, 52nt to 166nt, and 167nt to 224nt were designed to detect IRES activity (both purchased from Guangzhou Jisai Biotechnology Co., Ltd.).

[0096] 4. Western blot experimental method.

[0097] 1) Liquid preparation 1×TBST (Tris-HCl buffered saline + Tween-20): dilute 20×Tris-HCl buffered saline (TBS) buffer to 1×TBS buffer, add 500μLTween-20, and stir evenly. Electrophoresis buffer: dilute 10×Tris-glycine-sodium dodecyl sulfate (SDS) electrophoresis buffer to 1× concentration. Transfer buffer: mix 50mL 20×Y-Tec fast transfer buffer, 100mL anhydrous ethanol and 850mL ultrapure water. Protein extraction and quantification

[0098] 2) Cell treatment: digest cells, centrifuge (1000 rpm, 5 min), wash twice with PBS, centrifuge to remove supernatant, add IP lysis buffer (protease inhibitor 1:100), incubate on ice for 15 min, sonicate 5 times (10 sec each, 50 sec interval), centrifuge (12000 rpm, 4°C, 10 min).

[0099] 3) Protein quantification: Take 2 μL of supernatant and add 18 μL PBS to mix. Prepare bicinchininincacid (BCA) solution, dilute protein solution (10 times), mix well, incubate at 37°C for 30 min, and measure concentration by spectrophotometer. Add 5× loading buffer to dilute to 1× concentration, incubate at 95°C metal bath for 10 min to prepare loading solution.

[0100] 4) Electrophoresis and membrane transfer: Place the gel glass plate into the electrophoresis tank, add electrophoresis solution, and remove the comb. Add rainbow protein molecular weight standards and samples, and perform electrophoresis (120V, 50min-60min). Membrane transfer: Activate the polyvinylidene fluoride (PVDF) membrane in methanol for 3min-5min. Soak the membrane transfer sponge pad in the membrane transfer solution. Take out the gel after electrophoresis, place it in a "sandwich" model, and transfer it (400mA, 25min-35min).

[0101] 5) Blocking and incubating with antibodies: Wash the PVDF membrane 3 times with 1×TBST, 6 min each time. Block with 5% milk powder and incubate at room temperature for 2 h. Wash the PVDF membrane 3 times with 1×TBST again, spin dry, add primary antibody, and incubate at 4°C overnight. Recover the primary antibody, wash the PVDF membrane 3 times with 1×TBST, spin dry, add secondary antibody, and incubate for 2 h.

[0102] 6) Exposure: Wash the PVDF membrane three times and add fresh TBST. Prepare enhanced chemiluminescence (ECL) developer: mix 2 mL of reagent A and 2 mL of reagent B, soak the PVDF membrane, and expose. Save and record the exposure results.

[0103] 7) Use immunoprecipitation to pull down the target protein for mass spectrometry analysis. The sequence of ZNF131-354aa was identified as:

[0104] MYVSTVRNSLTILDILKNIFENIQSSPTAMEAEETMECLQEFPEHHKMILDRLNEQREQDRFTDITLIVDGHHFKAHKAVLAACSKFFYKFFQEFTQEPLVEIEGVSKMAFRHLIEFTYTAKLMIQGEEEANDVWKAAEFLQMLEAIKALEVRNKENSAPLEENTTGKNEAKKRKIAET SNVITESLPSAESEPVEIEVEIAEGTIEVEDEGIETLEEVASAKQSVKYIQSTGSSDDSALALLADITSKYRQGDRKGQIKEDGCPSDPTSKQEHMKSHSTESFKCEICNKRYLRESAWKQHLNCYHLEEGGVSKKQRTGKKIHVCQYCEKQFDHFGHFKEHLRKHTEQPDGHGG(SEQ ID NO:1)

[0105] 5. Verify that circZNF131 encodes a new protein ZNF131-354aa consisting of 354 amino acid residues.

[0106] According to the circRNADb database, it was predicted that the 967-nt circZNF131 might encode a protein consisting of 354 amino acid residues (named ZNF131-354aa; e.g. Figure 1 The ZNF131-354aa encoded across the cyclization site has a specific sequence at both ends. Based on the similar sequence segments between ZNF131-354aa and the full-length ZNF131 protein sequence, a ZNF131 (139aa to 238aa) antibody that can recognize ZNF131-354aa was found for experiments (as shown in Figure 1). Figure 1 After overexpressing circZNF131 in gastric cancer cell lines (AGS and HGC-27), protein extraction and Western blotting experiments revealed a band at 40.5 kDa that was consistent with the size of the predicted circZNF131-encoded protein ZNF131-354aa (as shown in Figure 2B). Figure 1 Next, after overexpressing circZNF131 in human embryonic kidney 293T cells, IP immunoprecipitation was performed using ZNF131 (139aa-238aa) antibody, and the pulled-down protein was subjected to gel electrophoresis and stained with Coomassie Brilliant Blue. It was found that the target protein (as shown in Figure 2) was also found at 40.5 kDa. Figure 1 As shown in D, the band at 55 kDa is heavy chain contamination, and the band at 27 kDa is light chain contamination). The IP-pulled protein was subjected to mass spectrometry analysis, as shown in Figure 1 As shown in E, red indicates specific peptides, and yellow indicates mass spectrometry detection sequences. The results identified multiple peptides on the ZNF131-354aa protein sequence, especially the specific peptide "YVSTVRNSLTILDILK" (SEQ ID NO: 3), proving that circZNF131 encodes the protein ZNF131-354aa.

[0107] 6. Verification of the ability of circZNF131 to encode the protein ZNF131-354aa.

[0108] The circular overexpression vector circZNF131-mut (“mut” is often used as an abbreviation for “mutant”, meaning mutant) with ATG mutation in the start codon of the circZNF131 protein encoding open reading frame and the linear overexpression vector ZNF131-354aa-GFP (such as Figure 2 Western blot experiments confirmed that the ATG-mutated circZNF131-mut did not encode ZNF131-354aa after overexpression (as shown in Figure 2A). Figure 2As shown in B), the linear overexpression vector ZNF131-354aa-GFP was verified to express the corresponding protein after incubation with GFP and ZNF131 antibodies. The IRES sequences with the highest prediction scores in the circRNADb and circBank databases were at 28nt-166nt and 51nt-224nt of circZNF131, respectively. Therefore, dual-luciferase reporter plasmids with three sequences of 28nt-51nt, 52nt-166nt, and 167nt-224nt were designed to verify the IRES activity. The results showed that the relative fluorescence intensity of the encephalomyocarditis virus (EMCV) IRES group increased, the relative fluorescence intensity of the IRES del-1 (28nt-51nt) and IRES del-2 (52nt-166nt) groups was weakened compared with the IRES group, and the relative fluorescence intensity of the IRES del-3 (167nt-224nt) group increased (as shown in B). Figure 2 (shown in C), data are expressed as mean ± standard deviation, n = 3. ****P < 0.0001. The above results show that circZNF131 can encode ZNF131-354aa protein through an active IRES sequence.

[0109] 7. ZNF131-354aa plays a tumor suppressor role in gastric cancer cells.

[0110] In gastric cancer cell lines, NC, circZNF131-mut, circZNF131, and ZNF131-354aa-GFP plasmids were transfected, and Western blotting was performed to verify the expression of ZNF131-354aa protein and to verify the transfection efficiency of circZNF131-mut, circZNF131, and ZNF131-354aa-GFP overexpression plasmids in AGS and HGC-27 cells (e.g. Figure 3 CCK-8 experiment, EdU experiment, Transwell migration and invasion experiment were performed to verify the biological function of ZNF131-354aa in gastric cancer cells. The results showed that the experimental results of CCK-8 experiment were as follows Figure 3 As shown in B, the experimental results of EdU experiment are as follows Figure 3 As shown in C, the proliferation ability of the circZNF131-mut group was decreased compared with the NC group, and the proliferation ability of the circZNF131 and ZNF131-354aa-GFP groups was decreased compared with the circZNF131-mut group. The data are expressed as mean ± standard deviation, n = 3. **P < 0.01, ***P < 0.001, ****P < 0.0001. In the Transwell migration and invasion assay, the Transwell migration results are shown in Figure 4As shown in A, the invasion results are as follows Figure 4 As shown in B, the migration and invasion abilities of the circZNF131-mut group were weakened, while the migration and invasion abilities of the circZNF131 and ZNF131-354aa-GFP groups were also weakened compared with the circZNF131-mut group.

[0111] 8. The mechanism of ZNF131-354aa's anti-tumor effect in gastric cancer cells: It regulates the expression of E-cadherin and PTEN (a tumor suppressor gene, whose Chinese name is the phosphatase and tensin homologous gene deleted from human chromosome 10) by inhibiting the transcription of carboxyl-terminal binding protein 2 (CTBP2).

[0112] Gastric cancer cell lines were transfected with NC, circZNF131-mut, circZNF131, and ZNF131-354aa-GFP plasmids, respectively, and Western blotting was performed for verification. Figure 5 As shown, the results showed that overexpression of circZNF131 and ZNF131-354aa-GFP reduced the level of CTBP2 protein compared with NC, while overexpression of circZNF131-mut had no significant change; E-cadherin levels increased after overexpression of circZNF131 and ZNF131-354aa-GFP in HGC-27 cells; and PTEN also increased after overexpression of circZNF131 and ZNF131-354aa-GFP in AGS and HGC-27 cells. Western blot detected the loss of E-cadherin expression in AGS cells (human gastric adenocarcinoma cells).

[0113] In summary, circZNF131 can encode the protein ZNF131-354aa. ZNF131-354aa has the function of inhibiting the proliferation of gastric cancer cells; mechanism studies have found that ZNF131-354aa inhibits the progression of gastric cancer by inhibiting the transcription of CTBP2 to regulate the expression of E-cadherin and PTEN.

[0114] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. znf131-354AA's use in preparing anti-tumor drugs, It is characterized by, The ZNF131-354AA is the target of anti-tumor drugs.

2. The use of ZNF131-354AA based on claim 1 in preparation of anti-tumor drugs, It is characterized by, The sequence of ZNF131-354AA is: MyVSTVRNSLTILDILKNIFENIQSSSSSPTAMETMECLQEHHKMILDRLNEQDRFTLIVGHFKAHKAVLAAVLAAVLACSKFFQEPLVSKMAFRHLIETTAFRHLIE GeeeandVWKAAEFLQMLEAIKALEVRNSAPLEENTGKNEAKKRKIAETSNVITESLPSAES EPVEIEVEGTEGIEVAKQSALALALALALALALALALALQGDRKDRKDRKDRKQGDRKDRKDRKDRKDRKDRKDRKDRQDRKDRQDRQDRK IKEDGCPSDPTSKSHMKSHSTSTESFKCEICNKRYLRESAWKQHLNCYHLEEGGVSKQRHVCQycekqfckhfkehlrkhteqpde (seq ID no: 1).

3. The ZNF131-354AA, which is based on claim 2, is used in the preparation of anti-tumor drugs. It is characterized by, The ZNF131-354AA is produced by the active IRS sequence of Circznf131.

4. ZNF131-354AA according to claim 3 in the preparation of anti-tumor drugs, It is characterized by, The CIRCZNF131 is made from the 2nd outer sub-appendage of the ZNF131 gene on the 5th chromosomal to the 6th outer appendage. AGCAGCCCGACGGCCATGGAGGCTGAAGAGACGATGGAATGCCTTCAGGAGTTCCCTGAACATCATAAAATGATCCTCGACCGATTGAATGAACAGCGAGAGCAGGACCGGTTTACTGACATCACCCTAATTGTCGACGGACACCATTTTAAGGCTCACAAGGCTGTTTTGGCTGCTTGTAGTAAGTTCTTCTACAAATTCTTTCAGGAGTTTACCCAAGAACCATTGGTGGAGATAGAAGGTGTTAGTAAAATGGCCTTTCGCCATTTAATTGAGTTCACATATACAGCAAAATTAATGATACAAGGAGAAGAAGAAGCCAATGATGTATGGAAAGCAGCAGAGTTTCTACAAATGCTAGAAGCTATCAAAGCCCTTGAAGTCAGGAACAAAGAAAACTCAGCTCCCTTAGAGGAAAATACCACAGGAAAAAATGAGGCCAAAAAAAGGAAGATTGCAGAAACTTCAAATGTTATCACTGAGTCATTGCCATCTGCAGAATCAGAACCTGTTGAAATTGAGGTAGAGATTGCCGAAGGCACCATTGAAGTGGAAGATGAAGGCATCGAAACATTAGAGGAAGTGGCTTCTGCCAAGCAGTCCGTAAAGTACATACAGAGCACAGGTTCCTCTGATGATTCTGCTCTAGCACTGTTGGCAGATATTACCAGCAAGTACCGTCAAGGTGACAGAAAAGGGCAGATTAAAGAAGATGGCTGTCCATCTGACCCCACGAGCAAACAGGAGCACATGAAATCACACTCCACTGAGAGTTTCAAGTGTGAAATATGCAATAAACGATATCTTCGAGAGAGCGCATGGAAACAGCACCTAAATTGTTACCACCTTGAAGAAGGTGGAGTCAGTAAGAAGCAAAGAACTGGGAAAAAAATTCATGTATGTCAGTACTGTGAGAAACAGTTTGACCATTTTGGACATTTTAAAGAACATCTTCGAAAACATACAG -3 ′ (SEQ ID NO: 2).

5. The use of ZNF131-354AA according to claim 4 in preparing for anti-tumor drugs, It is characterized by, The active IRES sequence is the 28 nucleotide-51 nucleotide, 52 nucleotide -166 nucleotide and 167 nucleotide nucleotide of the Circznf 131.

6. ZNF131-354AA based on claim 1 in preparing anti-tumor drugs, It is characterized by, The ZNF131-354AA can inhibit the transcription of the carboxyl binding protein 2 to regulate the expression of E-calcium viscosity and PTEN cancer suppression genes.

7. ZNF131-354AA based on claim 1 in preparing anti-tumor drugs, It is characterized by, The tumor includes gastric cancer.

8. The use of ZNF131-354AA according to claim 7 in preparation of anti-tumor drugs, It is characterized by, Anti-tumor drugs act on the target of ZNF131-354AA, which can affect the proliferation, migration and invasion of gastric cancer cells.

9. The ZNF131-354AA in the claim 7 in the preparation of anti-tumor drugs, It is characterized by, Anti-tumor drugs act on the target of ZNF131-354AA, which can affect the proliferation, migration and invasion of gastric cancer cells HGC-27.

10. ZNF131-354AA based on claim 1 in preparing for anti-tumor drugs, It is characterized by, The ZNF131-354AA includes specific peptide sequences: YVSTVRNSLTILDILK (SEQ ID NO: 3).

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