Use of a substance that inhibits the expression of a znf8 protein in the preparation of a product for the prevention and treatment of cancer

Abstract

The application discloses application of a substance for inhibiting expression of ZNF8 protein in preparation of a product for preventing and treating cancer. Experiments prove that the ZNF8 protein can be combined with p53 protein; the expression of the ZNF8 protein can increase transcriptional activity of the p53 protein, down-regulate expression of VEGF gene and ANGPLT4 gene, inhibit proliferation of tumor cells, inhibit migration of tumor cells, inhibit transfer of tumor cells, inhibit invasion and transfer of tumor cells, inhibit formation of blood vessels, inhibit growth of tumors, inhibit transfer of tumors, inhibit lung transfer of tumors and improve survival rate of tumor-bearing animals, so that the expression of the ZNF8 protein has important application value in inhibition of growth and invasion and transfer of tumors; the expression of the ZNF8 protein can prevent and treat cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical field, in particular to the application of a substance for inhibiting the expression of ZNF8 protein in the preparation of a product for preventing and treating cancer. BACKGROUND

[0002] The KRAB-type zinc finger protein (KZNF) family is the largest transcription factor / transcriptional regulatory factor family in mammals, and the protein structure thereof mainly comprises a KRAB (Krüppel-associated box) domain with strong transcription inhibition function at the N terminal, and a plurality of C2H2-type zinc finger structures for continuously recognizing continuous DNA sequences at the middle and C terminal. The KRAB domain combines with KAP-1 (KRAB-associated protein-1) to recruit various transcriptional inhibitors to form a transcriptional inhibition complex, and the C terminal zinc finger region is combined with DNA in the target gene regulatory region and / or other transcription factors to anchor the transcriptional inhibition complex formed with KAP-1 near the specific target sequence, thereby playing a specific transcriptional inhibition function and participating in cell differentiation, proliferation, apoptosis and other processes. The biggest feature of the KZNF family is that it appears late (begins to appear in quadruped vertebrates), evolves fast (more than 400 types have appeared in humans), and is closely related to the establishment of the fine regulation network of mammals and the maintenance of the stability of the genome.

[0003] ZNF8 protein is a member of the KRAB-Aa subfamily of the KZNF family, and the gene is located in the 19q13.43 region. The ORF thereof is 1728 bp, and the protein encoded thereby is composed of 575 amino acids. From the N terminal to the C terminal, the KRAB and the seven C2H2 domains are arranged in series on the ZNF8 protein. At present, its function and mechanism have not been reported.

[0004] RNA interference (RNAi) is an evolutionarily conserved defense mechanism against the invasion of transgenes or foreign viruses. Its essence is that siRNA is specifically combined with the target mRNA, and the degradation thereof is mediated by the RNA-induced silencing complex (RISC), so as to prevent the translation of mRNA and cause gene silencing. The use of exogenous or endogenous methods to introduce siRNA into specific cells for RNAi of specific genes can be used as an important tool for gene function research, gene therapy and targeted drug development. SUMMARY

[0005] The technical problem to be solved by the present application is how to prevent and / or treat cancer.

[0006] To solve the above technical problems, the present application first provides the use of a substance inhibiting the activity and / or expression amount of ZNF8 protein in the preparation of a product; the function of the product can be at least one of the following C1) to C15):

[0007] C1) preventing cancer; C2) treating cancer; C3) increasing the activity of p53 protein; C4) down-regulating the expression amount of VEGF gene; C5) down-regulating the expression amount of ANGPTL4 gene; C6) inhibiting the proliferation of tumor cells; C7) inhibiting the migration of tumor cells; C8) inhibiting the metastasis of tumor cells; C9) inhibiting the invasive metastasis of tumor cells; C10) inhibiting the formation of blood vessels; C11) inhibiting the growth of tumors; C12) inhibiting the metastasis of tumors; C13) inhibiting the lung metastasis of tumors; C14) increasing the survival rate of tumor-bearing animals; and C15) prolonging the survival time of tumor-bearing animals.

[0008] The present application also protects the use of a substance inhibiting the activity and / or expression amount of ZNF8 protein, which can be at least one of the following C1) to C15):

[0009] C1) preventing cancer; C2) treating cancer; C3) increasing the activity of p53 protein; C4) down-regulating the expression amount of VEGF gene; C5) down-regulating the expression amount of ANGPTL4 gene; C6) inhibiting the proliferation of tumor cells; C7) inhibiting the migration of tumor cells; C8) inhibiting the metastasis of tumor cells; C9) inhibiting the invasive metastasis of tumor cells; C10) inhibiting the formation of blood vessels; C11) inhibiting the growth of tumors; C12) inhibiting the metastasis of tumors; C13) inhibiting the lung metastasis of tumors; C14) increasing the survival rate of tumor-bearing animals; and C15) prolonging the survival time of tumor-bearing animals.

[0010] The present application also protects the use of ZNF8 protein as a drug target in the preparation of a product; the function of the product can be at least one of the following C1) to C15):

[0011] C1) preventing cancer; C2) treating cancer; C3) increasing the activity of p53 protein; C4) down-regulating the expression amount of VEGF gene; C5) down-regulating the expression amount of ANGPTL4 gene; C6) inhibiting the proliferation of tumor cells; C7) inhibiting the migration of tumor cells; C8) inhibiting the metastasis of tumor cells; C9) inhibiting the invasive metastasis of tumor cells; C10) inhibiting the formation of blood vessels; C11) inhibiting the growth of tumors; C12) inhibiting the metastasis of tumors; C13) inhibiting the lung metastasis of tumors; C14) increasing the survival rate of tumor-bearing animals; and C15) prolonging the survival time of tumor-bearing animals.

[0012] Any of the above-mentioned substances inhibiting the activity and / or expression amount of ZNF8 protein also falls within the protection scope of the present application.

[0013] The present application also protects the following X1) or X2):

[0014] X1) the use of ZNF8 protein in the preparation of a product; the function of the product can be at least one of A1) to A14);

[0015] X2) the use of ZNF8 protein, which can be at least one of A1) to A14);

[0016] A1) binds to p53 protein; A2) inhibits the activity of p53 protein; A3) up-regulates the expression of VEGF gene; A4) up-regulates the expression of ANGPTL4 gene; A5) promotes the proliferation of tumor cells; A6) promotes the migration of tumor cells; A7) promotes the metastasis of tumor cells; A8) promotes the invasion and metastasis of tumor cells; A9) promotes the formation of blood vessels; A10) promotes the growth of tumors; A11) promotes the metastasis of tumors; A12) promotes the lung metastasis of tumors; A13) reduces the survival rate of tumor-bearing animals; A14) shortens the survival time of tumor-bearing animals.

[0017] In any of the above-mentioned uses, the product can be a drug.

[0018] The present application also protects product A or product B;

[0019] The product A can contain any of the above-mentioned substances that inhibit the activity and / or expression of ZNF8 protein; the function of the product A can be at least one of the following C1) to C15):

[0020] C1) prevents cancer; C2) treats cancer; C3) increases the activity of p53 protein; C4) down-regulates the expression of VEGF gene; C5) down-regulates the expression of ANGPTL4 gene; C6) inhibits the proliferation of tumor cells; C7) inhibits the migration of tumor cells; C8) inhibits the metastasis of tumor cells; C9) inhibits the invasion and metastasis of tumor cells; C10) inhibits the formation of blood vessels; C11) inhibits the growth of tumors; C12) inhibits the metastasis of tumors; C13) inhibits the lung metastasis of tumors; C14) increases the survival rate of tumor-bearing animals; C15) prolongs the survival time of tumor-bearing animals;

[0021] The product B can contain ZNF8 protein; the function of the product B can be at least one of A1) to A14);

[0022] A1) binds to p53 protein; A2) inhibits the activity of p53 protein; A3) up-regulates the expression level of VEGF gene; A4) up-regulates the expression level of ANGPTL4 gene; A5) promotes the proliferation of tumor cells; A6) promotes the migration of tumor cells; A7) promotes the metastasis of tumor cells; A8) promotes the invasion and metastasis of tumor cells; A9) promotes the formation of blood vessels; A10) promotes the growth of tumor; A11) promotes the metastasis of tumor; A12) promotes the lung metastasis of tumor; A13) reduces the survival rate of tumor-bearing animals; A14) shortens the survival time of tumor-bearing animals.

[0023] The product A or the product B can be a drug.

[0024] The present application also protects the use of a substance that inhibits the activity and / or expression level of ZNF8 protein in the development or screening of an agent; the use of the agent can be at least one of the following C1) to C15):

[0025] C1) prevents cancer; C2) treats cancer; C3) increases the activity of p53 protein; C4) down-regulates the expression level of VEGF gene; C5) down-regulates the expression level of ANGPTL4 gene; C6) inhibits the proliferation of tumor cells; C7) inhibits the migration of tumor cells; C8) inhibits the metastasis of tumor cells; C9) inhibits the invasion and metastasis of tumor cells; C10) inhibits the formation of blood vessels; C11) inhibits the growth of tumor; C12) inhibits the metastasis of tumor; C13) inhibits the lung metastasis of tumor; C14) increases the survival rate of tumor-bearing animals; C15) prolongs the survival time of tumor-bearing animals.

[0026] Any of the above-mentioned "substances that inhibit the activity and / or expression level of ZNF8 protein" can be z1) or z2) or z3) or z4) or z5) or z6):

[0027] z1) oligonucleic acid siZNF8-1; the oligonucleic acid siZNF8-1 consists of a single-stranded nucleic acid molecule represented by sequence 5 in the sequence listing and a single-stranded nucleic acid molecule represented by sequence 6 in the sequence listing;

[0028] z2) oligonucleic acid siZNF8-2; the oligonucleic acid siZNF8-2 consists of a single-stranded nucleic acid molecule represented by sequence 7 in the sequence listing and a single-stranded nucleic acid molecule represented by sequence 8 in the sequence listing;

[0029] z3) an oligonucleic acid obtained by chemically modifying the oligonucleic acid siZNF8-1;

[0030] z4) an oligonucleic acid obtained by chemically modifying the oligonucleic acid siZNF8-2;

[0031] z5) shRNA synthesized by a shRNA expression system with the oligonucleic acid siZNF8-1 as a target;

[0032] z6) shRNA synthesized by a shRNA expression system targeting the oligonucleic acid siZNF8-2.

[0033] Any of the above-mentioned chemical modifications can be a phosphate backbone modification, a ribose modification, or a base modification. The stability of the oligonucleic acid siZNF8-1 or the oligonucleic acid siZNF8-2 subjected to chemical modification is increased, thereby effectively inhibiting the activity and / or expression amount of the ZNF8 protein.

[0034] The z5) can be specifically the shZNF8-1 plasmid mentioned in the examples; that is, the shRNA lentivirus expression plasmid of ZNF8 is constructed by Beijing Yingmaoshengye Biotechnology Co., Ltd. using the pLVshRNA-Puro plasmid as a carrier and targeting the siZNF8-1.

[0035] The z6) can be specifically the shZNF8-2 plasmid mentioned in the examples; that is, the shRNA lentivirus expression plasmid of ZNF8 is constructed by Beijing Yingmaoshengye Biotechnology Co., Ltd. using the pLVshRNA-Puro plasmid as a carrier and targeting the siZNF8-1.

[0036] The pLVshRNA-Puro plasmid can be a product of Beijing Yingmaoshengye Biotechnology Co., Ltd.

[0037] In any of the above-mentioned C1) or C2), the cancer can be colon cancer. In any of the above-mentioned C3), the "increasing the activity of p53 protein" can be increasing the activity of p53 protein in a cell; the cell can be a human colon cancer cell. In any of the above-mentioned C4), the "down-regulating the expression amount of VEGF gene" can be down-regulating the expression amount of VEGF gene in a cell; the cell can be a human colon cancer cell. In any of the above-mentioned C5), the "down-regulating the expression amount of ANGPTL4 gene" can be down-regulating the expression amount of ANGPTL4 gene in a cell; the cell can be a human colon cancer cell. In any of the above-mentioned C6), C7), C8), or C9), the tumor cell can be a human colon cancer cell. In any of the above-mentioned C10), the "inhibiting the formation of blood vessels" is inhibiting the formation of blood vessels by vascular endothelial cells. In any of the above-mentioned C11), C12), or C13), the tumor can be a tumor formed by human colon cancer cells in a mouse. In any of the above-mentioned C14) or C15), the tumor-bearing animal can be a mouse bearing a tumor formed by human colon cancer cells in a mouse. The vascular endothelial cell can be specifically a human umbilical vein endothelial cell.

[0038] In any of the above-mentioned A1), the "binding to p53 protein" can be binding to p53 protein in a cell; the cell can be a human colon cancer cell.

[0039] In any of the above-mentioned A1), the growth condition of the human colon cancer cell can be a DNA damage stress condition.

[0040] In any of the above-mentioned human colon cancer cell, the human colon cancer cell can be p53 + / + HCT116 cell.

[0041] In any of the above-mentioned ZNF8 protein, the ZNF8 protein can be a1) or a2) or a3) or a4):

[0042] a1) the protein having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0043] a2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of a1);

[0044] a3) a protein having the same function as a1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0045] a4) a protein having 80% or more identity to the amino acid sequence defined in SEQ ID NO: 1 in the sequence listing, derived from human and having the same function.

[0046] In any of the above-mentioned p53 protein, the p53 protein can be b1) or b2) or b3) or b4):

[0047] b1) the protein having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing;

[0048] b2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of a1);

[0049] b3) a protein having the same function as b1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing;

[0050] b4) a protein having 80% or more identity to the amino acid sequence defined in SEQ ID NO: 3 in the sequence listing, derived from human and having the same function.

[0051] wherein SEQ ID NO: 1 in the sequence listing consists of 575 amino acid residues and SEQ ID NO: 3 in the sequence listing consists of 393 amino acid residues.

[0052] In order to facilitate purification of the protein in a1), a tag shown in Table 1 can be linked to the amino terminal end or carboxyl terminal end of the protein shown in SEQ ID NO: 1 in the sequence listing. In order to facilitate purification of the protein in b1), a tag shown in Table 1 can be linked to the amino terminal end or carboxyl terminal end of the protein shown in SEQ ID NO: 3 in the sequence listing.

[0053] Table 1. Sequence of tag

[0054] Tag Residue Sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0055] The protein in a3) or b3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0056] The protein in a3) or b3) above can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0057] The gene encoding the protein in a3) above can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2 in the sequence listing, and / or performing a missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 at the 5' end and / or 3' end thereof.

[0058] The gene encoding the protein in b3) above can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 4 in the sequence listing, and / or performing a missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 at the 5' end and / or 3' end thereof.

[0059] In a4), the term "identity" refers to sequence similarity with the natural amino acid sequence. The "identity" includes an amino acid sequence having 80%, or 85% or more, or 90% or more, or 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing of the present application.

[0060] In b4), the term "identity" refers to sequence similarity with the natural amino acid sequence. The "identity" includes an amino acid sequence having 80%, or 85% or more, or 90% or more, or 95% or more identity with the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing of the present application.

[0061] Experiments have proved that the ZNF8 protein can bind to the p53 protein; inhibiting the expression of the ZNF8 protein can increase the transcriptional activity of the p53 protein, down-regulate the expression amount of the VEGF gene and the ANGPLT4 gene, inhibit the proliferation of tumor cells, inhibit the migration of tumor cells, inhibit the metastasis of tumor cells, inhibit the invasion and metastasis of tumor cells, inhibit the formation of blood vessels, inhibit the growth of tumors, inhibit the metastasis of tumors, inhibit the lung metastasis of tumors, and improve the survival rate of tumor-bearing animals, and therefore, inhibiting the expression of the ZNF8 protein has important application value in inhibiting the growth and invasion and metastasis of tumors; inhibiting the expression of the ZNF8 protein can prevent and / or treat cancer. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1Results for Example 1.

[0063] Figure 2 Results for Experiment 1 of Example 2.

[0064] Figure 3 Results for Experiment 2 of Example 2.

[0065] Figure 4 Results for Experiment 2 of Example 2.

[0066] Figure 5 Results for Experiment 1 of Example 3.

[0067] Figure 6 Results for Experiment 2 of Example 3.

[0068] Figure 7 Results for Experiment 1 of Example 4.

[0069] Figure 8 Results for Experiment 2 of Example 4.

[0070] Figure 9 Results for Experiment 3 of Example 4.

[0071] Figure 10 Results for Experiment 4 of Example 4.

[0072] Figure 11 Results for Example 5.

[0073] Figure 12 Results for Experiment 1 of Example 6.

[0074] Figure 13 Results for Experiment 2 of Example 6.

[0075] Figure 14 Results for Experiment 3 of Example 6. DETAILED DESCRIPTION

[0076] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the experimental materials are commercially available from a conventional biochemical reagent store unless otherwise specified. In the following examples, the quantitative experiments are set up in triplicate, and the results are averaged.

[0077] The amino acid sequence of ZNF8 protein is shown as SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence of the encoding gene (hereinafter referred to as ZNF8 gene) is shown as SEQ ID NO: 2 in the sequence listing. The amino acid sequence of p53 protein is shown as SEQ ID NO: 3 in the sequence listing, and the nucleotide sequence of the encoding gene (hereinafter referred to as p53 gene) is shown as SEQ ID NO: 4 in the sequence listing.

[0078] pG13L plasmid (also called pG13-Luc plasmid) is described in Chunyan Tian, et al. KRAB-type zinc-finger protein Apak specifically regulates p53-dependent apoptosis. Nat Cell Biol. 2009 May; 11(5): 580-91. This plasmid is a reporter plasmid, and 13 p53 binding elements are constructed in tandem upstream of its promoter region. If p53 protein exists, it binds to the p53 binding elements, causing the plasmid to express, and then emit fluorescence. The activity of p53 protein can be determined according to the fluorescence intensity.

[0079] pCMV-Myc plasmid was a product of Clontech, product catalog number PT3282-5. pCMV-Flag plasmid was a product of Sigma, product catalog number E3762. pRL-TK plasmid was a product of Promega, product catalog number E2241. Myc antibody was a product of Medical Biological Baboratoris, product catalog number M047-3. p53 antibody was a product of Calbiochem, product catalog number OP43L. GAPDH antibody was a product of proteintach, product catalog number 60004-1. Etoposide and cisplatin were products of Sigma, product catalog numbers E1383 and 106M4763V. ZNF8 antibody was a product of abcam, product catalog number ab87732. Dual luciferase reporter assay reagent was a product of Promega, product catalog number E1980. pLVshRNA-Puro plasmid was a product of Beijing Yingmaoshengye Biotechnology Co., Ltd., product catalog number VL3102. Protein-A / G Plus agrose was a product of Santa Cruz, product catalog number sc-2003. Mouse IgG antibody was a product of Santa Cruz, product catalog number sc-2025. Rabbit IgG antibody was a product of Santa Cruz, product catalog number sc-2027. Angiogenesis kit was a product of Cell Biolabs, product catalog number CBA-200. psPAX2 plasmid and pVSV-G plasmid were products of Addgene, product catalog numbers zt161 and zt160, respectively. Crystal violet was a product of Beijing Chemical Reagent Co., Ltd. Migration chamber was a product of Corning, product catalog number 353097. Invasion chamber was a product of Corning, product catalog number 354480. Myc-HRP antibody was a product of MBL, product catalog number 60004-1. Flag-HRP antibody was a product of Sigma, product catalog number SL12445. Human umbilical vein endothelial cells were a product of Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, product catalog number 3111C000CCC000437.

[0080] HCT116 cells with wild-type p53 (also referred to as p53 + / + HCT116 cells or p53 + / + HCT116 cells) and p53 null HCT116 cells (also referred to as p53 - / - HCT116 cells or p53 - / -HCT116 cells) are described in the following reference: Chunyan Tian, et al. KRAB-type zinc-finger protein Apak specifically regulates p53-dependent apoptosis. Nat Cell Biol. 2009 May; 11 (5): 580-91.

[0081] The nude mice used in the following examples are products of Beijing Vital River Laboratory Animal Technology Co., Ltd., and the production license is: SCXK (Jing) 2012-0001.

[0082] The transfection reagent used for transfection of plasmids in the following examples is TurboFect Transfection Reagent, and the specific steps refer to the instructions of TurboFect Transfection Reagent. TurboFect Transfection Reagent is a product of Thermo Scientific. The transfection reagent used for transfection of siRNA is Lipofectamine RNAi MAX Reagent, and the specific steps refer to the instructions of Lipofectamine RNAi MAX Reagent. Lipofectamine RNAi MAX Reagent is a product of invitrogen.

[0083] Blocking solution: 5 mg of skimmed milk powder was dissolved in 100 mL of TBST solution to obtain. TBST solution is a pH 7.5, 20 mM Tris-HCl buffer solution containing 140 mM NaCl and 0.1% (v / v) Tween-20.

[0084] 2x loading buffer: pH 8.0, 20 mM Tris-HCl buffer solution containing 200 mM DTT, 2% (2 g / 100 mL) SDS, 20% (v / v) glycerol and 0.016% (0.016 g / 100 mL) bromophenol blue.

[0085] The genebank number of VEGF gene is 7422. The genebank number of ANGPTL4 gene is 51129.

[0086] Example 1, construction of plasmid, preparation of oligonucleic acid and obtaining of cell

[0087] 1. Construction of Myc-ZNF8 plasmid

[0088] The small fragment between the recognition sequences of restriction enzymes EcoRI and Xho I of pCMV-Myc plasmid is replaced by the DNA molecule shown in sequence 2 in the sequence listing, and the obtained recombination plasmid is Myc-ZNF8 plasmid.

[0089] The ZNF8 protein shown in sequence 1 in the sequence listing of Myc-ZNF8 plasmid expression sequence is expressed.

[0090] 2. Construction of Flag-p53 plasmid

[0091] The small fragment between the recognition sequences of restriction enzymes EcoRI and BamHI of pCMV-Flag plasmid is replaced by the DNA molecule shown in sequence 4 in the sequence listing, and the obtained recombination plasmid is Flag-p53 plasmid.

[0092] The p53 protein shown in sequence 3 in the sequence listing of Flag-p53 plasmid expression sequence is expressed.

[0093] 3. Preparation of oligonucleic acid

[0094] The sense strand and the antisense strand shown in Table 2 are artificially synthesized. The sense strand is diluted with deionized water to obtain a sense strand diluent. The antisense strand is diluted with deionized water to obtain an antisense strand diluent. The sense strand diluent and the corresponding antisense strand diluent are taken to perform annealing reaction to form oligonucleic acid.

[0095] Three oligonucleic acids shown in Table 2 are prepared in this step. A, G, C and U in each oligonucleic acid represent adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide in turn, and T represents thymine deoxyribonucleotide.

[0096] The nucleotide sequence of siNC is a random sequence (as a negative control). The nucleotide sequences of siZNF8-1 and siZNF8-2 are designed according to the nucleotide sequence shown in sequence 2 in the sequence listing.

[0097] Table 2

[0098]

[0099]

[0100] 4. p53 - / - HCT116shZNF8-1 cells, p53 - / - HCT116shZNF8-2 cells, p53 + / + HCT116shZNF8-1 cells and p53 + / + Obtaining of HCT116shZNF8-2 cells

[0101] (1) Construction of shZNF8-1 plasmid, shZNF8-2 plasmid and shNC plasmid

[0102] The siZNF8-1 was taken as a target, the pLVshRNA-Puro plasmid was taken as a carrier, and the shRNA lentivirus expression plasmid of ZNF8 was constructed by Beijing Yingmaoshengye Biotechnology Co., Ltd., and was named as shZNF8-1 plasmid.

[0103] The siZNF8-2 was taken as a target, the pLVshRNA-Puro plasmid was taken as a carrier, and the shRNA lentivirus expression plasmid of ZNF8 was constructed by Beijing Yingmaoshengye Biotechnology Co., Ltd., and was named as shZNF8-2 plasmid.

[0104] The siNC was taken as a target, the pLVshRNA-Puro plasmid was taken as a carrier, and the negative control shRNA expression plasmid was constructed by Beijing Yingmaoshengye Biotechnology Co., Ltd., and was named as shNC plasmid.

[0105] (2) After step (1) is completed, 293T cells are inoculated in a culture flask containing 5 mL of DMEM medium (the specification of the culture flask is 25 cm 2 ; 5.0 x 10 5 cells per culture flask), and then is placed in a 37℃, 5% CO2 incubator for culture for 18 h (at this time, the fusion rate reaches 70-90%), and then is treated as follows:

[0106] The first bottle: 6 μg of shNC plasmid, 0.6 μg of pVSV-G plasmid and 5.4 μg of psPAX2 plasmid are added to each bottle, and are co-transfected for 24 h;

[0107] The second bottle: 6 μg of shZNF8-1 plasmid, 0.6 μg of pVSV-G plasmid and 5.4 μg of psPAX2 plasmid are added to each bottle, and are co-transfected for 24 h;

[0108] The third bottle: 6 μg of shZNF8-2 plasmid, 0.6 μg of pVSV-G plasmid and 5.4 μg of psPAX2 plasmid are added to each bottle, and are co-transfected for 24 h.

[0109] (3) After step (2) is completed, each bottle is replaced with fresh 5 mL DMEM medium, and is placed in a 37°C, 5% CO2 incubator for continued culture for 24 h; then 5 mL DMEM medium is added to each bottle, and is placed in a 37°C, 5% CO2 incubator for continued culture for 24 h, to obtain a culture solution; the culture solution is transferred to a centrifuge tube (specification 15 mL) respectively, and is centrifuged at 1000 rpm for 5 min, to collect the supernatant and filter (filter membrane with a diameter of 0.22 μm), to collect the filtrate. The filtrate collected from the first bottle is the control virus solution, the filtrate collected from the second bottle is the shZNF8-1 virus solution, and the filtrate collected from the third bottle is the shZNF8-2 virus solution.

[0110] (4) After step (3) is completed, p53 + / + HCT116 cells or p53 - / - HCT116 cells are inoculated in 3 holes of a 6-hole plate containing 1 mL DMEM medium (1.0 x 10 5 cells per hole), and are then placed in a 37°C, 5% CO2 incubator for culture. When the confluence rate reaches 70-90%, the following treatment is performed:

[0111] Hole 1: 5 mL of the control virus solution is added to each hole, and is placed in a 37°C, 5% CO2 incubator for culture for 24 h; the liquid phase is discarded, and 2 mL of DMEM medium is added for continued culture for 72 h;

[0112] Hole 2: 5 mL of the shZNF8-1 virus solution is added to each hole, and is placed in a 37°C, 5% CO2 incubator for culture for 24 h; the liquid phase is discarded, and 2 mL of DMEM medium is added for continued culture for 72 h;

[0113] Hole 3: 5 mL of the shZNF8-2 virus solution is added to each hole, and is placed in a 37°C, 5% CO2 incubator for culture for 24 h; the liquid phase is discarded, and 2 mL of DMEM medium is added for continued culture for 72 h.

[0114] (5) After step (4) is completed, the liquid phase is discarded, 4 ng / mL Puro-containing DMEM medium is added to each hole, and the cell line with the strongest luminescence is screened out by fluorescence microscopy.

[0115] (6) The cell line with the strongest luminescence screened out in step (5) is taken respectively, and the single cell strain with stronger luminescence is screened out by a flow cytometry sorting instrument.

[0116] The total protein of the single cell strain with stronger luminescence is extracted, and Western Blot is performed by taking ZNF8 antibody, p53 antibody or GAPDH antibody as a primary antibody (GAPDH protein is taken as an internal reference). The experimental results are shown in Figure 1 (ZNF8 is ZNF8 antibody, p53 is p53 antibody, and GAPDH is GAPDH antibody).

[0117] In p53 + / + HCT116 cells, the cells stably integrated with shNC plasmid were named as p53 + / + EV cells of HCT116 (hereinafter named as p53 + / + HCT116 EV cells), and as a control, the cells stably integrated with shZNF8-1 plasmid in which the expression level of ZNF8 protein was obviously reduced (hereinafter named as p53 + / + HCT116 shZNF8-1 cells) were selected, and the cells stably integrated with shZNF8-2 plasmid in which the expression level of ZNF8 protein was obviously reduced (hereinafter named as p53 + / + HCT116 shZNF8-2 cells) were selected.

[0118] In p53 - / - HCT116 cells, the cells stably integrated with shNC plasmid were named as p53 - / - EV cells of HCT116 (hereinafter named as p53 - / - HCT116 EV cells), and as a control, the cells stably integrated with shZNF8-1 plasmid in which the expression level of ZNF8 protein was obviously reduced (hereinafter named as p53 - / - HCT116 shZNF8-1 cells) were selected, and the cells stably integrated with shZNF8-2 plasmid in which the expression level of ZNF8 protein was obviously reduced (hereinafter named as p53 - / - HCT116 shZNF8-2 cells) were selected.

[0119] Example 2, Inhibition of the expression of ZNF8 protein promotes the activity of p53 protein

[0120] Experiment 1, Inhibition of the expression of ZNF8 protein promotes the transcriptional activity of endogenous p53 protein

[0121] The experiment was repeated three times and the average value was taken, and the steps of each repetition were as follows:

[0122] 1. The cells (p53 + / + HCT116 cells or p53 - / - HCT116 cells) were placed in 6 wells of a 24-well plate containing 0.5 mL of DMEM medium (8.0 x 10 4 cells per well), and then placed in a 37°C, 5% CO2 incubator for culture. When the confluence rate reached 70-90%, they were randomly divided into two groups, each group was set up in triplicate, and the following treatments were performed:

[0123] The first group: each well added 20 ng pG13L plasmid, 0.2 ng pRL-TK plasmid and 0.4 μg shNC plasmid, co-transfected for 36 h.

[0124] The second group: each well added 20 ng pG13L plasmid, 0.2 ng pRL-TK plasmid and 0.4 μg shZNF8-1 plasmid, co-transfected for 36 h.

[0125] 2、After completing step 1, the fluorescence intensity was detected by using the dual luciferase reporter gene kit, then the average value of each group was obtained respectively, and the fluorescence intensity of each group was obtained.

[0126] p53 - / - The fluorescence intensity of the first group of HCT116 cells was taken as 1, and the relative fluorescence intensity of other groups was calculated.

[0127] 3、After completing step 1, the total protein of each cell was extracted, and ZNF8 antibody, p53 antibody or GAPDH antibody was used as the primary antibody for Western Blot (GAPDH protein was used as the internal reference).

[0128] The experimental results are shown in Figure 2 (ZNF8 is ZNF8 antibody, p53 is p53 antibody, and GAPDH is GAPDH antibody). The results show that in p53 - / - In HCT116 cells, the expression of shZNF8-1 plasmid reduces the expression level of ZNF8 protein, but the transcriptional activity of endogenous p53 protein has no obvious change; in p53 + / + In HCT116 cells, the expression of shZNF8-1 plasmid reduces the expression level of ZNF8 protein, and promotes the transcriptional activity of endogenous p53 protein.

[0129] Experiment two, inhibiting the expression of ZNF8 protein, depending on p53 protein to down-regulate the expression of VEGF gene and ANGPTL4 gene

[0130] The experiment was repeated three times to take the average value, and each repetition was as follows:

[0131] 1、The cells (p53 + / + HCT116 cells or p53 - / - HCT116 cells) were placed in 9 wells of a 12-well plate containing 2 mL of DMEM medium (2.0 x 10 5 cells per well), and then placed in a 37℃, 5% CO2 incubator for culture. When the fusion rate reached 70-90%, they were randomly divided into three groups, each group set up three duplicate wells, and the following treatments were performed:

[0132] The first group: 0.5 μg shNC plasmid was added to each well, and co-transfected for 48 h.

[0133] The second group: 0.5 μg shZNF8-1 plasmid was added to each well, and co-transfection was performed for 48 h;

[0134] The third group: 0.5 μg shZNF8-2 plasmid was added to each well, and co-transfection was performed for 48 h.

[0135] 2. After step 1, total RNA of each cell was extracted, and then first strand cDNA was reverse transcribed by using reverse transcriptase to obtain cDNA of each cell. Then, relative expression amount of VEGF gene and ANGPTL4 gene was analyzed by using real-time fluorescence quantitative analysis (internal reference is GAPDH gene).

[0136] Primer for detecting VEGF gene: 5'-GGGCAGAATCATCACGAAGT-3' and 5'-TGGTGATGTTGGACTCCTCA-3'. Primer for detecting ANGPTL4 gene: 5'-GGACACGGCCTATAGCCTG-3' and 5'-CTCTTGGCGCAGTTCTTGTC-3'. Primer for detecting GAPDH gene: 5'-GGGAAGGTGAAGGTCGGAGT-3' and 5'-TTGAGGTCAATGAAGGGGTCA-3'.

[0137] p53 - / - Relative expression amount of VEGF gene in the first group of HCT116 cells was taken as 1, and relative expression amount of VEGF gene in other groups was calculated. Experimental results are shown in Table 1. Figure 3 (NC is shNC plasmid). The results show that, in p53 - / - In HCT116 cells, expression of shZNF8-1 plasmid or shZNF8-2 plasmid has no obvious influence on expression amount of VEGF gene; in p53 + / + In HCT116 cells, expression of shZNF8-1 plasmid or shZNF8-2 plasmid can significantly down-regulate expression amount of VEGF gene.

[0138] p53 - / - Relative expression amount of ANGPTL4 gene in the first group of HCT116 cells was taken as 1, and relative expression amount of ANGPTL4 gene in other groups was calculated. Experimental results are shown in Table 2. Figure 4 (NC is shNC plasmid). The results show that, in p53 - / - In HCT116 cells, expression of shZNF8-1 plasmid or shZNF8-2 plasmid has no obvious influence on expression amount of ANGPTL4 gene; in p53 + / +The expression of shZNF8-1 plasmid or shZNF8-2 plasmid in HCT116 cells can significantly down-regulate the expression of ANGPTL4 gene.

[0139] Example 3: ZNF8 protein and p53 protein interact with each other under normal conditions and DNA damage conditions

[0140] The interaction between ZNF8 protein and p53 protein under normal conditions and DNA damage conditions was detected by immunoprecipitation experiment.

[0141] Experiment 1: Endogenous ZNF8 protein can interact with p53 protein under normal conditions

[0142] The experiment was repeated three times, and the steps of each repetition were as follows:

[0143] 1. p53 + / + HCT116 cells were inoculated in a culture flask containing 30 mL of DMEM medium (the size of the culture flask was 25 cm 2 ; 3.0 x 10 6 cells per culture flask), and cultured in a 37°C, 5% CO2 incubator for 48 h (at this time, the confluence rate reached 100%).

[0144] 2. After completing step 1, add trypsin digestion solution to the culture flask (the purpose is to digest the cells), then transfer to a centrifuge tube (size 50 mL), centrifuge at 1000 rpm for 5 min, and collect the precipitate.

[0145] 3. Take the precipitate collected in step 2, wash it twice. Each washing step is as follows: add 1 mL of pH 7.4, 0.01 M PBS buffer, centrifuge at 1000 rpm for 5 min. The last time of washing needs to dry the pH 7.4, 0.01 M PBS buffer.

[0146] 4. Take the precipitate collected in step 3, add 2000 μL of pre-cooled NETN lysis solution, mix well, and incubate on ice for 30 min; then centrifuge at 4°C, 12000 rpm for 10 min, and collect the supernatant.

[0147] NETN lysis solution: add 0.02 mL of 50x protease inhibitor to 1 mL of pH 8.0, 20 mM Tris-Cl buffer containing 150 mM NaCl, 1 mM EDTA and 0.5% (m / m) NP-40, and mix well. 50x protease inhibitor is a water solution containing 1 mM DTT, 1 mM NaV3O4 and 1 mM NaF.

[0148] 5. Take 40 μL of the supernatant collected in step 4, add 40 μL of 2× loading buffer, mix well, and boil at 100°C for 10 minutes. The resulting solution is lysate.

[0149] 6. Take the supernatant collected in step 4, divide it into four equal parts and transfer them to centrifuge tubes (specification: 1.5 mL). Add antibody (1 μL of 1 mg / mL Mouse IgG antibody, 2 μL of 0.5 mg / mL p53 antibody, 1 μL of 1 mg / mL Rabbit IgG antibody, or 5 μL of 0.2 mg / mL ZNF8 antibody) to each tube and shake slowly at 4°C for 3 h. Then, add 40 μL of proein-A / G Plus agrose to each tube, place on a rotating mixer, and incubate at 4°C overnight.

[0150] 7. After completing step 6, collect the precipitates (i.e., agrose and antigen-antibody complexes) separately, then place on ice for 1 minute (to allow the agarose beads to settle), centrifuge at 4°C, 3000 rpm for 5 minutes, and collect the precipitates.

[0151] 8. Take the precipitate collected in step 7 and wash it four times. Each wash step is to add 1 mL of pre-cooled NETN lysis buffer and centrifuge at 3000 rpm at 4°C for 2 minutes.

[0152] 9. Take the precipitates collected in step 8, add 40 μL of NETN lysis buffer to resuspend, then add 40 μL of 2× loading buffer, mix well, and boil at 100°C for 10 minutes. The resulting solution is the IP sample.

[0153] Lysate and IP samples were subjected to Western blotting using p53 antibody diluent (obtained by diluting p53 antibody to 500-fold volume with blocking solution) or ZNF8 antibody diluent (obtained by diluting ZNF8 antibody to 400-fold volume with blocking solution) as the primary antibody.

[0154] The experimental results are shown in Figure 5 (WCL is whole cell lysate, Mouse IgG is Mouse IgG antibody, p53 is p53 antibody, Rabbit IgG is Rabbit IgG antibody, and ZNF8 is ZNF8 antibody.) The results showed that endogenous ZNF8 protein interacts with p53 protein.

[0155] Experiment 2: ZNF8 and p53 proteins interact under conditions of DNA damage

[0156] The experiment was repeated three times, and the steps for each repetition were as follows:

[0157] 1. p53 - / -HCT116 cells were inoculated in culture flasks (25 cm 2 in size) containing 5 mL of DMEM medium (5.0 x 10 5 cells per culture flask) and incubated in a 37°C, 5% CO2 incubator for 18 h (at which time the fusion rate reached 70-90%) and then treated as follows:

[0158] The first culture flask was co-transfected with 3.0 μg of pCMV-Myc plasmid and 3.0 μg of Flag-p53 plasmid for 48 h; then the liquid was discarded and the precipitate was washed three times with pre-cooled PBS buffer at pH 7.4, 0.01 M (the last time of washing required that the PBS buffer at pH 7.4, 0.01 M be aspirated) ;

[0159] The second culture flask was co-transfected with 3.0 μg of Myc-ZNF8 plasmid and 3.0 μg of Flag-p53 plasmid for 48 h; then the liquid was discarded and the precipitate was washed three times with pre-cooled PBS buffer at pH 7.4, 0.01 M (the last time of washing required that the PBS buffer at pH 7.4, 0.01 M be aspirated) ;

[0160] The third culture flask was co-transfected with 3.0 μg of pCMV-Myc plasmid and 3.0 μg of Flag-p53 plasmid for 36 h; then etoposide was added to obtain a treatment system (in the treatment system, the concentration of etoposide was 2 x 10 -5 mol / L) ; the treatment system was incubated in a 37°C, 5% CO2 incubator for 12 h, then the liquid was discarded and the precipitate was washed three times with pre-cooled PBS buffer at pH 7.4, 0.01 M (the last time of washing required that the PBS buffer at pH 7.4, 0.01 M be aspirated) ;

[0161] The fourth culture flask was co-transfected with 3.0 μg of Myc-ZNF8 plasmid and 3.0 μg of Flag-p53 plasmid for 36 h; then etoposide was added to obtain a treatment system (in the treatment system, the concentration of etoposide was 2 x 10 -5 mol / L) ; the treatment system was incubated in a 37°C, 5% CO2 incubator for 12 h, then the liquid was discarded and the precipitate was washed three times with pre-cooled PBS buffer at pH 7.4, 0.01 M (the last time of washing required that the PBS buffer at pH 7.4, 0.01 M be aspirated) ;

[0162] The fifth culture flask was co-transfected with 3.0 μg of pCMV-Myc plasmid and 3.0 μg of Flag-p53 plasmid for 36 h; then cisplatin was added to obtain a treatment system (in the treatment system, the concentration of cisplatin was 1 x 10-4 mol / L); the treatment system was placed in a 37°C, 5% CO2 incubator for 12 h, and then the liquid phase was discarded and the precipitate was washed with pre-cooled PBS buffer solution with pH 7.4 and 0.01 M for 3 times (in the last washing, the PBS buffer solution with pH 7.4 and 0.01 M was absorbed dry).

[0163] The 6th culture bottle: 3.0 μg of Myc-ZNF8 plasmid and 3.0 μg of Flag-p53 plasmid were added, and co-transfection was performed for 36 h; then cisplatin was added to obtain a treatment system (in the treatment system, the concentration of cisplatin was 1 x 10 -4 mol / L); the treatment system was placed in a 37°C, 5% CO2 incubator for 12 h, and then the liquid phase was discarded and the precipitate was washed with pre-cooled PBS buffer solution with pH 7.4 and 0.01 M for 3 times (in the last washing, the PBS buffer solution with pH 7.4 and 0.01 M was absorbed dry).

[0164] 2. After step 1 was completed, trypsin digestion solution was added to each culture bottle (for the purpose of cell digestion), and then was transferred to a centrifuge tube (10 mL) respectively, and was centrifuged at 1000 rpm for 5 min, and the precipitate was collected respectively.

[0165] 3. The precipitate collected in step 2 was taken respectively, and was washed for 2 times. Each time of washing was as follows: 1 mL of PBS buffer solution with pH 7.4 and 0.01 M was added, and was centrifuged at 1000 rpm for 5 min. In the last washing, the PBS buffer solution with pH 7.4 and 0.01 M was absorbed dry.

[0166] 4. The precipitate collected in step 3 was taken respectively, and 600 μL of pre-cooled NETN lysis solution was added, and was mixed uniformly, and was incubated on ice for 30 min; then was centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant was collected.

[0167] 5. 40 μL of the supernatant collected in step 4 was taken respectively, and 40 μL of 2x loading buffer was added, and was mixed, and was boiled at 100°C for 10 min, and the obtained solution was Lysate.

[0168] 6. The supernatant collected in step 4 was transferred to a centrifuge tube (1.5 mL) respectively, 1 μL of myc antibody was added to each tube, and was slowly shaken at 4°C for 3 h; then 40 μL of proein-A / G Plus agrose was added to each tube, and was placed on a rotary mixer, and was incubated at 4°C overnight.

[0169] 7. After step 6 was completed, the precipitate (i.e. the agrose and antigen-antibody complex) was collected respectively, and was placed on ice for 1 min (for the purpose of agarose bead sedimentation), and was centrifuged at 4°C, 3000 rpm for 5 min, and the precipitate was collected.

[0170] 8. Take the precipitate collected in step 7 and wash it four times. Each wash step is to add 1 mL of pre-cooled NETN lysis buffer and centrifuge at 3000 rpm at 4°C for 2 minutes.

[0171] 9. Take the precipitates collected in step 8, add 40 μL NETN lysis buffer to resuspend, then add 40 μL 2× loading buffer, mix well, and boil at 100°C for 10 minutes. The resulting solution is Myc-IP.

[0172] Lysate and Myc-IP were analyzed by Western blotting using Flag-HRP antibody diluent (obtained by diluting Flag-HRP antibody to 1000-fold volume with blocking solution) or Myc-HRP antibody diluent (obtained by diluting Myc-HRP antibody to 1000-fold volume with blocking solution) as the primary antibody.

[0173] The experimental results are shown in Figure 6 (Untreated refers to normal conditions; etoposide refers to treatment with etoposide; cisplatin refers to treatment with cisplatin; Flag refers to the Flag-HRP antibody; Myc refers to the Myc-HRP antibody.) The results showed that under conditions of DNA damage stress, ZNF8 and p53 proteins interact with each other, indicating that ZNF8 and p53 proteins have a certain binding ability in vivo.

[0174] Example 4: Inhibition of ZNF8 protein expression relies on p53 protein to inhibit tumor cell proliferation, migration, invasion and metastasis

[0175] Experiment 1: Inhibition of ZNF8 protein expression relies on p53 protein to inhibit tumor cell proliferation

[0176] 1. Take a 6-well plate, add 2 mL of DMEM medium to each well, and then inoculate each well with p53 in the logarithmic growth phase. + / + HCT116EV cells, p53 + / + HCT116shZNF8-1 cells, p53 + / + HCT116shZNF8-2 cells, p53 - / - HCT116EV cells, p53 - / - HCT116shZNF8-1 cells and p53 - / - HCT116shZNF8-2 cells (2000 cells per well) were then placed in a 37°C, 5% CO2 incubator and cultured for 10 days.

[0177] 2、After step 1, take the 6-well plate, discard the liquid phase, then add 1 mL of 0.5% (0.5 mg / 100 mL) crystal violet methanol solution to each well, stand for 20 min.

[0178] 3、After step 1, take the 6-well plate, rinse it gently with water, dry it, take a photo and count the number of cell clones formed in each well.

[0179] The experimental results are shown in Figure 7 . The results show that, compared with p53 - / - HCT116EV cells, the p53 - / - HCT116shZNF8-1 cells and p53 - / - HCT116shZNF8-2 cells have no significant difference in colony formation ability; compared with p53 + / + HCT116EV cells, the p53 + / + HCT116shZNF8-1 cells and p53 + / + HCT116shZNF8-2 cells have significantly reduced colony formation ability, that is, the inhibition of ZNF8 protein expression depends on the inhibition of p53 protein to inhibit tumor cell proliferation.

[0180] Experiment two, inhibition of ZNF8 protein expression depends on p53 protein to inhibit tumor cell migration

[0181] 1、Take a 6-well plate, first draw a horizontal line on the back of it with a marker pen, about every 0.5-1 cm, across the hole.

[0182] 2、After step 1, take the 6-well plate, first add 2 mL of DMEM medium to each well, then inoculate p53 + / + HCT116EV cells, p53 + / + HCT116shZNF8-1 cells, p53 + / + HCT116shZNF8-2 cells, p53 - / - HCT116EV cells, p53 - / - HCT116shZNF8-1 cells and p53 - / - HCT116shZNF8-2 cells (6x10 5 cells per well) in sequence, then place it in a 37℃, 5% CO2 incubator for 12 days (at this time the cells cover the plate).

[0183] 3. After completing step 2, scratch the cells in each well with the tip of the gun against a ruler, with the ruler perpendicular to the horizontal line on the back; then wash three times with pH 7.4, 0.01M PBS buffer (to remove scratched cells), and finally add DMEM medium containing 3% (v / v) fetal bovine serum and culture in a 37°C, 5% CO2 incubator; take pictures at 0 h, 24 h, and 48 h after scratching.

[0184] The experimental results are shown in Figure 8 The results showed that p53 - / - Compared with HCT116EV cells, p53 - / - HCT116shZNF8-1 cells and p53 - / - There was no significant difference in the healing ability of HCT116shZNF8-2 cells; + / + Compared with HCT116EV cells, p53 + / + HCT116shZNF8-1 cells and p53 + / + The scratch healing ability of HCT116shZNF8-2 cells was significantly weakened, indicating that the inhibition of ZNF8 protein expression depended on p53 protein to inhibit the migration of tumor cells.

[0185] Experiment 3: Inhibition of ZNF8 protein expression relies on p53 protein to inhibit tumor cell metastasis

[0186] 1. Take the cells to be tested (p53 + / + HCT116EV cells, p53 + / + HCT116shZNF8-1 cells, p53 + / + HCT116shZNF8-2 cells, p53 - / - HCT116EV cells, p53 - / - HCT116shZNF8-1 cells or p53 - / - HCT116shZNF8-2 cells) were starved with DMEM medium containing 3% (v / v) fetal bovine serum for 12-24 h.

[0187] 2. After completing step 1, take the cells to be tested, add trypsin digestion solution for digestion, then add appropriate amount of DMEM medium to terminate digestion, centrifuge at 4°C, 1000 rpm for 5 minutes, and collect the precipitate.

[0188] 3. After completing step 2, take the precipitate, wash it once with pH 7.4, 0.01M PBS buffer, and then add serum-free medium to resuspend it to obtain a concentration of 2×10 5 cells / mL of cell suspension.

[0189] 4. After completing step 3, add 200 μL of cell suspension to the migration chamber; take a 24-well plate and add 600 μL of DMEM culture medium containing 20% ​​(v / v) fetal bovine serum to each of the 6 wells; then place the migration chamber in the above 6 wells in sequence (requirement: no bubbles should be generated between the DMEM culture medium containing 20% ​​(v / v) fetal bovine serum and the migration chamber), and culture in a 37°C, 5% CO2 incubator for 25 h.

[0190] 5. After completing step 4, the migration chamber was removed, the liquid phase was discarded, and the non-migrated cells in the chamber were gently wiped off with a cotton swab, and then washed three times with pH 7.4, 0.01M PBS buffer. Finally, each migration chamber was placed in methanol for 15 minutes.

[0191] 6. After completing step 5, take out the migration chamber and air-dry; then add 0.5% (0.5 mg / 100 mL) crystal violet methanol solution to each migration chamber and let it stand for 20 minutes.

[0192] 7. After completing step 6, take the migration chamber, gently rinse the crystal violet with water, dry it, and observe the cells in five random fields under a 400x microscope, and take pictures, count and analyze.

[0193] The experimental results are shown in Figure 9 (The upper picture is the result of microscopy, and the lower picture is the result of counting.) The results show that p53 - / - Compared with HCT116EV cells, p53 - / - HCT116shZNF8-1 cells and p53 - / - There was no significant difference in the metastatic ability of HCT116shZNF8-2 cells; + / + Compared with HCT116EV cells, p53 + / + HCT116shZNF8-1 cells and p53 + / + The metastatic ability of HCT116shZNF8-2 cells was significantly weakened, indicating that the inhibition of ZNF8 protein expression depends on p53 protein to inhibit the metastasis of tumor cells.

[0194] Experiment 4: Inhibition of ZNF8 protein expression relies on p53 protein to inhibit tumor cell invasion and metastasis

[0195] 1. Take the cells to be tested (p53 + / + HCT116EV cells, p53 + / + HCT116shZNF8-1 cells, p53 + / + HCT116shZNF8-2 cells, p53 - / - HCT116EV cells, p53 - / -HCT116shZNF8-1 cells or p53 - / - HCT116shZNF8-2 cells), starved with DMEM medium containing 3% (v / v) fetal bovine serum for 12-24 h.

[0196] 2、After completing step 1, take the cells to be tested, add trypsin digestion solution for digestion, then add an appropriate amount of DMEM medium to terminate digestion, centrifuge at 4°C and 1000 rpm for 5 min, and collect the precipitate.

[0197] 3、After completing step 2, take the precipitate, first wash it once with PBS buffer at pH 7.4 and 0.01 M, then resuspend it in serum-free medium to obtain a cell suspension with a concentration of 2 x 10 5 individuals / mL.

[0198] 4、After completing step 3, add 200 μL of the cell suspension to the invasion chamber; take a 24-well plate and add 600 μL of DMEM medium containing 20% (v / v) fetal bovine serum to each of the 6 wells; then place the invasion chamber in the above 6 wells in turn (requirement: no air bubbles can be generated between the DMEM medium containing 20% (v / v) fetal bovine serum and the invasion chamber), and incubate in a 37°C, 5% CO2 incubator for 36 h.

[0199] 5、After completing step 4, remove the invasion chamber, discard the liquid, first gently wipe off the cells that have not migrated in the chamber with a cotton swab, then wash it 3 times with PBS buffer at pH 7.4 and 0.01 M, and finally place each invasion chamber in methanol for fixation for 15 min.

[0200] 6、After completing step 5, remove the invasion chamber, air dry it; then add 0.5% (0.5 mg / 100 mL) crystal violet methanol solution to each invasion chamber, and let it stand for 20 min.

[0201] 7、After completing step 6, take the invasion chamber, wash the crystal violet off with water, air dry it, and observe the cells under a 400x microscope in 5 random fields, take a photo, and count the cells.

[0202] The experimental results are shown in Figure 10 (the upper panel is the microscope photograph, and the lower panel is the counting result). The results show that, compared with p53 - / - HCT116EV cells, p53 - / - HCT116shZNF8-1 cells and p53 - / - HCT116shZNF8-2 cells had no significant difference in invasion ability; compared with p53 + / + HCT116EV cells, p53 + / + HCT116shZNF8-1 cells and p53+ / + The invasion ability of HCT116shZNF8-2 cells was significantly weakened, that is, the inhibition of ZNF8 protein expression depended on p53 protein to inhibit the invasion and metastasis of tumor cells.

[0203] Example 5, Inhibition of ZNF8 protein expression depends on p53 protein to inhibit the formation of blood vessels

[0204] 1. Take 6-hole plates, add 2 mL of DMEM medium to each hole, and inoculate the cells to be tested (p53 + / + HCT116EV cells, p53 + / + HCT116shZNF8-1 cells, p53 + / + HCT116shZNF8-2 cells, p53 - / - HCT116EV cells, p53 - / - HCT116shZNF8-1 cells or p53 - / - HCT116shZNF8-2 cells) in the logarithmic growth phase, and then place them in a 37°C, 5% CO2 incubator for 20h to obtain the culture solution.

[0205] 2. After step 1 is completed, transfer the culture solution to a centrifuge tube, centrifuge at 1000 rpm for 5 min, collect the supernatant and filter (filter membrane with a diameter of 0.22 μm), and collect the filtrate.

[0206] 3. Add the ECM matrix gel in a melted state in the blood vessel formation kit to the pre-cooled 48-hole plate with a pre-cooled gun head, 100 μL of ECM matrix gel per hole (ECM matrix gel is very easy to solidify at room temperature, and the whole process needs to be operated on ice).

[0207] 4. After step 3 is completed, place the 48-hole plate in a 37°C, 5% CO2 incubator for 1h (the purpose is to solidify the ECM matrix gel in a melted state).

[0208] 5. After step 4 is completed, take the 48-hole plate, add 300 μL of the filtrate collected in step 2 and human umbilical vein endothelial cells (5×10 4 per hole) to each hole, and place it in a 37°C, 5% CO2 incubator for 2h.

[0209] 6. After step 5 is completed, take the 48-hole plate, take a photo under a 400x microscope and count the number of human umbilical vein endothelial cells forming blood vessels in each hole.

[0210] The experimental results are shown in Figure 11 (left photo is the result of microscope photography, right photo is the statistical result). The results show that, compared with p53 - / - HCT116EV cells, p53 - / -HCT116shZNF8-1 cells or p53 - / - There was no significant difference in the ability of the culture medium of HCT116shZNF8-2 cells to promote new blood vessel formation; compared with p53 + / + HCT116EV cells, the culture of p53 + / + HCT116shZNF8-1 cells or p53 + / + The ability of the culture medium of HCT116shZNF8-2 cells to promote new blood vessel formation was significantly weakened, that is, the inhibition of the expression of ZNF8 protein depended on the p53 protein to inhibit the formation of blood vessels.

[0211] Example 6, Inhibition of the expression of ZNF8 protein depends on the p53 protein to inhibit the growth and metastasis of tumors

[0212] The cells to be tested were p53 + / + HCT116EV cells, p53 + / + HCT116shZNF8-1 cells, p53 - / - HCT116EV cells or p53 - / - HCT116shZNF8-1 cells.

[0213] Experiment 1, Inhibition of the expression of ZNF8 protein depends on the p53 protein to inhibit the growth of tumors

[0214] 1. Take the cells to be tested in the logarithmic growth phase, add trypsin digestion solution for digestion, then add an appropriate amount of DMEM medium to terminate digestion, centrifuge at 4°C, 1000 rpm for 5 min, and collect the precipitate.

[0215] 2. After completing step 1, take the precipitate, first wash it once with pH 7.4, 0.01M PBS buffer, then resuspend it with pH 7.4, 0.01M PBS buffer to obtain a cell suspension with a concentration of 3×10 7 cells / mL.

[0216] 3. Take 24 female BALB / c nude mice growing to 5 weeks of age, randomly divide them into two groups, 12 in each group, and then perform the following treatments:

[0217] The first group: subcutaneously inject 100 μL p53 - / - HCT116EV cell suspension (3×10 6 cells) into the skin of the left hind leg of each nude mouse, and subcutaneously inject 100 μL p53 - / - HCT116shZNF8-1 cell suspension (3×10 6 cells) into the skin of the right hind leg of each nude mouse;

[0218] The second group: subcutaneous injection of 100 μL p53 + / + HCT116 EV cell suspension (3 x 10 6 HCT116 EV cell suspension (3 x 10 + / + HCT116 shZNF8-1 cell suspension (3 x 10 6 HCT116 shZNF8-1 cell suspension (3 x 10

[0219] 4. After step 3, the nude mice were normally fed for 24 days, then executed by cervical vertebra dislocation and the tumors formed on the left and right sides were stripped out, fixed in 4% (v / v) paraformaldehyde solution, and the tumor volume was measured, the data was counted and analyzed.

[0220] The experimental results are shown in Figure 12 (left picture is the stripped tumor, right picture is the tumor volume counting result; shNT is EV, shZNF8 is shZNF8-1). The results show that in the nude mice, the tumor volume formed by p53 - / - HCT116 EV cells and p53 - / - HCT116 shZNF8-1 cells has no significant difference; compared with p53 + / + HCT116 EV cells, the tumor volume formed by p53 + / + HCT116 shZNF8-1 cells is significantly smaller.

[0221] Experiment two, inhibition of ZNF8 protein expression depends on p53 protein to inhibit lung metastasis of tumor

[0222] 1. Take the cells to be tested in the logarithmic growth phase, add trypsin digestive juice for digestion, then add appropriate amount of DMEM medium to terminate digestion, centrifuge at 4°C, 1000 rpm for 5 min, and collect the precipitate.

[0223] 2. After step 1, take the precipitate, first wash it once with pH 7.4, 0.01M PBS buffer, then resuspend it with pH 7.4, 0.01M PBS buffer to obtain a cell suspension to be tested with a concentration of 2 x 10 7 cells / mL.

[0224] 3. Take 24 female BALB / c nude mice growing to 5 weeks old, randomly divide them into four groups, 6 in each group, then perform the following treatments:

[0225] The first group: intravenous injection of 100 μL p53 - / - HCT116 EV cell suspension (2 x 10 6 HCT116 EV cell suspension (2 x 10

[0226] The second group: intravenous injection of 100 μL p53- / - HCT116shZNF8-1 cell suspension (2×10 6 cells);

[0227] Group 3: Each nude mouse was injected with 100 μL p53 via tail vein + / + HCT116EV cell suspension (2×10 6 cells);

[0228] Group 4: Each nude mouse was injected with 100 μL p53 via tail vein + / + HCT116shZNF8-1 cell suspension (2×10 6 cells).

[0229] 4. After completing step 3, the mice were raised normally for 90 days, then sacrificed by cervical dislocation and the lungs of each mouse were removed and fixed in 4% (v / v) paraformaldehyde solution. The lungs were observed for tumor growth, and the data were collected and analyzed.

[0230] The results showed that p53 was injected into nude mice - / - HCT116EV cells and p53 - / - HCT116shZNF8-1 cells, the number of visible tumor lesions produced in the lungs ( Figure 13 Middle left image), visible tumor surface area ( Figure 13 Middle Figure) and the number of tumor lesions under the microscope ( Figure 13 There was no significant difference between the two groups (middle right figure); + / + Compared with HCT116EV cells, p53 + / + The number of visible tumor lesions produced by HCT116shZNF8-1 cells in the lungs ( Figure 13 Middle left image), visible tumor surface area ( Figure 13 Middle Figure) and the number of tumor lesions under the microscope ( Figure 13 The expression of ZNF8 protein was significantly reduced (middle right figure), indicating that the inhibition of ZNF8 protein expression depends on p53 protein to inhibit lung metastasis of tumor.

[0231] Experiment 3: Inhibiting ZNF8 protein expression increases the survival rate of tumor-bearing mice in a p53-dependent manner

[0232] 1. Take the cells to be tested in the logarithmic growth phase, add trypsin digestion solution for digestion, then add appropriate amount of DMEM medium to terminate the digestion, centrifuge at 4°C, 1000 rpm for 5 minutes, and collect the precipitate.

[0233] 2. After completing step 1, take the precipitate, wash it once with pH 7.4, 0.01M PBS buffer, and then resuspend it in pH 7.4, 0.01M PBS buffer to obtain a concentration of 2×10 72x105cells / mL of the cells to be tested.

[0234] 3. 24 female BALB / c nude mice growing to 5 weeks of age were randomly divided into four groups, 6 in each group, and then treated as follows:

[0235] The first group: each nude mouse was injected with 100 μL p53 - / - HCT116EV cell suspension (2x10 6 cells) through the tail vein.

[0236] The second group: each nude mouse was injected with 100 μL p53 - / - HCT116shZNF8-1 cell suspension (2x10 6 cells) through the tail vein.

[0237] The third group: each nude mouse was injected with 100 μL p53 + / + HCT116EV cell suspension (2x10 6 cells) through the tail vein.

[0238] The fourth group: each nude mouse was injected with 100 μL p53 + / + HCT116shZNF8-1 cell suspension (2x10 6 cells) through the tail vein.

[0239] 4. After completing step 3, the growth of each nude mouse was closely observed and the death time of each nude mouse was recorded, and the data was counted and analyzed.

[0240] The experimental results are shown in Figure 14 . The results show that there is no significant difference in survival rate between p53 - / - HCT116EV cells and p53 - / - HCT116shZNF8-1 cells injected into nude mice; and compared with p53 + / + HCT116EV cells, the survival rate of p53 + / + HCT116shZNF8-1 cells injected into nude mice is significantly improved. <110> Beijing Proteome Research Center <120> Use of a substance inhibiting the expression amount of ZNF8 protein in the preparation of products for preventing and treating cancer <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 575 <212> PRT <213> Artificial sequence <220> <223> <400> 1 Met Asp Pro Glu Asp Glu Gly Val Ala Gly Val Met Ser Val Gly Pro 1 5 10 15 Pro Ala Ala Arg Leu Gln Glu Pro Val Thr Phe Arg Asp Val Ala Val 20 25 30 Asp Phe Thr Gln Glu Glu Trp Gly Gln Leu Asp Pro Thr Gln Arg Ile 35 40 45 Leu Tyr Arg Asp Val Met Leu Glu Thr Phe Gly His Leu Leu Ser Ile 50 55 60 Gly Pro Glu Leu Pro Lys Pro Glu Val Ile Ser Gln Leu Glu Gln Gly 65 70 75 80 Thr Glu Leu Trp Val Ala Glu Arg Gly Thr Thr Gln Gly Cys His Pro 85 90 95 Ala Trp Glu Pro Arg Ser Glu Ser Gln Ala Ser Arg Lys Glu Glu Gly 100 105 110 Leu Pro Glu Glu Glu Pro Ser His Val Thr Gly Arg Glu Gly Phe Pro 115 120 125 Thr Asp Ala Pro Tyr Pro Thr Thr Leu Gly Lys Asp Arg Glu Cys Gln 130 135 140 Ser Gin Ser Leu Ala Leu Lys Glu Gin Asn Asn Leu Lys Gin Leu Glu 145 150 155 160 Phe Gly Leu Lys Glu Ala Pro Val Gin Asp Gin Gly Tyr Lys Thr Leu 165 170 175 Arg Leu Arg Glu Asn Cys Val Leu Ser Ser Ser Pro Asn Pro Phe Pro 180 185 190 Glu He Ser Arg Gly Glu Tyr Leu Tyr Thr Tyr Asp Ser Gin He Thr 195 200 205 Asp Ser Glu His Asn Ser Ser Leu Val Ser Gin Gin Thr Gly Ser Pro 210 215 220 Gly Lys Gin Pro Gly Glu Asn Ser Asp Cys His Arg Asp Ser Ser Gin 225 230 235 240 Ala He Pro He Thr Glu Leu Thr Lys Ser Gin Val Gin Asp Lys Pro 245 250 255 Tyr Lys Cys Thr Asp Cys Gly Lys Ser Phe Asn His Asn Ala His Leu 260 265 270 Thr Val His Lys Arg He His Thr Gly Glu Arg Pro Tyr Met Cys Lys 275 280 285 Glu Cys Gly Lys Ala Phe Ser Gin Asn Ser Ser Leu Val Gin His Glu 290 295 300 Arg Ile His Thr Gly Asp Lys Pro Tyr Lys Cys Ala Glu Cys Gly Lys 305 310 315 320 Ser Phe Cys His Ser Thr His Leu Thr Val His Arg Arg Ile His Thr 325 330 335 Gly Glu Lys Pro Tyr Glu Cys Gln Asp Cys Gly Arg Ala Phe Asn Gln 340 345 350 Asn Ser Ser Leu Gly Arg His Lys Arg Thr His Thr Gly Glu Lys Pro 355 360 365 Tyr Thr Cys Ser Val Cys Gly Lys Ser Phe Ser Arg Thr Thr Cys Leu 370 375 380 Phe Leu His Leu Arg Thr His Thr Glu Glu Arg Pro Tyr Glu Cys Asn 385 390 395 400 His Cys Gly Lys Gly Phe Arg His Ser Ser Ser Leu Ala Gln His Gln 405 410 415 Arg Lys His Ala Gly Glu Lys Pro Phe Glu Cys Arg Gln Arg Leu Ile 420 425 430 Phe Glu Gln Thr Pro Ala Leu Thr Lys His Glu Trp Thr Glu Ala Leu 435 440 445 Gly Cys Asp Pro Pro Leu Ser Gln Asp Glu Arg Thr His Arg Ser Asp 450 455 460 Arg Pro Phe Lys Cys Asn Gln Cys Gly Lys Cys Phe Ile Gln Ser Ser 465 470 475 480 His Leu Ile Arg His Gln Ile Thr His Thr Arg Glu Glu Gln Pro His 485 490 495 Gly Arg Ser Arg Arg Arg Glu Gln Ser Ser Ser Arg Asn Ser His Leu 500 505 510 Val Gln His Gln His Pro Asn Ser Arg Lys Ser Ser Ala Gly Gly Ala 515 520 525 Lys Ala Gly Gln Pro Glu Ser Arg Ala Leu Ala Leu Phe Asp Ile Gln 530 535 540 Lys Ile Met Gln Glu Lys Asn Pro Val His Val Ile Gly Val Glu Glu 545 550 555 560 Pro Ser Val Gly Ala Ser Met Leu Phe Asp Ile Arg Glu Ser Thr 565 570 575 <210> 2 <211> 1728 <212> DNA <213> 人工序列 <220> <223> <400> 2 atggaccccg aggacgaagg ggtagcggga gtgatgtctg tggggccgcc ggcggcccgg 60 cttcaggaac cagtgacctt ccgggatgtg gctgtggact ttacccagga ggaatggggg 120 cagctggacc ctacccagag gatcctctac cgtgacgtga tgctggagac ctttggtcac 180 ctgctctcca taggtcctga gcttccgaag cctgaagtca tctcccagct ggagcaaggg 240 accgagctat gggtggctga gagaggaacc acccagggct gccatccagc ctgggagcct 300 cgatctgaaa gccaagcatc acgcaaggaa gagggcctgc ctgaagagga gccatcccat 360 gtcacgggaa gggaaggatt cccgacagat gctccttatc ccaccacgtt agggaaagac 420 agggagtgtc agagccagag tctggcactc aaggagcaga ataacttgaa gcagttggaa 480 tttggcctca aggaagcacc agttcaagat caaggctaca aaactctcag actcagggaa 540 aactgcgtcc tgagttcaag cccaaatcca ttcccagaga tctctagagg ggagtatttg 600 tatacttacg actcacagat tacagactca gaacataact ccagcttagt cagtcagcag 660 acaggctccc caggaaaaca gcccggtgaa aacagtgact gtcacagaga ttccagtcag 720 gccattccaa ttacggaact cacaaaaagc caggtgcagg acaaacccta caaatgtact 780 gactgtggga agtcgtttaa ccataacgca cacctcaccg tgcacaagag gattcatacg 840 ggagaaagac cttatatgtg caaggagtgt gggaaagcct tcagccagaa ctcctccctc 900 gtccagcatg agcgcatcca cactggagac aagccctaca agtgtgccga atgtgggaag 960 tctttctgcc atagtacaca ccttaccgtc catcggagga ttcacactgg ggagaagccc 1020 tatgagtgtc aggactgtgg gagggccttc aaccagaact cctccctggg gcggcacaag 1080 aggacacaca ctggggagaa gccatacacc tgcagtgtgt gtgggaaatc cttctctcgg 1140 accactlgcc ttttcctgca cctgagaact cacaccgagg agaggcccta cgagtgtaac 1200 cactgcggga agggcttcag gcacagctca tccctggccc agcaccagcg gaagcacgcg 1260 ggggagaagc cctttgagtg ccgccagagg ctgatctttg agcagacgcc agctctcaca 1320 aagcatgaat ggacagaagc cctgggctgt gacccacctt tgagtcaaga tgagaggact 1380 caccgaagcg acagaccctt caaatgtaat cagtgtggga agtgtttcat tcagagctct 1440 cacctcatcc ggcaccagat aactcacacc agagaggagc agccccatgg gcgaagccgg 1500 cggcgtgaac aatcctcgag caggaactca cacctggttc agcatcaaca cccgaactcc 1560 agaaagagct ctgcaggcgg agcaaaggca gggcagccgg aaagcagagc cctggctttg 1620 tttgacatcc aaaaaatcat gcaagagaaa aaccctgtgc acgttattgg ggtggaagag 1680 ccttctgtgg gtgcttccat gttatttgac atcagagaat ccacatag 1728 <210> 3 <211> 393 <212> PRT <213>人工序列 <220> <223> <400> 3 Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Dear Pro Leu Pro Dear Gln Ala Master Asp Asp Leu Master Leu Dear Pro Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser Gin Lys Thr Tyr Gin Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gin Leu Ala Lys Thr Cys Pro Val Gin 130 135 140 Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala He Tyr Lys Gin Ser Gin His Met Thr Gin Val Val Arg Arg Cys 165 170 175 Pro His His Gin Arg Cys Ser Asp Ser Asp Gin Leu Ala Pro Pro Gin 180 185 190 His Leu He Arg Val Gin Gin Gin Leu Gin Gin Gin Gin Gin Gin Gin Gin 195 200 205 Arg Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 210 215 220 Val Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390 <210> 4 <211> 1182 <212> DNA <213> Artificial Sequence <220> <223> <400> 4 atggaggagc cgcagtcaga tcctagcgtc gagccccctc tgagtcagga aacattttca 60 gacctatgga aactacttcc tgaaaacaac gttctgtccc ccttgccgtc ccaagcaatg 120 gatgatttga tgctgtcccc ggacgatatt gaacaatggt tcactgaaga cccaggtcca 180 gatgaagctc ccagaatgcc agaggctgct ccccccgtgg cccctgcacc agcagctcct 240 acaccggcgg cccctgcacc agccccctcc tggcccctgt catcttctgt cccttcccag 300 aaaacctacc agggcagcta cggtttccgt ctgggcttct tgcattctgg gacagccaag 360 tctgtgactt gcacgtactc ccctgccctc aacaagatgt tttgccaact ggccaagacc 420 tgccctgtgc agctgtgggt tgattccaca cccccgcccg gcacccgcgt ccgcgccatg 480 gccatctaca agcagtcaca gcacatgacg gaggttgtga ggcgctgccc ccaccatgag 540 cgctgctcag atagcgatgg tctggcccct cctcagcatc ttatccgagt ggaaggaaat 600 ttgcgtgtgg agtatttgga tgacagaaac acttttcgac atagtgtggt ggtgccctat 660 gagccgcctg aggttggctc tgactgtacc accatccact acaactacat gtgtaacagt 720 tcctgcatgg gcggcatgaa ccggaggccc atcctcacca tcatcacact ggaagactcc 780 agtggtaatc tactgggacg gaacagcttt gaggtgcgtg tttgtgcctg tcctgggaga 840 gaccggcgca cagaggaaga gaatctccgc aagaaagggg agcctcacca cgagctgccc 900 ccagggagca ctaagcgagc actgcccaac aacaccagct cctctcccca gccaaagaag 960 aaaccactgg atggagaata tttcaccctt cagatccgtg ggcgtgagcg cttcgagatg 1020 ttccgagagc tgaatgaggc cttggaactc aaggatgccc aggctgggaa ggagccaggg 1080 gggagcaggg ctcactccag ccacctgaag tccaaaaagg gtcagtctac ctcccgccat 1140 aaaaaactca tgttcaagac agaagggcct gactcagact ga 1182 <210> 5 <211> 21 <212> DNA / RNA <213> Artificial Sequence <220> <223> <400> 5 aguaauuuau gguagaguat t 21 <210> 6 <211> 21 <212> DNA / RNA <213> Artificial Sequence <220> <223> <400> 6 uacucuacca uaaauuacut t 21 <210> 7 <211> 21 <212> DNA / RNA <213> Artificial Sequence <220> <223> <400> 7 acauagugua cucauggaat t 21 <210> 8 <211> 21 <212> DNA / RNA <213> Artificial Sequence <220> <223> <400> 8 uuccaugagu acacuaugut t 21

Claims

1. Use of a substance that inhibits the activity and / or expression of ZNF8 protein in the preparation of a product; the function of the product is to inhibit the lung metastasis of tumors; The tumor is formed by human colon cancer cells; The "substance that inhibits the activity and / or expression of ZNF8 protein" is z1) or z2) or z3): z1) oligonucleic acid siZNF8-1; the oligonucleic acid siZNF8-1 is formed by annealing the single-stranded nucleic acid molecule represented by sequence 5 in the sequence listing and the single-stranded nucleic acid molecule represented by sequence 6 in the sequence listing; z2) chemically modifying the oligonucleic acid siZNF8-1 to obtain an oligonucleic acid; z3) shRNA synthesized by the shRNA expression system using the oligonucleic acid siZNF8-1 as the target.

2. The use according to claim 1, characterized in that: The tumor is a tumor formed by human colon cancer cells in mice.

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