CN106620650B

CN106620650B - Application of ZNF383 protein in preparing product for inhibiting activity of p53 protein

Info

Publication number: CN106620650B
Application number: CN201710062820.7A
Authority: CN
Inventors: 田春艳; 王建; 贺福初; 张秀园; 原艳芝
Original assignee: Institute of Radiation Medicine of CAMMS
Current assignee: Institute of Radiation Medicine of CAMMS
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2022-01-18
Anticipated expiration: 2037-01-25
Also published as: CN106620650A

Abstract

The invention discloses an application of ZNF383 protein in preparing a product for inhibiting the activity of p53 protein. Experiments prove that the ZNF383 protein can inhibit the activity of p53 protein, can reduce the expression quantity of target genes related to apoptosis (such as puma gene or p53AIP1 gene), can reduce the expression quantity of target genes related to immunity (such as IRF5 gene, ISG15 gene or IFN-beta gene), can reduce the expression level of puma protein, can inhibit a signal path related to p53 protein and can be combined with p53 protein. Therefore, the ZNF383 protein has important application value in preparing products for inhibiting the activity of the p53 protein.

Description

Application of ZNF383 protein in preparing product for inhibiting activity of p53 protein

Technical Field

The invention relates to the field of biomedicine, in particular to application of ZNF383 protein in preparing a product for inhibiting the activity of p53 protein.

Background

The KRAB-containing zinc finger protein (KZNF) family is the largest transcription factor/transcription regulatory factor family in mammals, and the protein structure mainly comprises a conserved KRAB (Krauppel-associated box) structure domain with strong transcription inhibition function at the N end and a plurality of C2H2 type zinc fingers continuously recognizing continuous DNA sequences at the middle part and the C end. The KRAB domain binds to KAP-1(KRAB-associated protein-1) to recruit various transcription repressing factors to form a transcription repressing complex, and the C-terminal zinc finger region binds to DNA and/or other transcription factors in the regulatory region of the target gene to anchor the transcription repressing complex formed with KAP-1 near a specific target sequence to exert a specific transcription repressing function.

The p53 gene is the gene which is found to be most related to human tumor, the mutation of the p53 gene is found in more than 50 percent of tumor tissues of human, and most of the tumors in which the p53 gene is not mutated have defects of the p53 gene regulation mechanism. It not only acts as an anti-cancer gene to play an anti-proliferation role, but also generates different cell effects aiming at different stress signals under different time spaces to determine the 'birth' or 'death' of cells.

The complicated biological effects of the p53 gene are mainly achieved by the regulation of expression of hundreds of target genes as transcription factors, and the functions of the target genes relate to cycle regulation, apoptosis, angiogenesis, tumor metastasis, metabolism, immunity, reproduction, DNA damage repair and the like.

Disclosure of Invention

The technical problem to be solved by the invention is how to inhibit the activity of the p53 protein.

In order to solve the technical problems, the invention firstly provides the application of ZNF383 protein in preparing products; the function of the product may be at least one of the following a1) to a 11):

A1) inhibiting the activity of p53 protein; A2) down-regulating the expression level of an apoptosis-related target gene; A3) the expression quantity of the puma gene is reduced; A4) down-regulating the expression level of p53AIP1 gene; A5) down-regulating the expression level of an immune-related target gene; A6) the expression level of IRF5 gene is reduced; A7) down-regulating the expression level of ISG15 gene; A8) down-regulating the expression level of IFN-beta gene; A9) reducing the expression level of puma protein; A10) inhibiting the signaling pathway associated with the p53 protein; A11) binding with p53 protein.

The application of ZNF383 protein also belongs to the protective scope of the invention. The application of the ZNF383 protein can be at least one of A1) to A11) as follows:

The A1), the "inhibiting activity of p53 protein" is inhibiting activity of p53 protein in cells; the cell is a human colon cancer cell, a cervical cancer cell or an embryonic fibroblast.

The a2), the apoptosis-related target gene can be an apoptosis-related target gene (i.e. an apoptosis-related target gene regulated by a p53 gene) downstream of the p53 gene.

In the a3), the "down-regulated puma gene expression level" may be a down-regulated puma gene expression level downstream of the p53 gene in the cell. The cells are human colon cancer cells.

The A4), the "down-regulation of the expression level of the p53AIP1 gene" may be the down-regulation of the expression level of the p53AIP1 gene downstream of the p53 gene in the cell. The cells are human colon cancer cells.

The a5), the immune-related target gene can be an immune-related target gene downstream of the p53 gene (i.e. an immune-related target gene regulated by the p53 gene).

The a6), the "down-regulating the expression level of the IRF5 gene" may be down-regulating the expression level of the IRF5 gene downstream of the p53 gene in the cell. The cells may be human colon cancer cells or embryonic fibroblasts.

The a7), the "downregulating the expression level of the ISG15 gene" may be downregulating the expression level of the ISG15 gene downstream of the p53 gene in the cell. The cell may be a human colon cancer cell.

The A8), the "down-regulated expression level of IFN- β gene" may be down-regulated expression level of IFN- β gene in cells. The cell may be an embryonic fibroblast.

The a9), the "reducing the expression level of puma protein" may be reducing the expression level of puma protein in the cell. The cell may be a human colon cancer cell.

The A10), the "signal path related to the p53 protein" can be an apoptosis path related to the p53 protein and/or an IFN-beta path related to the p53 protein.

The A1), ZNF383 protein can inhibit the activity of p53 protein in a dose-dependent mode. The growth condition of the human colon cancer cell may be a normal growth condition or a DNA damage stress condition. The growth condition of the cervical cancer cell may be a normal growth condition. The growth conditions of the embryonic fibroblasts may be virus infection stress conditions.

The growth conditions of the human colon cancer cells in A3), a4), a6), and a7) may be normal growth conditions.

The A6) or A8), the growth condition of the embryonic fibroblasts can be a virus infection stress condition.

The growing condition of the human colon cancer cell in A9) may be a normal growing condition or a DNA damage stress condition.

The application of the substance for inhibiting the activity and/or the expression quantity of the ZNF383 protein in the preparation of products also belongs to the protection scope of the invention; the function of the product may be at least one of the following C1) to C11):

C1) increasing the activity of p53 protein; C2) up-regulating the expression level of an apoptosis-related target gene; C3) up-regulating the expression level of puma gene; C4) up-regulating the expression level of p53AIP1 gene; C5) up-regulating the expression level of an immune-related target gene; C6) up-regulating the expression level of IRF5 gene; C7) up-regulating the expression level of ISG15 gene; C8) up-regulating the expression level of IFN-beta gene; C9) increasing the expression level of puma protein; C10) increase the signaling pathway associated with the p53 protein; C11) binding with p53 protein.

The C1), the "increasing activity of p53 protein" may be increasing activity of p53 protein in a cell. The cell may be a human colon cancer cell or a cervical cancer cell.

Said C9), said "increasing the expression level of puma protein" may be increasing the expression level of puma protein in the cell. The cell may be a human colon cancer cell.

The C1), the growth condition of the human colon cancer cell may be a normal growth condition or a DNA damage stress condition. The growth condition of the cervical cancer cell may be a normal growth condition.

The C9), the growth condition of the human colon cancer cell may be a DNA damage stress condition.

The C10), the "signal pathway related to p53 protein" may be an apoptosis pathway related to p53 protein and/or an IFN- β pathway related to p53 protein.

In any of the above applications, the product may be a medicament.

In order to solve the technical problems, the invention also provides a product A or a product B.

The product A contains ZNF383 protein; the function of the product A can be at least one of the following A1) to A11):

The product B contains substances for inhibiting the activity and/or expression quantity of ZNF383 protein; the function of the product B can be at least one of the following C1) to C11):

The product A or product B can be a medicine.

Any one of the above-mentioned "substances inhibiting the activity and/or expression of ZNF383 protein" may be z1) or z2) or z3) or z 4):

z1) oligomeric nucleic acid siRNA 1; the oligomeric nucleic acid siRNA1 consists of a single-stranded RNA molecule shown in a sequence 5 in a sequence table and a single-stranded RNA molecule shown in a sequence 6 in the sequence table;

z2) oligomeric nucleic acid siRNA 2; the oligomeric nucleic acid siRNA2 is composed of a single-stranded RNA molecule shown in a sequence 7 in a sequence table and a single-stranded RNA molecule shown in a sequence 8 in the sequence table.

z3) using the oligo-nucleic acid siRNA1 as a target point, and synthesizing shRNA by a shRNA expression system;

z4) and shRNA synthesized by shRNA expression system by using the oligonucleotide siRNA2 as target spot.

Any of the human colon cancer cells described above may be p53^+/+HCT116cells or p53^-/-HCT116 cells.

Any of the cervical cancer cells described above may be Hela cells.

Any of the embryonic fibroblasts described above may be p53^-/-MDM2^-/-MEF cells.

Any one of the ZNF383 proteins described above may be a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 1 in a sequence table;

a2) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of a 1);

a3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table.

Any one of the p53 proteins above may be b1) or b2) or b 3):

b1) the amino acid sequence is protein shown as a sequence 3 in a sequence table;

b2) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of b 1);

b3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3 in the sequence table.

The ZNF383 protein also belongs to the protection scope of the invention.

The nucleotide molecule for encoding the ZNF383 protein also belongs to the protection scope of the invention.

The nucleotide molecule encoding the ZNF383 protein can be a DNA molecule shown as (k1) or (k2) or (k3) or (k 4):

(k1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(k2) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table;

(k3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in (k1) or (k2) and encoding the ZNF383 protein;

(k4) a DNA molecule which hybridizes with the nucleotide sequence defined by (k1) or (k2) under strict conditions and encodes the ZNF383 protein.

Wherein, the nucleotide molecule for encoding the ZNF383 protein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 2 in the sequence table consists of 1428 nucleotides, and the nucleotide of the sequence 2 in the sequence table encodes an amino acid sequence shown as the sequence 1 in the sequence table.

The nucleotide sequence encoding the ZNF383 protein of the invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of ZNF383 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode ZNF383 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in sequence 1 of the sequence listing of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines comprising nucleic acid molecules encoding the ZNF383 protein also belong to the scope of protection of the present invention.

The recombinant vector can be specifically Myc-ZNF383 plasmid. The Myc-ZNF383 plasmid is a recombinant plasmid obtained by replacing a small fragment between recognition sequences of restriction enzymes SalI and Not I of a pCMV-Myc plasmid with a DNA molecule shown in a sequence 2 in a sequence table.

The oligomeric nucleic acid siRNA1 also belongs to the protection scope of the invention.

The oligomeric nucleic acid siRNA2 also belongs to the protection scope of the invention.

Experiments prove that the ZNF383 protein can inhibit the activity of p53 protein, can reduce the expression quantity of target genes related to apoptosis (such as puma gene or p53AIP1 gene), can reduce the expression quantity of target genes related to immunity (such as IRF5 gene, ISG15 gene or IFN-beta gene), can reduce the expression level of puma protein, can inhibit a signal path related to p53 protein and can be combined with p53 protein. Therefore, the ZNF383 protein has important application value in preparing products for inhibiting the activity of the p53 protein.

Drawings

Fig. 1 shows the results of experiment one of example 2.

FIG. 2 shows the results of experiment two of example 2.

Fig. 3 shows the results of experiment one of example 3.

FIG. 4 shows the results of experiment two of example 3.

Fig. 5 shows the results of experiment one of example 4.

FIG. 6 shows the results of experiment two of example 4.

Fig. 7 shows the results of experiment three in example 4.

Fig. 8 shows the results of experiment four in example 4.

Fig. 9 shows the results of experiment five of example 4.

Fig. 10 shows the results of experiment one of example 5.

FIG. 11 shows the results of experiment two in example 5.

Fig. 12 shows the results of experiment three in example 5.

Fig. 13 shows the results of experiment four in example 5.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The amino acid sequence of the ZNF383 protein is shown as a sequence 1 in a sequence table, and the nucleotide sequence of a coding gene (hereinafter, referred to as ZNF383 gene) is shown as a sequence 2 in the sequence table. The amino acid sequence of the p53 protein is shown as sequence 3 in the sequence table, and the nucleotide sequence of the coding gene (hereinafter referred to as p53 gene) is shown as sequence 4 in the sequence table.

The pG13L plasmid (also known as pG13-Luc plasmid) is described in the following documents: chunyan Tian, et al, KRAB-type zinc-finger protein Apak specific regulations p53-dependent apoptosis, Nat Cell biol.2009May; 11(5): 580-91, available to the public from the institute of radiology and radiology medicine, the national institute of liberty military medical sciences. The plasmid is a reporter gene plasmid, 13 tandem p53 binding elements are constructed at the upstream of a promoter region of the reporter gene plasmid, if p53 protein exists, the p53 binding elements can be combined with the p53 binding elements to cause the expression of the plasmid, and then fluorescence is emitted, and then the activity of the p53 protein is judged according to the fluorescence intensity.

The pCMV-Myc plasmid is a product of Clontech. The pCMV-Flag plasmid is a product of Sigma company and has a product catalog number of E3762. The pRL-TK plasmid is a product of Promega corporation, and the product catalog number is E2241. The dual-luciferase reporter gene kit is a product of Promega company, and the catalog number is E1980. Myc antibody is a product of Medical Biological laboratories, Inc., catalog No. M047-3. The p53 antibody is available from Calbiochem under the catalog number OP 43L. The Puma antibody is a product of Cell Signaling Technology, inc, under catalog number 4976S. The GAPDH antibody is a product of proteintach company, and the catalog number of the product is 60004-1. Etoposide is a product from Sigma, catalog No. E1383. The proein-A/G Plus agrose is a product of Santa Cruz, Inc., and the catalog number is sc-2003. Poly (I: C) is a product from Sigma, catalog number P0913.

Hela cells were deposited in American type culture Collection (ATCC; accession number: http:// www.atcc.org /), accession number PTA-5659.

P53 wild type HCT116cell (also called p 53)^+/+HCT116cells or p53^+/+HCT116 cell) and p 53-deficient HCT116cell (also known as p 53)^-/-HCT116cells or p53^-/-HCT116 cells) are described in the following references: chunyan Tian, et al, KRAB-type zinc-finger protein Apak specific regulations p53-dependent apoptosis, Nat Cell biol.2009May; 11(5): 580-91, available to the public from the institute of radiology and radiology medicine, the national institute of liberty military medical sciences.

MEF cell (also called p 53) with p53 and MDM2 deletion type^-/-HDM2^-/-MEF cells or p53^-/-HDM2^-/-MEF cells) are described in the following documents: string Nie, et al, Smad ubiquitin alignment factor 1/2(Smurf1/2) proteins p53 degradation by y stabilizing the E3 strain MDM2.J Biol chem.2010Jul 23; 285(30): 22818-30, publicly available from the institute of radiology and radiology, national institute of military medical sciences.

In the following examples, the Transfection Reagent used in the co-Transfection was TurboFect Transfection Reagent, and the specific steps are described in the specification of TurboFect Transfection Reagent. TurboFect transformation Reagent is a product of Thermo Scientific.

Sealing liquid: 5mg of skim milk powder was dissolved in 100mL of TBST solution. The TBST solution was 20mM Tris-HCl buffer, pH7.5, containing 140mM NaCl and 0.1% (v/v) Tween-20.

2 × loading buffer: a Tris-HCl buffer pH8.0, 20mM, containing 200mM DTT, 2% (2g/100mL) SDS, 20% (v/v) glycerol and 0.016% (0.016g/100mL) bromophenol blue.

The puma gene has genebank number 27113. The genebank number of the p53AIP1 gene is 63970. The genebank number of the p21 gene is 1026. The genebank number of the 14-3-3 sigma gene is 2810. The genebank number of the p53R2 gene is 50484. The genebank number of the DDB2 gene is 1643. The gene XPC has the genebank number of 7508. The genebank number of the TLR3 gene is 7098. The genebank number of the IRF5 gene is 3663. The genebank number of the ISG15 gene is 9636. The genebank number of the PML gene is 5371. The genebank number of the HDM2 gene is 4193. The genebank number of the MDR gene is 5243. The genebank number of CDC25c gene is 995. The genebank number of the IFN-beta gene is 3456.

Example 1 construction of plasmids and preparation of oligo-nucleic acids

1. Construction of Myc-ZNF383 plasmid

And replacing a small fragment between the recognition sequences of restriction enzymes SalI and Not I of the pCMV-Myc plasmid with a DNA molecule shown as a sequence 2 in a sequence table to obtain a recombinant plasmid, namely the Myc-ZNF383 plasmid.

The Myc-ZNF383 plasmid expresses ZNF383 protein shown in a sequence 1 in a sequence table.

2. Construction of Flag-p53 plasmid

Replacing a small fragment between the recognition sequences of restriction enzymes EcoRI and BamHI of the pCMV-Flag plasmid with a DNA molecule shown in a sequence 4 in a sequence table to obtain a recombinant plasmid, namely the Flag-p53 plasmid.

The Flag-p53 plasmid expresses p53 protein shown in sequence 3 in the sequence table.

3. Preparation of oligo-nucleic acids

Sense and antisense strands shown in Table 1 were synthesized artificially. And diluting the sense strand by using deionized water to obtain a sense strand diluent. And diluting the antisense strand by using deionized water to obtain an antisense strand diluent. And taking the sense strand diluent and the corresponding antisense strand diluent to carry out annealing reaction to form the oligomeric nucleic acid.

This step prepares 3 oligo-nucleic acids shown in Table 1. A, G, C and U in each oligonucleotide represent adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide in sequence, and T represents thymine deoxynucleotide.

The nucleotide sequence of siRNA-con is a random sequence (as a negative control). The nucleotide sequences of siRNA1 and siRNA2 are designed according to sequence 2 in the sequence table.

TABLE 1

The Myc-ZNF383 plasmid, the Flag-p53 plasmid, the siRNA1, the siRNA2 and the siRNA-con were subjected to subsequent experiments.

Example 2 inhibition of the Activity of the endogenous p53 protein by the ZNF383 protein in a dose-dependent manner

One, experiment one

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

1. mixing cells (p 53)^+/+HCT116cells or Hela cells) were seeded in 24-well plates (8.0 × 10 per well) containing 0.5mL of DMEM medium⁴Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, randomly dividing the mixture into four groups when the fusion rate reaches 70-90%, setting three composite holes in each group, and performing the following treatment:

a first group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.4. mu.g pCMV-Myc plasmid into each well, and co-transfecting;

second group: adding 0.1. mu.g of Myc-ZNF383 plasmid, 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid and 0.3. mu.g of pCMV-Myc plasmid into each well, and performing cotransfection;

third group: adding 0.2 mu g of Myc-ZNF383 plasmid, 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid and 0.2 mu g of pCMV-Myc plasmid into each hole for cotransfection;

and a fourth group: mu.g of Myc-ZNF383 plasmid, 20ng of pG13L plasmid and 0.2ng of pRL-TK plasmid were added per well and co-transfected.

2. After the step 136 h is finished, detecting the fluorescence intensity by adopting a dual-luciferase reporter gene kit, and then respectively averaging according to the groups to obtain the fluorescence intensity of each group.

The fluorescence intensity of the first group was regarded as 1 (i.e., 1 with respect to the activity of p53 protein), and the fluorescence intensity of the other groups (i.e., with respect to the activity of p53 protein) was calculated.

The experimental results are shown in FIG. 1 (left panel is p 53)^+/+HCT116cells, right panel Hela cells). The results show that at p53^+/+In HCT116cells or Hela cells, ZNF383 protein obviously inhibits the activity of endogenous p53 protein in a dose-dependent manner.

Second, experiment two

1. mixing cells (p 53)^+/+HCT116cells or Hela cells) were seeded in 24-well plates (8.0 × 10 per well) containing 0.5mL of DMEM medium⁴Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, randomly dividing into three groups when the fusion rate reaches 70-90%, setting three complex holes in each group, and performing the following treatment:

a first group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.5. mu.g siRNA-con into each well, and co-transfecting;

second group: 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid and 0.5. mu.g of siRNA1 were added to each well for co-transfection;

third group: 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid and 0.5. mu.g of siRNA2 were added to each well and co-transfected.

The experimental results are shown in A in FIG. 2 (left panel is p 53)^+/+HCT116cells, right panel Hela cells).

3. After the step 136 h is finished, total RNA of each cell is extracted respectively, and then reverse transcription is carried out on the first strand cDNA by adopting reverse transcriptase to obtain cDNA of each cell. Then, the relative expression amount of ZNF383 gene (internal reference is GAPDH gene) in cDNA of each cell is analyzed by real-time fluorescence quantification. Primers for detecting ZNF383 gene were 5'-GAGAACGGCCTCAAGGAAGA-3' and 5'-TGTATAAGTCCCTCTGAACAGG-3'. Primers for detecting GAPDH gene were 5'-GGGAAGGTGAAGGTCGGAGT-3' and 5'-TTGAGGTCAATGAAGGGGTCA-3'.

The relative expression level of the ZNF383 gene in the cDNA of the first group of cells was regarded as 1, and the relative expression level of the ZNF383 gene in the cDNAs of the second and third groups of cells was calculated.

p53^+/+The results of the experiment with HCT116cells are shown in FIG. 2B.

The results show that both siRNA1 and siRNA2 can obviously inhibit the expression of endogenous ZNF383 gene; at p53^+/+In HCT116cells or Hela cells, after siRNA1 and siRNA2 obviously inhibit the expression of an endogenous ZNF383 gene, the activity of the endogenous p53 protein can be obviously increased.

Example 3 ZNF383 protein-dependent p53 protein selectively downregulating expression level of downstream apoptosis and immune-related target genes of p53 gene

One, experiment one

1. p53^+/+HCT116cells were seeded in 6-well plates (2.0X 10 per well) containing 2mL of DMEM medium⁵Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, randomly dividing the mixture into two groups when the fusion rate reaches 70-90%, setting three complex holes in each group, and performing the following treatment:

a first group: 2.0 mug pCMV-Myc plasmid is added into each hole for cotransfection;

second group: 2.0. mu.g of Myc-ZNF383 plasmid was added to each well and co-transfected.

2. After the completion of step 148 h, total RNA of each cell is extracted, and then reverse transcription is performed to obtain first strand cDNA by using reverse transcriptase, so as to obtain cDNA of each cell. Then, the relative expression of the target gene downstream of the p53 gene (internal reference is GAPDH gene) was quantitatively analyzed by real-time fluorescence. The downstream target gene of the p53 gene is a puma gene, a p53AIP1 gene, a p21 gene, a 14-3-3 sigma gene, a p53R2 gene, a DDB2 gene, an XPC gene, a TLR3 gene, an IRF5 gene, an ISG15 gene, a PML gene, an HDM2 gene, an MDR gene or a CDC25c gene.

Primers for detecting puma gene: 5'-GGACGACCTCAACGCACAGT-3', and 5'-AATTGGGCTCCATCTCGGGG-3'. Primers for detecting p53AIP1 gene: 5'-TCTTCCTCTGAGGCGAGCT-3', and 5'-AGGGTGCTGCGAAGCTGACGC-3'. Primers for detecting p21 gene: 5'-CACCGAGACACCACTGGAGG-3', and 5'-GAGAAGATCAGCCGGCGTTT-3'. Primers for detecting 14-3-3 sigma gene: 5'-GGCCATGGACATCAGCAAGAA-3', and 5'-CGAAAGTGGTCTTGGCCAGAG-3'. Primers for detecting p53R2 gene: 5'-TGGCTTCGTCGTTGCGAGCG-3', and 5'-TCTGAAGATGATCTCCCGGCCT-3'. Primers for detecting the DDB2 gene: 5'-CTGAACCCATGCTGTGATTG-3', and 5'-AAACTCGGATCTCGCTCTTC-3'. The primer for detecting the XPC gene comprises the following components: 5'-ACGGGCCCAAGAGTGAGGC-3', and 5'-TTGAGGCCAGGAGGCAGCCA-3'. Primers for detecting the TLR3 gene: 5'-CAGCATCAAAAGAAGCAGAAAA-3', and 5'-CAATAGCTTGTTGAACTGCATGA-3'. Primers for detecting IRF5 gene: 5'-CCAGTGACAAGCAGCGCTTCTACAC-3', and 5'-TCTGGCCCTTTTGGAACAGGATGAG-3'. Primers for detecting the ISG15 gene: 5'-CTGAGAGGCAGCGAACTCAT-3', and 5'-AGCATCTTCACCGTCAGGTC-3'. Primers for detection of PML gene: 5'-CGCCCTGGATAACGTCTTTTT-3', and 5'-TCCACAATCTGCCGGTACAC-3'. Primers for detecting the HDM2 gene: 5'-ATCTTGGCCAGTATATTATG-3', and 5'-GTTCCTGTAGATCATGGTAT-3'. Primers for detection of MDR gene: 5'-ATAATGCGACAGGAGATAGG-3', and 5'-CCAAAATCACAAGGGTTAGC-3'. Primers for detecting CDC25c gene: 5'-GAACAGGCCAAGACTGAAGC-3', and 5'-GCCCCTGGTTAGAATCTTCC-3'. Primers for detection of GAPDH gene: 5'-GGGAAGGTGAAGGTCGGAGT-3', and 5'-TTGAGGTCAATGAAGGGGTCA-3'.

The relative expression amounts of the genes in the first group of cells were taken as 1, and the relative expression amounts of the corresponding genes in the second group of cells were shown in FIG. 3 ("-" is pCMV-Myc plasmid; "+" is Myc-ZNF383 plasmid; "is puma gene, B is 53AIP1 gene, C is p21 gene, D is 14-3-3. sigma. gene, E is p53R2 gene, F is DDB2 gene, G is XPC gene, H is TLR3 gene, I is IRF5 gene, J is ISG15 gene, K is PML gene, L is HDM2 gene, M is MDR gene, and N is CDC25C gene).

The result shows that ZNF383 protein selectively reduces the expression quantity of apoptosis related target genes (puma genes and p53AIP1 genes) and immunity related target genes (IRF5 genes and ISG15 genes) at the downstream of the p53 gene.

Second, test two

1. p53^-/-HCT116cells were seeded in 4 wells of 24-well plates (8.0X 10 per well) filled with 0.5mL of DMEM medium⁴Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, and when the fusion rate reaches 70-90%, performing the following treatment:

1 st well: adding 0.2. mu.g Flag-p53 plasmid and 0.4. mu.g pCMV-Myc plasmid, and co-transfecting;

well 2: adding 0.2 mu g Flag-p53 plasmid and 0.4 mu g Myc-ZNF383 plasmid for cotransfection;

well 3: adding 0.6 mu g of pCMV-Myc plasmid, and cotransfecting;

4 th well: 0.4. mu.g of the Myc-ZNF383 plasmid and 0.2. mu.g of the pCMV-Myc plasmid were added and co-transfected.

2. After completion of step 136 h, the total protein of each cell was extracted, and Western Blot was performed using a Puma antibody, a p53 antibody, a Myc antibody or a GAPDH antibody as a primary antibody (using GAPDH protein as an internal reference).

The results of the experiment are shown in FIG. 4. The results show that the reaction is carried out at P53^-/-In HCT116cells, ZNF383 protein depended on p53 protein to reduce the expression level of puma protein (Gene ID: 27113).

Example 4 increase of the ability of ZNF383 protein to inhibit the binding of p53 protein under stress conditions of DNA damage

In this example, cells were treated with etoposide to mimic DNA damage.

One, experiment one

The in vivo binding capacity of ZNF383 protein and p53 protein is detected by a co-immunoprecipitation experiment, which comprises the following steps:

1. p53^-/-HCT116cells were transferred into a flask containing 5mL of DMEM medium (25 cm gauge flask)²(ii) a 5.0X 10 of each culture flask⁵Individual cells) were incubated at 37 ℃ with 5% CO₂Culturing for 18h in an incubator (the fusion rate reaches 70-90 percent at the moment), and then carrying out the following treatment:

the 1 st flask: adding 6.0 mu g of pCMV-Myc plasmid and 1.5 mu g of Flag-p53 plasmid, and co-transfecting for 48 h;

the 2 nd flask: adding 6.0 mu g of Myc-ZNF383 plasmid and 1.5 mu g of Flag-p53 plasmid, and co-transfecting for 48 h;

3 rd flask: adding 6.0 mu g of pCMV-Myc plasmid and 1.5 mu g of Flag-p53 plasmid, and cotransfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing in incubator for 12h, discarding the liquid phase, washing with pre-cooled PBS buffer (pH7.4, 0.01M) for 3 times (PBS buffer (pH7.4, 0.01M is blotted for the last 1 washing);

culture flask 4: adding 6.0 mu g of Myc-ZNF383 plasmid and 1.5 mu g of Flag-p53 plasmid, and co-transfecting for 36 h; then etoposide is added) to obtain a treatment system (the concentration of etoposide in the treatment system is 2X 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂After 12h incubation in the incubator, the liquid phase was discarded and washed 3 times with pre-cooled PBS buffer (pH7.4, 0.01M) (the final 1 time PBS buffer (pH7.4, 0.01M) was blotted).

2. After completing step 1, pancreatin digest (for cell digestion) was added to each flask, and then transferred to a centrifuge tube (10 mL) for 5min at 1000rpm, and the pellet was collected.

3. And (4) taking the precipitate collected in the step (2) respectively, and washing for 2 times. The steps of each washing are as follows: 1mL of 0.01M PBS buffer, pH7.4, was added, and the mixture was centrifuged at 1000rpm for 5 min. The final 1 wash was performed by blotting 0.01M PBS buffer, pH 7.4.

4. Adding 600 mu L of precooled NETN lysate into the precipitates respectively collected in the step 3, uniformly mixing, and incubating on ice for 30 min; then, the mixture was centrifuged at 12000rpm for 10min at 4 ℃ to collect the supernatant.

NETN lysate: to 1mL of 20mM Tris-Cl buffer pH8.0 containing 150mM NaCl, 1mM EDTA and 0.5% (m/m) NP-40 was added 0.02mL of 50 XProtease inhibitor, and the mixture was mixed well. 50 Xthe protease inhibitor is 1mM DTT, 1mM NaV₃O4 and 1mM NaF in water.

5. And (3) adding 40 mu L of the supernatant collected in the step (4) into 40 mu L of 2 Xloading buffer solution, uniformly mixing, and boiling at 100 ℃ for 10min to obtain a solution, namely Lysate.

6. Respectively transferring the supernatants collected in the step 4 to centrifuge tubes (the specification is 1.5mL), adding 1 mu L of myc antibody into each tube, and slowly shaking for 3h at 4 ℃; then 40. mu.L of proein-A/G Plus agrose were added to each tube and placed on a rotary mixer overnight at 4 ℃.

7. After completion of step 6, the precipitates (i.e., the complex of agrose and antigen-antibody) were collected, respectively, and then placed on ice for 1min (in order to allow the agarose beads to settle), centrifuged at 4 ℃ and 3000rpm for 5min, and the precipitates were collected.

8. And (4) taking the precipitate collected in the step (7) and washing the precipitate for 4 times. The steps of each washing are as follows: 1mL of precooled NETN lysate was added and centrifuged at 4 ℃ and 3000rpm for 2 min.

9. And (3) taking the precipitates respectively collected in the step (8), adding 40 mu L of NETN lysate for resuspension, adding 40 mu L of 2 Xloading buffer solution, mixing uniformly, and boiling for 10min at 100 ℃ to obtain a solution, namely Myc-IP.

Western Blots were performed on Lysate and Myc-IP using either Flag-HRP antibody dilution (obtained by diluting Flag-HRP antibody with blocking solution to 1000 volumes) or Myc-HRP antibody dilution (obtained by diluting Myc-HRP antibody with blocking solution to 1000 volumes) as the primary antibody.

The results of the experiment are shown in FIG. 5. The results show that after etoposide treatment (i.e. under DNA injury stress conditions), ZNF383 protein has increased binding capacity with p53 protein.

Second, experiment two

1. p53^+/+HCT116cells were seeded in 24-well plates (8.0X 10 per well) with 0.5mL DMEM medium⁴Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, randomly dividing the mixture into four groups when the fusion rate reaches 70-90%, setting three composite holes in each group, and performing the following treatment:

a first group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.4. mu.g pCMV-Myc plasmid into each well, and co-transfecting for 48 h;

second group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.3 mu g Myc-ZNF383 plasmid into each hole, and co-transfecting for 48 h;

third group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.4. mu.g pCMV-Myc plasmid into each well, and co-transfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing for 12h in an incubator;

and a fourth group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.3. mu.g Myc-ZNF383 plasmid into each well, and co-transfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^- ⁵mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing in an incubator for 12 h.

2. After the step 1 is completed, detecting the fluorescence intensity by adopting a dual-luciferase reporter gene kit, and then respectively averaging according to the groups to obtain the fluorescence intensity of each group.

The results of the experiment are shown in FIG. 6(1 is the first group, 2 is the second group, 3 is the third group, and 4 is the fourth group). The results show that at p53^+/+In HCT116cells, the activity of p53 protein is increased after etoposide treatment, and the inhibition capability of ZNF383 protein on the activity of endogenous p53 protein is increased.

Third, experiment three

a first group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.5 ug siRNA-con into each well, and co-transfecting for 48 h;

second group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.5. mu.g siRNA1 into each well, and co-transfecting for 48 h;

third group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.5. mu.g siRNA2 into each well, and co-transfecting for 48 h;

and a fourth group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.5 ug siRNA-con into each well, and co-transfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing for 12h in an incubator;

and a fifth group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.5. mu.g siRNA1 into each well, and co-transfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing for 12h in an incubator;

a sixth group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.5. mu.g siRNA2 into each well, and co-transfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing in an incubator for 12 h.

The results of the experiment are shown in FIG. 7(1 is the first group, 2 is the second group, 3 is the third group, 4 is the fourth group, 5 is the fifth group, and 6 is the sixth group). The results show that etoposide treatment p53^+/+The activity of p53 protein is increased after HCT116 cells; the siRNA1 and siRNA2 can obviously increase the activity of endogenous p53 protein and are compared with p53 which is not treated by etoposide^+/+Etoposide treated p53 in comparison to HCT116cells^+/+The activity of the endogenous p53 protein was significantly increased in HCT116 cells.

Fourth, experiment four

1. p53^+/+HCT116cellsSeeded in 4 wells of 24-well plates (8.0X 10 per well) filled with 0.5mL of DMEM medium⁴Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, and when the fusion rate reaches 70-90%, performing the following treatment:

1 st well: adding 0.4 mu g of pCMV-Myc plasmid, and cotransfecting for 48 h;

well 2: adding 0.4 mu g of Myc-ZNF383 plasmid, and co-transfecting for 48 h;

well 3: adding 0.4 mu g of pCMV-Myc plasmid, and cotransfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing for 12h in an incubator;

4 th well: adding 0.4 mu g of Myc-ZNF383 plasmid, and co-transfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing in an incubator for 12 h.

2. After completion of step 1, the total protein of each cell was extracted, and Western Blot was performed using a Puma antibody, a p53 antibody, a Myc antibody or a GAPDH antibody as a primary antibody (using GAPDH protein as an internal control).

The results of the experiment are shown in FIG. 8. The results show that p53 is not treated with etoposide^+/+Etoposide treated p53 in comparison to HCT116cells^+/+The expression level of puma protein in HCT116cells was significantly reduced.

Fifth, experiment fifth

1. p53^+/+HCT116cells were seeded in 6 wells (8.0X 10 per well) of 24-well plates containing 0.5mL of DMEM medium⁴Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, and when the fusion rate reaches 70-90%, performing the following treatment:

1 st well: adding 0.5 mu g of siRNA-con, and cotransfecting for 48 h;

well 2: adding 0.5 mu g of siRNA1, and co-transfecting for 48 h;

well 3: adding 0.5 mu g of siRNA2, and co-transfecting for 48 h;

4 th well: adding 0.5 mu g of siRNA-con, and cotransfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing for 12h in an incubator;

5 th well: adding 0.5 mu g of siRNA1, and cotransfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing for 12h in an incubator;

6 th well: adding 0.5 mu g of siRNA2, and cotransfecting for 36 h; then etoposide is added to obtain a treatment system (the concentration of etoposide in the treatment system is 2 multiplied by 10)^-5mol/L); the treatment system was placed at 37 ℃ in 5% CO₂Culturing in an incubator for 12 h.

The results of the experiment are shown in FIG. 9(1 is the 1 st well, 2 is the 2 nd well, 3 is the 3 rd well, 4 is the 4 th well, 5 is the 5 th well, 6 is the 6 th well). The results show that p53 is not treated with etoposide^-/-Etoposide treated p53 in comparison to HCT116cells^-/-The expression level of puma protein in HCT116cells was significantly increased.

Example 5 ZNF383 protein can inhibit the activity of p53 protein under the stress condition of virus infection

In this example, cells were treated with Poly (I: C) to mimic virus-infected cells.

One, experiment one

1. p53^-/-MDM2^-/-MEF cells were transferred into a flask containing 5mL of DMEM medium (25 cm gauge)²(ii) a 5.0X 10 of each culture flask⁵Individual cells) were plated at 37 deg.C、5％CO₂Culturing for 18h in an incubator (the fusion rate reaches 70-90 percent at the moment), and then carrying out the following treatment:

3 rd flask: adding 6.0 mu g of pCMV-Myc plasmid and 1.5 mu g of Flag-p53 plasmid, and co-transfecting for 24 h; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h; finally, the liquid phase was discarded, and washed 3 times with pre-cooled PBS buffer (pH7.4, 0.01M) (the final 1 wash was performed by blotting out PBS buffer (pH7.4, 0.01M);

culture flask 4: adding 6.0 mu g of Myc-ZNF383 plasmid and 1.5 mu g of Flag-p53 plasmid, and co-transfecting for 24 h; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h; the liquid phase was finally discarded and washed 3 times with pre-cooled 0.01M PBS buffer, pH7.4 (the final 1 wash was performed by blotting 0.01M PBS buffer, pH 7.4).

2. The same as 2 in the first step of example 4.

3. The same as 3 in the first step of example 4.

4. Same as 4 in the first step of example 4.

5. The same as 5 in the first step of example 4.

6. The same as 6 in the first step of example 4.

7. Same as 7 in the first step of example 4.

8. The same as 8 in the first step of example 4.

9. The same as 9 in the first step of example 4.

Western Blot is carried out on Lysate and Myc-IP by taking Flag-HRP antibody diluent or Myc-HRP antibody diluent as a primary antibody.

The results of the experiment are shown in FIG. 10. The results show that ZNF383 protein still has certain binding capacity with p53 protein after Poly (I: C) treatment (namely, under the condition of stress infection of the virus).

Second, experiment two

1. p53^-/-MDM2^-/-MEF cells were seeded in 24-well plates (8.0X 10 per well) with 0.5mL DMEM medium⁴Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, when the fusion rate reaches 70-90%, randomly dividing the mixture into eight groups, setting three composite holes in each group, and performing the following treatment:

a first group: adding 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid and 0.6 mu g of pCMV-Myc plasmid, and co-transfecting for 48 h;

second group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid, 0.2 μ g pCMV-Myc plasmid and 0.3 μ g Myc-ZNF383 plasmid, and co-transfecting for 48 h;

third group: adding 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid and 0.6 mu g of pCMV-Myc plasmid, and co-transfecting for 24 h; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h;

and a fourth group: adding 20ng pG13L plasmid, 0.2ng pRL-TK plasmid, 0.2 μ g pCMV-Myc plasmid and 0.3 μ g Myc-ZNF383 plasmid, and co-transfecting for 24 h; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h;

and a fifth group: adding 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid, 0.4 mu g of pCMV-Myc plasmid and 0.2 mu g of Flag-p53 plasmid, and co-transfecting for 48 h;

a sixth group: adding 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid, 0.2 mu g of Flag-p53 plasmid and 0.3 mu g of Myc-ZNF383 plasmid, and co-transfecting for 48 h;

a seventh group: adding 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid, 0.4 mu g of pCMV-Myc plasmid and 0.2 mu g of Flag-p53 plasmid, and co-transfecting for 24 h; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h;

and an eighth group: adding 20ng of pG13L plasmid, 0.2ng of pRL-TK plasmid, 0.2 mu g of Flag-p53 plasmid and 0.3 mu g of Myc-ZNF383 plasmid, and co-transfecting for 24 hours; then 5. mu.g of Poly (I: C) was added and co-transfected for 24 h.

The experimental results are shown inFig. 11. The results show that at p53^-/-MDM2^-/-In MEF cells, the activity of p53 protein is increased after Poly (I: C) treatment; the ZNF383 protein can still inhibit the activity of endogenous p53 protein.

Third, experiment three

1. p53^-/-MDM2^-/-MEF cells were seeded in 8 wells of 6-well plates (2.0X 10 per well) filled with 2mL of DMEM medium⁵Individual cells) and then placed at 37 ℃ in 5% CO₂Culturing in an incubator, and when the fusion rate reaches 70-90%, performing the following treatment:

1 st well: adding 0.6 mu g of pCMV-Myc plasmid, and cotransfecting for 48 h;

well 2: adding 0.4 mu g of Myc-ZNF383 plasmid and 0.2 mu g of pCMV-Myc plasmid, and co-transfecting for 48 h;

well 3: adding 0.4 mu g of pCMV-Myc plasmid and 0.2 mu g of Flag-p53 plasmid, and cotransfecting for 48 h;

4 th well: adding 0.4 mu g of Myc-ZNF383 plasmid and 0.2 mu g of Flag-p53 plasmid, and co-transfecting for 48 h;

5 th well: adding 0.6 mu g of pCMV-Myc plasmid, and cotransfecting for 24 h; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h;

6 th well: adding 0.4 mu g of Myc-ZNF383 plasmid and 0.2 mu g of pCMV-Myc plasmid, and co-transfecting for 24 hours; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h;

7 th well: adding 0.4 mu g of pCMV-Myc plasmid and 0.2 mu g of Flag-p53 plasmid, and cotransfecting for 24 h; then adding 5 mu g of Poly (I: C), and cotransfecting for 24 h;

8 th well: adding 0.4 mu g of Myc-ZNF383 plasmid and 0.2 mu g of Flag-p53 plasmid, and co-transfecting for 24 h; then 5. mu.g of Poly (I: C) was added and co-transfected for 24 h.

2. After step 1 is completed, total RNA of each cell is extracted respectively, and then reverse transcription is carried out to obtain first strand cDNA by adopting reverse transcriptase, so as to obtain cDNA of each cell. The relative expression level of IRF5 gene (internal reference GAPDH gene) in cDNA from each cell was then quantified using real-time fluorescence. Primers for detecting IRF5 gene: 5'-CCAGTGACAAGCAGCGCTTCTACAC-3', and 5'-TCTGGCCCTTTTGGAACAGGATGAG-3'. Primers for detecting GAPDH gene were 5'-GGGAAGGTGAAGGTCGGAGT-3' and 5'-TTGAGGTCAATGAAGGGGTCA-3'.

The relative expression level of IRF5 gene in the 1 st well was defined as 1, and the relative expression level of IRF5 gene in the other wells was calculated.

The results of the experiment are shown in FIG. 12 (1 st well, 2 nd well, 3 rd well, 4 th well, 5 th well, 6 th well, 7 th well and 8 th well in this order from left to right). The result shows that ZNF383 protein depends on p53 protein to reduce the expression quantity of IRF5 gene; poly (I: C) treatment of p53^-/-MDM2^-/-After MEF cells, the expression level of IRF5 gene is increased, and ZNF383 protein can still reduce the expression level of IRF5 gene depending on p53 protein.

Fourth, experiment four

1 st well: adding 0.6 mu g of pCMV-Myc plasmid, and cotransfecting for 48 h;

2. After step 1 is completed, total RNA of each cell is extracted respectively, and then reverse transcription is carried out to obtain first strand cDNA by adopting reverse transcriptase, so as to obtain cDNA of each cell. The relative expression of IFN-. beta.gene (internal reference GAPDH gene) in cDNA from each cell was then quantified using real-time fluorescence. Primers for detecting IFN- β gene: 5'-AAGAGTTACACTGCCTTTGCCATC-3', and 5'-CACTGTCTGCTGGTGGAGTTCATC-3'. Primers for detecting GAPDH gene were 5'-GGGAAGGTGAAGGTCGGAGT-3' and 5'-TTGAGGTCAATGAAGGGGTCA-3'.

The relative expression amount of IFN-. beta.gene in the 1 st well was taken as 1, and the relative expression amounts of IFN-. beta.gene in the other wells were calculated.

The results of the experiment are shown in FIG. 13 (1 st well, 2 nd well, 3 rd well, 4 th well, 5 th well, 6 th well, 7 th well and 8 th well in this order from left to right). The results show that Poly (I: C) treatment p53^-/-MDM2^-/-After MEF cells and after Poly (I: C) treatment of the cells, ZNF383 protein can reduce the expression quantity of IFN-beta gene depending on p53 protein.

<110> institute of radiology and radiology medical science institute of military medical science institute of people's liberation force of China

Application of <120> ZNF383 protein in preparation of product for inhibiting activity of p53 protein

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 475

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 1

Met Ala Glu Gly Ser Val Met Phe Ser Asp Val Ser Ile Asp Phe Ser

1 5 10 15

Gln Glu Glu Trp Asp Cys Leu Asp Pro Val Gln Arg Asp Leu Tyr Arg

20 25 30

Asp Val Met Leu Glu Asn Tyr Gly Asn Leu Val Ser Met Gly Leu Tyr

35 40 45

Thr Pro Lys Pro Gln Val Ile Ser Leu Leu Glu Gln Gly Lys Glu Pro

50 55 60

Trp Met Val Gly Arg Glu Leu Thr Arg Gly Leu Cys Ser Asp Leu Glu

65 70 75 80

Ser Met Cys Glu Thr Lys Leu Leu Ser Leu Lys Lys Glu Val Tyr Glu

85 90 95

Ile Glu Leu Cys Gln Arg Glu Ile Met Gly Leu Thr Lys His Gly Leu

100 105 110

Glu Tyr Ser Ser Phe Gly Asp Val Leu Glu Tyr Arg Ser His Leu Ala

115 120 125

Lys Arg Leu Gly Tyr Pro Asn Gly His Phe Ser Gln Glu Ile Phe Thr

130 135 140

Pro Glu Tyr Met Pro Thr Phe Ile Gln Gln Thr Phe Leu Thr Leu His

145 150 155 160

Gln Ile Ile Asn Asn Glu Asp Arg Pro Tyr Glu Cys Lys Lys Cys Gly

165 170 175

Lys Ala Phe Ser Gln Asn Ser Gln Phe Ile Gln His Gln Arg Ile His

180 185 190

Ile Gly Glu Lys Ser Tyr Glu Cys Lys Glu Cys Gly Lys Phe Phe Ser

195 200 205

Cys Gly Ser His Val Thr Arg His Leu Lys Ile His Thr Gly Glu Lys

210 215 220

Pro Phe Glu Cys Lys Glu Cys Gly Lys Ala Phe Ser Cys Ser Ser Tyr

225 230 235 240

Leu Ser Gln His Gln Arg Ile His Thr Gly Lys Lys Pro Tyr Glu Cys

245 250 255

Lys Glu Cys Gly Lys Ala Phe Ser Tyr Cys Ser Asn Leu Ile Asp His

260 265 270

Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Val Cys Gly

275 280 285

Lys Ala Phe Thr Lys Ser Ser Gln Leu Phe Gln His Ala Arg Ile His

290 295 300

Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Thr

305 310 315 320

Gln Ser Ser Lys Leu Val Gln His Gln Arg Ile His Thr Gly Glu Lys

325 330 335

Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Ser Ser Gly Ser Ala

340 345 350

Leu Thr Asn His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Asp Cys

355 360 365

Lys Glu Cys Gly Lys Ala Phe Thr Gln Ser Ser Gln Leu Arg Gln His

370 375 380

Gln Arg Ile His Ala Gly Glu Lys Pro Phe Glu Cys Leu Glu Cys Gly

385 390 395 400

Lys Ala Phe Thr Gln Asn Ser Gln Leu Phe Gln His Gln Arg Ile His

405 410 415

Thr Asp Glu Lys Pro Tyr Glu Cys Asn Glu Cys Gly Lys Ala Phe Asn

420 425 430

Lys Cys Ser Asn Leu Thr Arg His Leu Arg Ile His Thr Gly Glu Lys

435 440 445

Pro Tyr Asn Cys Lys Glu Cys Gly Lys Ala Phe Ser Ser Gly Ser Asp

450 455 460

Leu Ile Arg His Gln Gly Ile His Thr Asn Lys

465 470 475

<210> 2

<211> 1428

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 2

atggctgagg gatcagtgat gttcagtgat gtgtccatag acttctctca ggaggagtgg 60

gactgcctgg accctgttca gagggactta tacagagatg tgatgttgga gaactacggc 120

aatctggttt caatgggact ttacactcct aagcctcaag tgatctcctt attggaacaa 180

gggaaagagc cctggatggt tggcagagag cttacaagag gcctgtgttc agatctggaa 240

tcgatgtgtg aaaccaagtt attatctcta aagaaggaag tttatgaaat agaattatgc 300

cagagggaga taatgggact tacaaagcat ggccttgagt actccagttt tggagatgtt 360

ttggaatata gaagccacct tgcaaaacga ctgggatatc caaatgggca ttttagtcaa 420

gaaatattca ctcctgaata catgcccaca tttattcaac agacattcct tactctccat 480

caaataatta ataatgaaga cagaccctat gaatgtaaga aatgtggaaa ggcctttagt 540

cagaactcac aatttattca acatcagaga attcatattg gtgaaaaatc ttatgaatgt 600

aaagagtgtg ggaaattctt tagttgtggt tcacatgtta ctcggcatct gaaaattcat 660

actggcgaaa aaccctttga atgtaaggaa tgtggaaagg ccttcagttg tagctcatac 720

ctttctcaac atcagagaat ccataccggt aagaaaccct atgaatgtaa ggaatgtggg 780

aaggccttta gttattgctc aaatcttatt gaccatcagc gaattcacac tggtgaaaaa 840

ccttatgaat gtaaagtatg tgggaaagcc tttactaaga gctcacaact ttttcagcat 900

gcacgaattc atacaggtga gaaaccctat gaatgtaagg aatgtggcaa agcctttacc 960

cagagctcaa agcttgttca acatcagaga attcatactg gtgagaaacc ctatgagtgc 1020

aaggaatgtg gcaaagcctt tagtagtggc tcagcactta ctaatcatca gagaattcac 1080

actggtgaga aaccctatga ttgtaaggaa tgtggaaagg cttttactca gagctcacag 1140

cttcgtcaac atcagagaat tcacgctggt gagaaaccct ttgaatgtct tgaatgtggg 1200

aaggccttta ctcagaactc acaacttttc cagcatcaga gaattcatac agatgaaaaa 1260

ccatatgaat gtaatgaatg tggaaaggcc tttaataaat gctcaaacct tactcgacat 1320

ctgagaattc acactggtga aaagccctat aactgtaagg aatgtgggaa ggcttttagt 1380

agtggctcgg atctcattcg tcatcaggga attcatacta ataaataa 1428

<210> 3

<211> 393

<212> PRT

<213> Artificial sequence

<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

Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser 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 Gln Lys Thr Tyr Gln 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 Gln Leu Ala Lys Thr Cys Pro Val Gln

130 135 140

Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met

145 150 155 160

Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys

165 170 175

Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln

180 185 190

His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp

195 200 205

Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu

210 215 220

Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser

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

gggauaucca aaugggcaut t 21

<210> 6

<211> 21

<212> DNA/RNA

<213> Artificial sequence

<220>

<223>

<400> 6

augcccauuu ggauauccct t 21

<210> 7

<211> 21

<212> DNA/RNA

<213> Artificial sequence

<220>

<223>

<400> 7

gcucacagcu ucgucaacat t 21

<210> 8

<211> 21

<212> DNA/RNA

<213> Artificial sequence

<220>

<223>

<400> 8

uguugacgaa gcugugagct t 21

Claims

1. The application of the oligomeric nucleic acid siRNA1 in preparing products; the product has the function of increasing the activity of p53 protein in human cervical carcinoma cells;

the oligomeric nucleic acid siRNA1 consists of a single-stranded RNA molecule shown in a sequence 5 in a sequence table and a single-stranded RNA molecule shown in a sequence 6 in the sequence table;

the p53 protein amino acid sequence is a protein shown in a sequence 3 in a sequence table.