CN107812192A

CN107812192A - Suppress application of the material of ZNF498 expressing quantities in the product for preparing prevention and treatment cancer

Info

Publication number: CN107812192A
Application number: CN201711156034.XA
Authority: CN
Inventors: 田春艳; 王建; 令吉明; 张秀园; 原艳芝
Original assignee: BEIJING PROTEOME RESEARCH CENTER
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2017-11-20
Filing date: 2017-11-20
Publication date: 2018-03-20
Anticipated expiration: 2037-11-20
Also published as: CN107812192B

Abstract

The invention discloses suppress application of the material of ZNF498 expressing quantities in the product for preparing prevention and treatment cancer.It is demonstrated experimentally that ZNF498 albumen with p53 protein bindings, can suppress the transcriptional activity of p53 albumen, reduces the expression of Puma albumen and lower the expression quantity of puma genes.The expression of suppression ZNF498 albumen can increase the growth of the propagation and tumour of the transcriptional activity of p53 albumen, the expression for promoting Puma albumen and Gadd45 albumen, the expression quantity for raising puma genes, the phosphorylation level for increasing p53 the 46th serine of albumen, the apoptosis for promoting p53 positive tumor cells and suppression tumour cell.Therefore, the expression for suppressing ZNF498 albumen has important application value in the apoptosis for promoting tumour cell, the propagation for suppressing tumour cell and the growth for suppressing tumour；Suppressing the expression of ZNF498 albumen can prevent and/treating cancer.

Description

The material for suppressing ZNF498 expressing quantities is preparing the product of prevention and treatment cancer In application

Technical field

The present invention relates to biomedical sector, and in particular to suppress ZNF498 expressing quantities material prepare prevention and Application in the product for the treatment of cancer.

Background technology

KRAB types zinc finger protein (KRAB-containing zinc finger protein, KZNF) family is that lactation is moved Maximum transcription factor/transcription regulatory factor family in thing, its protein structure mainly contains conservative having by N-terminal relatively transcribes by force KRAB (Kr ü ppel-associated box) domain, middle part and the C-terminal for suppressing function contains lasting identification continuous DNA sequence Multiple C2H2 types zinc fingerses composition.Wherein KRAB domains by with KAP-1 (KRAB-associated protein-1) With reference to a variety of transcription inhibitory factors are raised, form Transcription inhibition complex, C-terminal zinc finger area and the DNA in target gene control region and/ Or after other transcription factors combine, the Transcription inhibition complex formed with KAP-1 is anchored near specific target sequence, play Special Transcription inhibition function, so as to participate in the processes such as cell differentiation, propagation, apoptosis.

ZNF498 albumen is also known as ZSCAN25, is a member of KZNF families SCAN subfamilies, the assignment of genes gene mapping is in 7q22.1 Area, its ORF are 1635bp, encode the protein being made up of 544 amino acid.From N-terminal to C-terminal, the SCAN on ZNF498 albumen (SRE-ZBP, CTfin51, AW-1 and Number18cDNA), KRAB and 7 C2H2 domain arranged in series.At present its function and Mechanism of action there is no report.

P53 genes are discovery so far and human tumor correlation highest gene, in the tumor tissues of the mankind more than 50% In be found that the mutation of p53 genes, and lacking for p53 mechanisms of gene regulation largely be present in the unmutated tumour of p53 genes Fall into.P53 albumen can mediate different downstream work(as the sequence-specific transcription factor by adjusting the expression of a large amount of target genes Can, participate in many cell events such as cell-cycle arrest, apoptosis, aging and DNA damage reparation.Wherein p53 is to Apoptosis work( The regulation and control of energy are played in the occurrence and development that p53 suppresses tumour and its important effect.

RNA interference (RNA interference, RNAi) be in a kind of evolution guard resist transgenosis or adventitious viruses The defense mechanism of infringement.Its essence is siRNA and targeting mRNA specific bonds, and by the silencing complex (RNA of RNA inductions Induced silencing complex, RISC) its degraded is mediated, so as to prevent mRNA translation, cause gene silencing.Profit By the use of external source or endogenous mode using siRNA import specific cells specific gene is carried out RNAi can as gene functional research, Gene therapy and the important tool of targeted drug exploitation.

The content of the invention

The technical problems to be solved by the invention be how pre- anti-cancer and/or treating cancer.

In order to solve the above technical problems, present invention firstly provides the thing for suppressing ZNF498 protein actives and/or expression quantity Application of the matter in product is prepared；At least one of the function of the product can be following C1) to C12)：

C1) pre- anti-cancer；C2) treating cancer；C3 the propagation of tumour cell) is suppressed；C4 the growth of tumour) is suppressed；C5) promote Enter the apoptosis of tumour cell；C6 the apoptosis of p53 positive tumor cells) is promoted；C7 it is horizontal) to increase p53 protein phosphorylations；C8) increase Add the phosphorylation level of the 46th serine of p53 albumen；C9 the expression of Puma albumen) is increased；C10 Gadd45 eggs) are increased White expression；C11 the expression quantity of puma genes) is raised；C12 the activity of p53 albumen) is increased.

The present invention also protection suppresses the application of the material of ZNF498 protein actives and/or expression quantity, can be following C1) extremely At least one of C12)：

The present invention also protects application of the ZNF498 albumen as drug target in product is prepared；The function of the product can At least one of for following C1) to C12)：

The material of any of the above-described the suppression ZNF498 protein actives and/or expression quantity falls within the protection model of the present invention Enclose.

The present invention also protects following X1) or X2)：

X1) application of the ZNF498 albumen in product is prepared；The function of the product can be A1) to A11) at least one Kind；

At least one of X2) the application of ZNF498 albumen, can be A1) to A11)；

A1 the activity of p53 albumen) is suppressed；A2 the expression of Puma albumen) is suppressed；A3 the expression of puma genes) is lowered Amount；A4) with p53 protein bindings；A5 the propagation of tumour cell) is promoted；A6 the growth of tumour) is promoted；A7 tumour cell) is suppressed Apoptosis；A8 the apoptosis of p53 positive tumor cells) is suppressed；A9 it is horizontal) to suppress p53 protein phosphorylations；A10 p53 albumen the) is suppressed The phosphorylation level of 46 serines；A11 the expression of Gadd45 albumen) is suppressed.

In any of the above-described described application, the product can be medicine.

The present invention also protects product first or product second；

Material of the product first containing any of the above-described suppression ZNF498 protein actives and/or expression quantity；It is described At least one of the function of product first can be following C1) to C12)：

C1) pre- anti-cancer；C2) treating cancer；C3 the propagation of tumour cell) is suppressed；C4 the growth of tumour) is suppressed；C5) promote Enter the apoptosis of tumour cell；C6 the apoptosis of p53 positive tumor cells) is promoted；C7 it is horizontal) to increase p53 protein phosphorylations；C8) increase Add the phosphorylation level of the 46th serine of p53 albumen；C9 the expression of Puma albumen) is increased；C10 Gadd45 eggs) are increased White expression；C11 the expression quantity of puma genes) is raised；C12 the activity of p53 albumen) is increased；

The product second can contain ZNF498 albumen；The function of the product second can be following A1) to A11) at least It is a kind of：

The product first or product second can be medicine.

The present invention also protection suppresses material the answering in exploitation or screening reagent of ZNF498 protein actives and/or expression quantity With；At least one of the purposes of the reagent can be following C1) to C12)：

Any of the above-described " material for suppressing ZNF498 protein actives and/or expression quantity " can be z1) or z2) or z3) or Z4) or z5) or z6)：

Z1) nucleic acid oligomer siRNAa；Nucleic acid oligomer siRNAa single stranded nucleic acid molecules as shown in the sequence 5 in sequence table With the single stranded nucleic acid molecule composition shown in the sequence 6 of sequence table；

Z2) nucleic acid oligomer siRNAb；Nucleic acid oligomer siRNAb single stranded nucleic acid molecules as shown in the sequence 7 in sequence table Formed with single stranded nucleic acid molecule shown in the sequence 8 of sequence table；

Z3) the nucleic acid oligomer siRNAa is chemically modified, obtained nucleic acid oligomer；

Z4) the nucleic acid oligomer siRNAb is chemically modified, obtained nucleic acid oligomer；

Z5) using the nucleic acid oligomer siRNAa as target spot, the shRNA that is synthesized by shRNA expression systems；

Z6) using the nucleic acid oligomer siRNAb as target spot, the shRNA that is synthesized by shRNA expression systems.

Any of the above-described chemical modification can be phosphate backbones modification, ribose modification or base modification.It is chemically modified Nucleic acid oligomer siRNAa or nucleic acid oligomer siRNAb stability increase, so as to effectively suppress ZNF498 albumen activity and/ Or expression quantity.

The z5) shRNA-ZNF498 plasmids that concretely embodiment refers to；I.e. using siRNAa as target spot, with PLVshRNA-EGFP (2A) Puro plasmids are carrier, and by Beijing English, luxuriant industry Bioisystech Co., Ltd builds ZNF498's ShRNA slow virus expression plasmids.PLVshRNA-EGFP (2A) Puro plasmids can be the luxuriant industry Bioisystech Co., Ltd of Beijing English Product.

Any of the above-described C1) or the C2) in, the cancer can be liver cancer, lung cancer or colon cancer.It is any of the above-described described C3 in), the tumour cell can be human liver cancer cell.Any of the above-described C4) in, the tumour can be human liver cancer cell small The tumour formed in mouse body.Any of the above-described C5) in, the tumour cell can be human liver cancer cell.Any of the above-described C6) In, the tumour cell can be human liver cancer cell.Any of the above-described C7) in, " increase p53 protein phosphorylations are horizontal " can It is horizontal for p53 protein phosphorylations in increase cell；The cell can be human liver cancer cell.Any of the above-described C8) in, it is described " phosphorylation level of increase p53 the 46th serine of albumen " can be the phosphoric acid of the 46th serine of p53 albumen in increase cell Change horizontal；The cell can be human liver cancer cell.Any of the above-described C9) in, " expression of increase Puma albumen " It can be the expression of Puma albumen in increase cell；The cell can be human liver cancer cell.Any of the above-described C10) in, institute State the expression that " expression of increase Gadd45 albumen " can be Gadd45 albumen in increase cell；The cell can be people Liver cancer cells.Any of the above-described C11) in, " expression quantity of up-regulation puma genes " can be puma genes in up-regulation cell Expression quantity；The cell can be human liver cancer cell.Any of the above-described C12) in, " activity of increase p53 albumen " can For the activity of p53 albumen in increase cell；The cell can be human liver cancer cell.

Any of the above-described A1) in, " activity for suppressing p53 albumen " can be the activity for suppressing p53 albumen in cell； The cell can be human liver cancer cell.Any of the above-described A2) in, " expression for suppressing Puma albumen " can be to suppress The expression of Puma albumen in cell；The cell can be human liver cancer cell.Any of the above-described A3) in, it is described " to lower The expression quantity of puma genes " can be the expression quantity for lowering puma genes in cell；The cell can be human liver cancer cell.Above-mentioned One A4) in, " with p53 protein bindings " can be in cell with p53 protein bindings；The cell can be that human lung cancer is thin Born of the same parents.Any of the above-described A7) in, " apoptosis for suppressing tumour cell " can be the apoptosis for suppressing tumour cell in cell；Institute It can be human colon cancer cell to state cell.

Any of the above-described A3) in, the growth conditions of the human liver cancer cell can be DNA damage stressed condition.

Any of the above-described A4) in, the growth conditions of the human lung carcinoma cell can be that normal growing conditions or DNA damage should Swash condition.

Any of the above-described C9) and C10) in, the growth conditions of the human liver cancer cell can be DNA damage stressed condition.

Any of the above-described C5) and C6) in, the growth conditions of the human liver cancer cell can be normal growing conditions or DNA Damage stressed condition.

Any of the above-described human liver cancer cell can be p53^+/+HepG2 cells or p53^-/-Hep3B cells.

Any of the above-described lung cancer can be non-small cell lung cancer.

Any of the above-described human lung carcinoma cell can be p53^-/-H1299 cells.

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

Any of the above-described ZNF498 albumen can be a1) or a2) or a3) or a4)：

A1) amino acid sequence is the protein shown in sequence 1 in sequence table；

A2) in a1) N-terminal or/and the obtained fused protein of C-terminal connection label；

A3) by the amino acid sequence shown in sequence in sequence table 1 by one or several amino acid residues substitution and/or The protein with identical function that missing and/or addition obtain；

A4) amino acid sequence limited with sequence 1 in sequence table has 80% or more than 80% homogeneity, from people and Protein with identical function.

Any of the above-described p53 albumen can be b1) or b2) or b3) or b4)：

B1) amino acid sequence is the protein shown in sequence 3 in sequence table；

B2) in a1) N-terminal or/and the obtained fused protein of C-terminal connection label；

B3) by the amino acid sequence shown in sequence in sequence table 3 by one or several amino acid residues substitution and/or The protein with identical function that missing and/or addition obtain；

B4) amino acid sequence limited with sequence 3 in sequence table has 80% or more than 80% homogeneity, from people and Protein with identical function.

Wherein, sequence 1 is made up of 544 amino acid residues in sequence table, and sequence 3 is residual by 393 amino acid in sequence table Base forms.

In order that a1) in protein be easy to purify, the amino terminal of protein that can be in sequence table shown in sequence 1 or The upper label as shown in table 1 of carboxyl terminal connection.In order that b1) in protein be easy to purify, can in sequence table the institute of sequence 3 The upper label as shown in table 1 of amino terminal or carboxyl terminal connection of the protein shown.

The sequence of the label of table 1.

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

Above-mentioned a3) or b3) in protein, the substitution of one or several amino acid residues and/or missing and/or add Add as the substitution and/or missing and/or addition for being no more than 10 amino acid residues.

Above-mentioned a3) or b3) in protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression Obtain.

Above-mentioned a3) in the encoding gene of protein can be by the way that one will be lacked in the DNA sequence dna shown in sequence in sequence table 2 The codon of individual or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.

Above-mentioned b3) in the encoding gene of protein can be by the way that one will be lacked in the DNA sequence dna shown in sequence in sequence table 4 The codon of individual or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.

Above-mentioned a4) in, the term " homogeneity " used refers to the sequence similarity with natural acid sequence." homogeneity " is wrapped Include has 80%, or 85% or higher, or 90% or higher with the amino acid sequence in the sequence table of the present invention shown in sequence 1, Or 95% or higher homogeneity amino acid sequence.

Above-mentioned b4) in, the term " homogeneity " used refers to the sequence similarity with natural acid sequence." homogeneity " is wrapped Include has 80%, or 85% or higher, or 90% or higher with the amino acid sequence in the sequence table of the present invention shown in sequence 3, Or 95% or higher homogeneity amino acid sequence.

It is demonstrated experimentally that ZNF498 albumen with p53 protein bindings, can suppress the transcriptional activity of p53 albumen, reduce Puma eggs White expression and the expression quantity of downward puma genes.The transcription of p53 albumen can be increased by suppressing the expression of ZNF498 albumen Activity, the expression for promoting Puma albumen and Gadd45 albumen, the expression quantity for raising puma genes, increase p53 the 46th silk of albumen The growth of the propagation and tumour of the phosphorylation level of propylhomoserin, the apoptosis for promoting p53 positive tumor cells and suppression tumour cell.Cause This, suppresses the expression of ZNF498 albumen in the apoptosis for promoting tumour cell, the propagation for suppressing tumour cell and the life for suppressing tumour There is important application value in length；Suppressing the expression of ZNF498 albumen can prevent and/treating cancer.

Brief description of the drawings

Fig. 1 is the result of the experiment one of embodiment 2.

Fig. 2 is the result of the experiment two of embodiment 2.

Fig. 3 is the result of the experiment three of embodiment 2.

Fig. 4 is the result of the experiment one of embodiment 3.

Fig. 5 is the result of the experiment two of embodiment 3.

Fig. 6 is the result of the experiment three of embodiment 3.

Fig. 7 is the result of embodiment 4.

Fig. 8 is the result of embodiment 5.

Fig. 9 is the result of the experiment one of embodiment 6.

Figure 10 is the result of the experiment two of embodiment 6.

Figure 11 is the result of the experiment one of embodiment 7.

Figure 12 is the result of the experiment two of embodiment 7.

Embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even Average.

The amino acid sequence of ZNF498 albumen is as shown in sequence 1 in sequence table, its encoding gene (hereinafter referred to as ZNF498 base Cause) nucleotide sequence as shown in sequence 2 in sequence table.The amino acid sequence of p53 albumen as shown in sequence 3 in sequence table, its The nucleotide sequence of encoding gene (hereinafter referred to as p53 genes) is as shown in sequence 4 in sequence table.

PG13L plasmids (also known as pG13-Luc plasmids) are recorded in following document：Chunyan Tian, et al.KRAB- type zinc-finger protein Apak specifically regulates p53-dependent apoptosis.Nat Cell Biol.2009May；11(5)：580-91..The plasmid is a kind of reporter plasmid, is opened at it Mover area upstream construct 13 series connection p53 combination original papers, if p53 albumen exist can with p53 combination original paper with reference to so that Cause the expression of the plasmid, and then send fluorescence, the activity of p53 albumen is then judged according to fluorescence intensity.

PCMV-Myc plasmids are the product of Clontech companies.PCMV-Flag plasmids be Sigma companies product, product Catalog number (Cat.No.) is E3762.PRL-TK plasmids be Promega companies product, catalog number E2241.Dual-Luciferase is reported Kit gene be Promega companies product, catalog number E1980.Myc antibody is Medical Biological The product of Baboratoris companies, catalog number M047-3.Myc-HRP antibody be MBL companies product, catalogue Number it is 60004-1.P53 antibody be Calbiochem companies product, catalog number OP43L.HDM2 antibody is won for Huaxing The product of wound company, catalog number HX15828.P53R2 antibody and Gadd45 antibody are the production of Santa Cruz companies Product, catalog number are respectively I1009 and I2210.Flag antibody is the product of SantaCruz companies, and catalog number is 9E10.Flag-HRP antibody be sigma companies product, catalog number SL12445.Puma antibody is Cell The product of Signaling Technology companies, catalog number 4976S.GAPDH antibody is proteintach companies Product, catalog number 60004-1.Etoposide and cisplatin is the product of Sigma companies, and catalog number is E1383 and 106M4763V.PLVshRNA-EGFP (2A) Puro plasmids are the production of the luxuriant industry Bioisystech Co., Ltd of Beijing English Product, catalog number VL3103.P53S46 positions phospho-AB is the product of abcam companies, and catalog number is GR116931-5.Proein-A/G Plus agrose be Santa Cruz companies product, catalog number sc-2003. PsPAX2 and pVSV-G is the product of Addgene companies, and catalog number is respectively zt161 and zt160.

Apoptosis detection kit be invitrogen companies product, catalog number BMS500FI-100；Annexin V-APC combinations liquid, Annexin-APC dyestuffs and propidium iodide are the component in apoptosis detection kit.

HCT116 cells (the also known as p53 of p53 wild types^+/+HCT116cells or p53^+/+HCT116 cells), p53 missing HCT116 cells (the also known as p53 of type^-/-HCT116cells or p53^-/-HCT116 cells) and p53 defects non-small cell lung cancer H1299 cells (also known as p53^-/-H1299cells or p53^-/-H1299 cells) it is recorded in following document：Chunyan Tian, et al.KRAB-type zinc-finger protein Apak specifically regulates p53- dependent apoptosis.Nat Cell Biol.2009May；11(5)：580-91..

Hepatoma Hep G 2 cells (the also known as p53 of p53 wild types^+/+HepG2cells or p53^+/+HepG2 cells) it is preserved in State's Academy of Medical Sciences Institute of Basic Medical Sciences preclinical medicine cell centre, resource number：3111C0001CCC000035.

Liver cancer Hep3B cells (the also known as p53 of p53 defects^-/-Hep3B cells or p53^-/-Hep3B cells) be recorded in as In Publication about Document：Liu X et al.Synergistic inhibitory effects on hepatocellular carcinoma with recombinant human adenovirus Aspp2 and oxaliplatin via p53- independent pathway in vitro and in vivo.Int J Oncol.2017Oct；51(4)：1291- 1299.。

Nude mice in following embodiments is the product of Beijing Vital River Experimental Animals Technology Co., Ltd., production permit Card：SCXK (capital) 2012-0001.

The transfection reagent that the transfection of plasmid uses in following embodiments for TurboFect Transfection Reagent, Specific steps refer to TurboFect Transfection Reagent specification.TurboFect Transfection Reagent is the product of Thermo Scientific companies.The transfection reagent that siRNA transfection uses is Lipofectamine RNAi MAX Reagent, specific steps refer to Lipofectamine RNAi MAX Reagent specification. Lipofectamine RNAi MAX Reagent are the product of invitrogen companies.

Confining liquid：5mg skimmed milk powers are dissolved in into 100mL TBST solution to obtain.TBST solution is to contain 140mM NaCl With 0.1% (v/v) Tween-20 pH7.5,20mM Tris-HCl buffer solutions.

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

No. genebank of ZNF498 genes is 221785.No. genebank of puma genes is 27113.P53R2 genes No. genebank be 50484.No. genebank of Gadd45 genes is 1647.No. genebank of HDM2 genes is 4193.

ZNF498 antibody is standby by Jin Sirui company systems, comprises the following steps that：(1) synthesis polypeptide, the amino acid sequence of polypeptide For (from N-terminal to C-terminal)：GGGSKEKEAKPPQEC；(2) large ear rabbit of the polypeptide immune health prepared with step (1), is collected Serum, obtain ZNF498 antibody.

Embodiment 1, the structure of plasmid, the preparation of nucleic acid oligomer and the acquisition of HepG2 shRNA-ZNF498 cells

1st, the structure of Myc-ZNF498 plasmids

Small fragment between the restriction enzyme EcoR I of pCMV-Myc plasmids and Xho I recognition sequence is replaced with into sequence DNA molecular in table shown in sequence 2, obtained recombinant plasmid are Myc-ZNF498 plasmids.

ZNF498 albumen in Myc-ZNF498 plasmid expression sequence tables shown in sequence 1.

2nd, the structure of Flag-p53 plasmids

Small fragment between the restriction enzyme EcoR I of pCMV-Flag plasmids and BamH I recognition sequence is replaced with into sequence DNA molecular in list shown in sequence 4, obtained recombinant plasmid are Flag-p53 plasmids.

P53 albumen in Flag-p53 plasmid expression sequence tables shown in sequence 3.

3rd, the preparation of nucleic acid oligomer

Positive-sense strand and antisense strand shown in artificial synthesized table 2.Positive-sense strand is diluted with deionized water, obtains positive-sense strand dilution Liquid.Antisense strand is diluted with deionized water, obtains antisense strand dilution.Positive-sense strand dilution and corresponding antisense strand is taken to dilute Liquid, annealing reaction is carried out, form nucleic acid oligomer.

This step prepares 3 nucleic acid oligomers shown in table 2 altogether.A, G, C and U in each nucleic acid oligomer represent that gland is fast successively Purine ribonucleotide, guanosine ribonucleoside acid, cytosine ribonucleotides acid and uracil ribonucleotide, T represent that thymus gland is phonetic Pyridine deoxynucleotide.

SiRNA-con nucleotides sequence is classified as random sequence (as negative control).SiRNAa and siRNAb nucleotides Nucleotide sequence design of the sequence according to sequence in sequence table 2.

Table 2

4th, the acquisition of HepG2 shRNA-ZNF498 cells

(1) structure of shRNA-ZNF498 plasmids and shRNA-con plasmids

Using siRNAa as target spot, using pLVshRNA-EGFP (2A) Puro plasmids as carrier, by the luxuriant industry biology skill of Beijing English Art Co., Ltd builds ZNF498 shRNA slow virus expression plasmids, is named as shRNA-ZNF498 plasmids.

Using siRNA-con as target spot, using pLVshRNA-EGFP (2A) Puro plasmids as carrier, given birth to by the luxuriant industry of Beijing English Thing Technology Co., Ltd. builds negative control shRNA expression plasmids, is named as shRNA-con plasmids.

(2) after completing step (1), 293T cells are inoculated in (blake bottle in the blake bottle equipped with 5mL DMEM culture mediums Specification be 25cm²；Each blake bottle 5.0 × 10⁵Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂18h (this is cultivated in incubator When fusion rate reach 70~90%), be then handled as follows：

First bottle：Every bottle adds 6 μ g shRNA-con plasmids, 4.5 μ g psPAX2,1.5 μ g pVSV-G, cotransfection 24h；

Second bottle：Every bottle adds 6 μ g shRNA-ZNF498 plasmids, 4.5 μ g psPAX2,1.5 μ g pVSV-G, cotransfection 24h。

(3) after completing step (2), every bottle is changed fresh DMEM culture mediums, is placed in 37 DEG C, 5%CO₂Continue in incubator 24-36h is cultivated, obtains nutrient solution；Nutrient solution 1000rpm is centrifuged into 5min, supernatant is collected and (diameter is housed with cell filter Filter membrane for 0.22 μm) filtering, collect filtrate.The filtrate of first bottle of collection is comparison virus liquid, the filtrate of second bottle of collection As experimental virus liquid.

(4) after completing step (3), by p53^+/+HepG2 cells are inoculated in 2 of 6 orifice plates equipped with 0.5mL DMEM culture mediums (per hole 8.0 × 10 in individual hole⁴Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂To be cultivated in incubator, rate to be fused reaches 70~ When 90%, it is handled as follows：

1st hole：3mL comparison virus liquid, 37 DEG C, 5%CO are added per hole₂24h is cultivated in incubator；Change afterwards fresh DMEM culture mediums continue cultivate 48h (whether green fluorescence is sent using fluorescence microscope cell during culture).

2nd hole：3mL experimental virus liquid, 37 DEG C, 5%CO are added per hole₂24h is cultivated in incubator；Change afterwards fresh DMEM culture mediums continue cultivate 48h (whether green fluorescence is sent using fluorescence microscope cell during culture).

(5) treat that more than the 30% of the cells on total cells of green fluorescence can be sent in step (4), Puro added per hole, Luminous most strong cell line is filtered out using fluorescence microscope.

(6) the luminous most strong cell line that step (5) filters out is taken respectively, is filtered out by flow cell sorter luminous Stronger unicellular strain (also will can again pick out unicellular strain after luminous most strong cell line dilution to a certain extent, continue to train Foster screening obtains).

The total protein of the luminous stronger unicellular strain of extraction respectively, is carried out using ZNF498 antibody or GAPDH antibody as primary antibody Western Blot (using GAPDH albumen as internal reference).(it is named as with the unicellular strain that comparison virus liquid inductance contaminates to obtain HepG2 shRNA-con cells) to compare, screen stabilization checking ZNF498 in the unicellular strain for contaminating to obtain from experimental virus liquid inductance The unicellular strain of protein expression (i.e. the expression quantity of ZNF498 albumen significantly reduces), is named as HepG2 shRNA-ZNF498 Cell.

Embodiment 2, ZNF498 albumen can suppress endogenous p53 protein actives

Experiment one, ZNF498 albumen can suppress the transcriptional activity of endogenous p53 albumen

Experiment is averaged in triplicate, and the step of repetition is as follows every time：

1st, by p53^+/+HepG2 cells are inoculated in 12 holes of 24 orifice plates equipped with 0.5mL DMEM culture mediums (per hole 4.0×10⁴Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂Cultivated in incubator, when rate to be fused reaches 70~90%, random point Into four groups, three multiple holes of every group of setting, it is handled as follows：

First group：Each hole adds 20ng pG13L plasmids, 0.2ng pRL-TK plasmids and 0.4 μ g pCMV-Myc plasmids, altogether Transfect 36h.

Second group：Each hole add 20ng pG13L plasmids, 0.2ng pRL-TK plasmids, 0.3 μ g pCMV-Myc plasmids and 0.1 μ g Myc-ZNF498 plasmids, cotransfection 36h.

3rd group：Each hole add 20ng pG13L plasmids, 0.2ng pRL-TK plasmids, 0.2 μ g pCMV-Myc plasmids and 0.2 μ g Myc-ZNF498 plasmids, cotransfection 36h.

4th group：Each hole adds 20ng pG13L plasmids, 0.2ng pRL-TK plasmids and 0.4 μ g Myc-ZNF498 plasmids, Cotransfection 36h.

2nd, after completing step 1, using luciferase reporter gene kit fluorescence intensity, then taken respectively by group Average value, obtain the fluorescence intensity of each group.

Using first group of fluorescence intensity as 1, other groups of relative intensity of fluorescence is calculated.

3rd, after completing step 1, the total protein of each cell is extracted respectively, is entered using myc antibody or GAPDH antibody as primary antibody Row Western Blot (using GAPDH albumen as internal reference).

Experimental result is shown in Fig. 1 (myc is myc antibody, and GAPDH is GAPDH antibody).As a result show, in p53^+/+HepG2 is thin In born of the same parents, ZNF498 albumen can substantially suppress the transcriptional activity of endogenous p53 albumen, and with ZNF498 protein expression levels Increase, the rejection ability increase to p53 protein transcriptions activation capability activity.

Experiment two, ZNF498 albumen rely on the expression of p53 albumen reduction puma albumen

1st, by p53^-/-Hep3B cells are inoculated in 3 holes of 24 orifice plates equipped with 0.5mL DMEM culture mediums (per hole 4.0 ×10⁴Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂Cultivate in incubator, when rate to be fused reaches 70~90%, located as follows Reason：

1st hole：Add 0.7 μ g pCMV-Myc and 0.3 μ g pCMV-Flag plasmids, cotransfection 36h.

2nd hole：Add 0.7 μ g pCMV-Myc and 0.3 μ g Flag-p53 plasmids, cotransfection 36h.

3rd hole：Add 0.7 μ g Myc-ZNF498 plasmids and 0.3 μ g Flag-p53 plasmids, cotransfection 36h.

2nd, after completing step 1, extract the total protein of each cell respectively, with Myc antibody, Flag antibody, Puma antibody or GAPDH antibody is that primary antibody carries out Western Blot (using GAPDH albumen as internal reference).

Experimental result is shown in that (Myc is Myc antibody to Fig. 2, and Flag is Flag antibody, and puma is Puma antibody, GAPDH GAPDH Antibody).As a result show, in p53^-/-In Hep3B cells, ZNF498 albumen, which relies on p53 albumen, reduces puma albumen (Gene ID For：27113) expression.

ZNF498 albumen can suppress the expression of puma genes under the conditions of experiment three, DNA damage

In the present embodiment, cell is handled using Etoposide to simulate DNA damage.

1st, by p53^+/+HepG2 cells are inoculated in 4 holes of 24 orifice plates equipped with 0.5mL DMEM culture mediums (per hole 4.0 ×10⁴Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂Cultivate in incubator, when rate to be fused reaches 70~90%, located as follows Reason：

1st hole：36h without any processing.

2nd hole：24h without any processing；Add Etoposide, obtain system for handling (in system for handling, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator；

3rd hole：Add 0.7 μ g pCMV-Myc plasmids, cotransfection 24h；Then Etoposide is added, obtains handling body System (in system for handling, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂Cultivated in incubator 12h；

4th hole：Add 0.7g Myc-ZNF498 plasmids, cotransfection 24h；Then Etoposide is added, is handled System (in system for handling, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂Trained in incubator Support 12h.

2nd, after completing step 1, extract the total protein of each cell respectively, with Puma antibody, p53 antibody, Myc antibody or GAPDH antibody is that primary antibody carries out Western Blot (using GAPDH albumen as internal reference).

Experimental result is shown in that (puma is puma antibody to Fig. 3, and p53 is p53 antibody, and Myc is Myc antibody, and GAPDH resists for GAPDH Body).As a result show, in p53^+/+After Etoposide processing is added in HepG2 cells, the expression increase of endogenous p53 albumen, ZNF498 albumen can still suppress the expression of endogenous Puma albumen.As can be seen here, ZNF498 albumen can suppress puma genes Expression.

Embodiment 3, the expression for expressing increase Puma albumen for suppressing ZNF498 albumen

After experiment one, the expression of suppression ZNF498 albumen, increase the expression of Puma albumen and Gadd45 albumen

1st, by p53^+/+HepG2 cells are inoculated in 3 holes of 24 orifice plates equipped with 0.5mL DMEM culture mediums (per hole 4.0 ×10⁴Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂Cultivate in incubator, when rate to be fused reaches 70~90%, located as follows Reason：

1st hole：Add 0.5 μ g siRNA-con, cotransfection 36h；

2nd hole：Add 0.5 μ g siRNAa, cotransfection 36h；

3rd hole：Add 0.5 μ g siRNAb, cotransfection 36h.

2nd, after completing step 1, extract the total protein of each cell respectively, with Puma antibody, p53 antibody, ZNF498 antibody, Anti-p53R2, anti-Gadd45, anti-HDM2 or GAPDH antibody are that primary antibody progress Western Blot (use GAPDH eggs It is used as internal reference in vain).

Experimental result is shown in that (puma is Puma antibody to Fig. 4, and p53 is p53 antibody, and ZNF498 is ZNF498 antibody, and p53R2 is P53R2 antibody, Gadd45 are Gadd45 antibody, and Hdm2 is HDM2 antibody, and GAPDH is GAPDH antibody).As a result show, in p53^+/+In HepG2 cells, it is suppressed that after the expression of ZNF498 albumen, on the expression of Puma albumen and Gadd45 albumen Rise；The protein level of other target genes (such as Hdm2, p53R2) of p53 albumen does not change.

Experiment two, the expression for suppressing ZNF498 albumen, raise the expression quantity of puma genes

1st, by p53^+/+HepG2 cells are inoculated in 3 holes of 6 orifice plates equipped with 0.5mL DMEM culture mediums (per hole 2.0 ×10⁵Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂Cultivate in incubator, when rate to be fused reaches 70~90%, located as follows Reason：

1st hole：Add 0.5 μ g siRNA-con, cotransfection 48h；

2nd hole：Add 0.5 μ g siRNAa, cotransfection 48h；

3rd hole：Add 0.5 μ g siRNAb, cotransfection 48h.

2nd, after completing step 1, the total serum IgE of each cell is extracted respectively, the first chain is then gone out using reverse transcriptase reverse transcription CDNA, obtain the cDNA of each cell.Then using Real time PCR puma genes and the relative table of ZNF498 genes Up to amount (internal reference is GAPDH genes).

Detect the primer of Puma genes：5 '-GACCTCAACGCACAGTACGAG-3 ' and 5 '- AGGAGTCCCATGATGAGATTGT-3’.Detect the primer of ZNF498 genes：5 '-GCAGCAGTTGGGTATTCCTGT-3 ' and 5’-GCCGAAAAGTCTCTGGACTAGG-3’.Detect the primer of GAPDH genes：5 '-GGGAAGGTGAAGGTCGGAGT-3 ' and 5’-TTGAGGTCAATGAAGGGGTCA-3’。

Using the relative expression quantity of ZNF498 genes in the cell in the 1st hole as 1, the thin of the 2nd hole and the 3rd hole is calculated The relative expression quantity of ZNF498 genes in born of the same parents.Experimental result is shown in left figure in Fig. 5.

Using the relative expression quantity of Puma genes in the cell in the 1st hole as 1, the cell in the 2nd hole and the 3rd hole is calculated The relative expression quantity of middle Puma genes.Experimental result is shown in right figure in Fig. 5.

As a result show, in p53^+/+In HepG2 cells, siRNAa and siRNAb can effectively suppress ZNF498 genes Expression quantity；After expression by reducing endogenous ZNF498 albumen, the expression quantity of puma genes is raised.

Suppress the expression of the expression increase Puma albumen of ZNF498 albumen under the conditions of experiment three, DNA damage

Using Etoposide processing cell simulation DNA damage conditions in this experiment.

1st hole：Add 0.5 μ g siRNA-con, cotransfection 36h；

2nd hole：Add 0.5 μ g siRNA-con, cotransfection 24h；Then Etoposide is added, obtains system for handling (in system for handling, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂Cultivated in incubator 12h；

3rd hole：Add 0.5 μ g siRNAa, cotransfection 24h；Then Etoposide is added, obtains system for handling (place In reason system, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator；

4th hole：Add 0.5 μ g siRNAb, cotransfection 24h；Then Etoposide is added, obtains system for handling (place In reason system, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator.

2nd, after completing step 1, extract the total protein of each cell respectively, with Puma antibody, p53 antibody, ZNF498 antibody, P53R2 antibody, Gadd45 antibody, HDM2 antibody or GAPDH antibody are that primary antibody progress Western Blot (use GAPDH albumen As internal reference).

Experimental result is shown in that (puma is Puma antibody to Fig. 6, and p53 is p53 antibody, and ZNF498 is ZNF498 antibody, and p53R2 is P53R2 antibody, Gadd45 are Gadd45 antibody, and Hdm2 is HDM2 antibody, and GAPDH is GAPDH antibody).As a result show, in p53^+/+In HepG2 cells, under conditions of DNA damage, endogenous p53 protein levels increase；After ZNF498 protein expressions suppress, Puma The expression of albumen and Gadd45 albumen rises；The expression of Hdm2 albumen and p53R2 albumen does not change.

Embodiment 4, under conditions of normal condition and DNA damage, interaction be present in ZNF498 and p53

Under the conditions of detecting normal condition and DNA damage by co-immunoprecipitation experiment, ZNF498 albumen is with p53 albumen in body Interior binding ability.In triplicate, the step of repetition is as follows every time for experiment：

1st, by p53^-/-(specification of blake bottle is in the incoming blake bottle equipped with 5mL DMEM culture mediums of H1299 cells 25cm²；Each blake bottle 5.0 × 10⁵Individual cell), it is placed in 37 DEG C, 5%CO₂18h is cultivated in incubator, and (now fusion rate reaches 70~90%), then it is handled as follows：

1st blake bottle：Add 5.0 μ g pCMV-Myc plasmids and 3.0 μ g Flag-p53 plasmids, cotransfection 48h；Then Liquid phase is abandoned, washing precipitation 3 times with pH7.4,0.01M of precooling PBS, (last 1 washing need to be by pH7.4,0.01M PBS blots)；

2nd blake bottle:Add 5.0 μ g Myc-ZNF498 plasmids and 3.0 μ g Flag-p53 plasmids, cotransfection 48h；So After abandon liquid phase, with pH7.4,0.01M of precooling PBS wash precipitation 3 times (it is last 1 time washing need to be by pH7.4,0.01M PBS blot)；

3rd blake bottle：Add 5.0 μ g pCMV-Myc plasmids and 3.0 μ g Flag-p53 plasmids, cotransfection 36h；Then Add Etoposide, obtain system for handling (in system for handling, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator, then abandons liquid phase, precipitation 3 is washed with pH7.4,0.01M of precooling PBS Secondary (last 1 washing need to blot pH7.4,0.01M PBS)；

4th blake bottle:Add 5.0 μ g Myc-ZNF498 plasmids and 3.0 μ g Flag-p53 plasmids, cotransfection 36h；So After add Etoposide, obtain system for handling (in system for handling, Etoposide concentration is 30 μM)；The system for handling is put In 37 DEG C, 5%CO₂12h is cultivated in incubator, then abandons liquid phase, it is heavy to be washed with pH7.4,0.01M of precooling PBS Form sediment 3 times (last 1 washing need to blot pH7.4,0.01M PBS)；

5th blake bottle：Add 5.0 μ g pCMV-Myc plasmids and 3.0 μ g Flag-p53 plasmids, cotransfection 36h；Then Add cisplatin, obtain system for handling (in system for handling, cisplatin concentration is 20 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator, then abandons liquid phase, precipitation 3 is washed with pH7.4,0.01M of precooling PBS Secondary (last 1 washing need to blot pH7.4,0.01M PBS)；

6th blake bottle:Add 5.0 μ g Myc-ZNF498 plasmids and 3.0 μ g Flag-p53 plasmids, cotransfection 36h；So After add cisplatin, obtain system for handling (in system for handling, cisplatin concentration is 20 μM)；The system for handling is put In 37 DEG C, 5%CO₂12h is cultivated in incubator, then abandons liquid phase, it is heavy to be washed with pH7.4,0.01M of precooling PBS Form sediment 3 times (last 1 washing need to blot pH7.4,0.01M PBS).

2nd, after completing step 1, pancreatin digestive juice (purpose is to be digested to cell) is separately added into each blake bottle, Then centrifuge tube (specification 10mL) is transferred to successively, 1000rpm centrifugation 5min, collects precipitation.

3rd, the precipitation for taking step 2 to collect respectively, wash 2 times.Every time washing the step of be：Add 1mL pH7.4,0.01M PBS, 1000rpm centrifugation 5min.Last 1 washing need to blot pH7.4,0.01M PBS.

4th, the precipitation for taking step 3 to collect respectively, the NETN lysates of 600 μ L precoolings is added, are incubated on ice after well mixed 30min；Then 4 DEG C, 12000rpm centrifugation 10min, collect supernatant.

NETN lysates：To 1mL NaCl containing 150mM, 1mM EDTA and 0.5% (m/m) NP-40 pH8.0,20mM 50 × protease inhibitors of 0.02mL is added in Tris-Cl buffer solutions, is mixed.50 × protease inhibitors be DTT containing 1mM, 1mM NaV₃O4 the and 1mM NaF aqueous solution.

5th, the supernatant for taking 40 μ L steps 4 to collect respectively, 40 μ 2 × sample-loading buffers of L are added, mixed, 100 DEG C are boiled 10min, obtained solution are Lysate.

6th, the supernatant for respectively collecting step 4 is transferred to centrifuge tube (specification 1.5mL), and often pipe adds 1 μ L myc and resisted Body, 4 DEG C are slowly shaken 3h；Then often pipe adds 40 μ L proein-A/G Plus agrose, is placed on rotary mixer, 4 DEG C Overnight.

7th, after completing step 6, precipitation (i.e. agrose and antigen antibody complex) is collected respectively, then places 1min on ice (purpose is to settle sepharose 4B), 4 DEG C, 3000rpm centrifugation 5min, collects precipitation.

8th, the precipitation for taking step 7 to collect respectively, wash 4 times.Every time washing the step of be：The NETN for adding 1mL precoolings splits Solve liquid, 4 DEG C, 3000rpm centrifugations 2min.

9th, the precipitation for taking step 8 to collect respectively, the resuspension of 40 μ L NETN lysates is first added, adds 40 μ 2 × loadings of L Buffer solution, mix, 100 DEG C are boiled 10min, and obtained solution is Myc-IP.

With Flag-HRP antibody diluents (being obtained with confining liquid dilution Flag-HRP antibody to 1000 times of volumes) or Myc- HRP antibody diluents (being obtained with confining liquid dilution Myc-HRP antibody to 1000 times of volumes) are primary antibody, to Lysate and Myc-IP Carry out Western Blot.

Experimental result is shown in that (untreated is normal condition to Fig. 7, and etoposide is to add Etoposide into system to enter Row processing, cisplatin are that cisplatin processing is added into system；Flag is Flag-HRP antibody, Myc Myc- HRP antibody).As a result show, under conditions of normal condition and DNA damage, ZNF498 and p53 have interaction, i.e., ZNF498 albumen has certain binding ability in vivo with p53 albumen.

Embodiment 5, the phosphorylation level for expressing increase p53 the 46th serine of albumen for suppressing ZNF498 albumen

1st, by p53^+/+HepG2 cells are inoculated in 6 holes of 24 orifice plates equipped with 0.5mL DMEM culture mediums (per hole 4.0 ×10⁴Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂18h (now fusion rate reaches 70~90%) is cultivated in incubator, is carried out such as Lower processing：

1st hole：0.5 μ g siRNA-con are added, transfect 36h；

2nd hole：0.5 μ g siRNAa are added, transfect 36h；

3rd hole：0.5 μ g siRNAb are added, transfect 36h；

4th hole：0.5 μ g siRNA-con are added, transfect 24h；Then Etoposide is added, obtains system for handling (place In reason system, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator；

5th hole：0.5 μ g siRNAa are added, transfect 24h；Then Etoposide is added, obtains system for handling (processing In system, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator；

6th hole：0.5 μ g siRNAb are added, transfect 24h；Then Etoposide is added, obtains system for handling (processing In system, Etoposide concentration is 30 μM)；The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator.

2nd, after completing step 1, the total protein of each cell is extracted respectively, is resisted with p53S46 positions phospho-AB, ZNF498 Body, p53 antibody or GAPDH antibody are that primary antibody carries out Western Blot (using GAPDH albumen as internal reference).

Experimental result is shown in that (untreated is normal condition to Fig. 8, and etoposide is to add Etoposide into system to enter Row processing, P-p53-Ser46 is p53S46 positions phospho-AB, and ZNF498 is ZNF498 antibody, and p53 is p53 antibody, GAPDH For GAPDH antibody).As a result show, under the conditions of the normal and DNA damage under the conditions of, siRNAa and siRNAb can effectively press down The expression of ZNF498 albumen processed, and after the expression inhibiting of ZNF498 albumen, can increase the phosphorus of the 46th serine of p53 albumen Acidifying is horizontal.

Embodiment 6, the apoptosis for expressing promotion p53 positive tumor cells for suppressing ZNF498 albumen

Experiment one, ZNF498 albumen suppress the apoptosis of tumour cell by regulating and controlling p53 albumen

1st, 6 orifice plates are taken, 2mL DMEM culture mediums are added per hole, then add p53 into the 1st hole and the 2nd hole^+/+ HCT116 cells are (per hole 2.0 × 10⁵Individual cell), add p53 into the 3rd hole, the 4th hole, the 5th hole and the 6th hole^-/- HCT116 cells are (per hole 2.0 × 10⁵Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂18h is cultivated in incubator, and (now fusion rate reaches To 70~90%) when, it is handled as follows：

1st hole：Add 2 μ g pCMV-Myc plasmids, cotransfection 48h；

2nd hole：Add 2 μ g Myc-ZNF498 plasmids, cotransfection 48h；

3rd hole, add 2 μ g pCMV-Myc plasmids and 0.4 μ g pCMV-Flag plasmids, cotransfection 48h.

4th hole, add 2 μ g Myc-ZNF498 plasmids and 0.4 μ g pCMV-Flag plasmids, cotransfection 48h.

5th hole, add 2 μ g pCMV-Myc plasmids and 0.4 μ g Flag-p53 plasmids, cotransfection 48h.

6th hole, add 2 μ g Myc-ZNF498 plasmids and 0.4 μ g Flag-p53 plasmids, cotransfection 48h.

2nd, complete step 1 after, take 6 orifice plates, abandon liquid phase (if having the dead cell largely to suspend in liquid phase, suspension it is dead Cell will be also collected), then add pancreatin digestive juice to each hole and digested, add appropriate DMEM culture mediums termination and disappear Change；Centrifuge tube (specification 10mL) is finally transferred to, 4 DEG C, 1000rpm centrifugation 5min, collects cell precipitation.

3rd, the cell precipitation for taking step 2 to collect respectively, the PBS for adding pH7.4,0.01M of precooling suspend, 4 DEG C, 1000rpm centrifuges 5min, regathers cell precipitation.

4th, the cell precipitation for taking step 3 to collect respectively, the Annexin V-APC combinations liquid for first adding 200 μ L suspend, then 5 μ L Annexin-APC dyestuffs and propidium iodide are added, is incubated at room temperature 15min.

5th, the system after step 4 is taken into, using the change of flow cytomery apoptosis.

Experimental result is shown in Fig. 9.As a result show, to p53^+/+It is transferred in HCT116 cells the (the i.e. the 2nd after Myc-ZNF498 plasmids Individual hole), the apoptosis rate of cell is less than control group (i.e. the 1st hole)；To p53^-/-Myc-ZNF498 matter is only transferred in HCT116 cells Grain, does not influence on the apoptosis rate of cell；To p53^-/-After Flag-p53 plasmids are transferred in HCT116 cells, the apoptosis rate of cell Substantially increase, while be transferred to Myc-ZNF498 plasmids can then suppress the apoptosis of cell.ZNF498 albumen is by regulating and controlling p53 albumen Suppress the apoptosis of tumour cell.

Experiment two, the apoptosis for expressing promotion p53 positive tumor cells for suppressing ZNF498

1st, 6 orifice plates are taken, 2mL DMEM culture mediums are added per hole, are then added into the 1st hole and the 2nd hole HepG2shRNA-ZNF498 cells are (per hole 2.0 × 10⁵Individual cell), add HepG2 into the 3rd hole and the 4th hole ShRNA-con cells are (per hole 2.0 × 10⁵Individual cell), it is subsequently placed in 37 DEG C, 5%CO₂18h is cultivated in incubator (now to merge Rate reaches 70~90%) when, it is handled as follows：

1st hole：Without handling 12h；

2nd hole：Add Etoposide, obtain system for handling (in system for handling, Etoposide concentration is 30 μM)； The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator；

3rd hole：Without handling 12h；

4th hole：Add Etoposide, obtain system for handling (in system for handling, Etoposide concentration is 30 μM)； The system for handling is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator.

2nd, with step 12.

3rd, with step 13.

4th, with step 14.

5th, with step 15.

Experimental result is shown in that (untreated is normal condition to Figure 10, and etoposide is to add Etoposide into system to enter Row processing).As a result show, under normal operation, it is suppressed that after the expression of ZNF498 albumen, the apoptosis rate of cell rises；In DNA Under conditions of damage, the apoptosis rate increase of cell, then suppress the apoptosis rate rising increase of cell after the expression of ZNF498 albumen.By This is visible, and ZNF498 albumen can suppress the apoptosis of p53 positive tumor cells under the conditions of normal condition and DNA damage.Suppression ZNF498 processed expression promotes the apoptosis of p53 positive tumor cells.

Embodiment 7, suppress ZNF498 albumen expression inhibiting tumour cell propagation and tumour growth

Test the propagation of the expression inhibiting tumour cell of one, suppression ZNF498 albumen

1st, 96 orifice plates are taken, 100 μ L DMEM culture mediums are added per hole, then add HepG2 in 18 holes thereto ShRNA-ZNF498 cells are (per hole 1.0 × 10⁵Individual cell), it is (every that HepG2 shRNA-con cells are added into other 18 holes Hole 1.0 × 10⁵Individual cell).Above-mentioned 36 holes are randomly divided into 6 groups, it is every group thin including 3 addition HepG2 shRNA-ZNF498 The hole of born of the same parents and the hole of 3 addition HepG2 shRNA-con cells.

2nd, after completing step 1,96 orifice plate is placed in 37 DEG C, 5%CO₂16h is cultivated in incubator, and (now fusion rate reaches To 70~90%) when, then it is handled as follows：

First group：10 μ L CCK-8 is added, detects 450nm absorbance after 2h with ELIASA.

Second group：37 DEG C, 5%CO₂Continue to cultivate 24h in incubator, then add 10 μ L CCK-8, with enzyme mark after 2h Instrument detects 450nm absorbance.

3rd group：37 DEG C, 5%CO₂Continue to cultivate 48h in incubator, then add 10 μ L CCK-8, with enzyme mark after 2h Instrument detects 450nm absorbance.

4th group：37 DEG C, 5%CO₂Continue to cultivate 72h in incubator, then add 10 μ L CCK-8, with enzyme mark after 2h Instrument detects 450nm absorbance.

5th group：37 DEG C, 5%CO₂Continue to cultivate 96h in incubator, then add 10 μ L CCK-8, with enzyme mark after 2h Instrument detects 450nm absorbance.

6th group：37 DEG C, 5%CO₂Continue to cultivate 120h in incubator, then add 10 μ L CCK-8, with enzyme mark after 2h Instrument detects 450nm absorbance.

The absorbance that six groups of statistical analysis, then calculate the average value and error of each group of absorbance.With first group Mean light absorbency be 1, calculate second group, the 3rd group, the 4th group, the 5th group and the 6th group of Relative Absorbance value respectively. Analyze the multiplication capacity of HepG2 shRNA-con and shRNA-ZNF498 cells.

Experimental result is shown in Figure 11.As a result show, compared with HepG2 shRNA-con cells, HepG2 shRNA-ZNF498 The ability of cell proliferation of cell reduces.As can be seen here, the propagation of the expression inhibiting tumour cell of ZNF498 albumen, ZNF498 are suppressed Albumen can promote the propagation of tumour cell.

Test the growth of the expression inhibiting tumour of two, suppression ZNF498 albumen

1st, HepG2 shRNA-con cells or HepG2 shRNA-ZNF498 cells are seeded to and cultivated equipped with appropriate DMEM In the Tissue Culture Dish (specification 20cm) of base, 37 DEG C, 5%CO₂Culture to fusion rate reaches 70~90% in incubator.

2nd, after completing step 1, the Tissue Culture Dish is taken, first addition pancreatin is digested to unicellular, adds appropriate DMEM Culture medium terminates digestion, is transferred to centrifuge tube, 4 DEG C, 1000rpm centrifugation 5min, collects cell precipitation.

3rd, the cell precipitation for taking step 2 to collect, the PBS for adding pH7.4,0.01M of precooling suspend, 4 DEG C, 1000rpm centrifuges 5min, regathers cell precipitation.

4th, the cell precipitation for taking step 3 to collect, the PBS for adding pH7.4,0.01M of precooling suspend, and obtain cell Suspension.

5th, 10 nude mices for growing to 4 week old are taken, every nude mice left dorsal injects the thin of HepG2 shRNA-con cells Born of the same parents' suspension (contains 2 × 10⁶Individual cell), the cell suspension of right side injection HepG2 shRNA-ZNF498 cells (contains 2 × 10⁶It is individual thin Born of the same parents), the volume of tumor mass is then measured week about, stops measurement after tumor mass grows to proper volume.Statistical analysis is injected The change of the tumor mass of HepG2shRNA-con cells and HepG2 shRNA-ZNF498 cells.

Experimental result is shown in Figure 12.As a result show, the volume of the tumor mass grown up to dorsal injection HepG2 shRNA-con cells Compare, the volume for the tumor mass that dorsal injection HepG2 shRNA-ZNF498 cells grow up to is significantly smaller.As can be seen here, suppress The growth of the expression inhibiting tumour of ZNF498 albumen, ZNF498 albumen suppress the speed that tumor mass grows after expressing and slowed down.

<110>Beijing Proteome Research Center

<120>Suppress application of the material of ZNF498 expressing quantities in the product for preparing prevention and treatment cancer

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 544

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<213>Artificial sequence

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<223>

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Arg Tyr Gln Glu Ala Ala Gly Pro Gln Glu Ala Leu Arg Glu Leu Gln

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Glu Leu Cys Arg Arg Trp Leu Arg Pro Glu Leu His Thr Lys Glu Gln

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Ile Leu Glu Leu Leu Val Leu Glu Gln Phe Leu Thr Ile Leu Pro Arg

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Glu Phe Tyr Ala Trp Ile Arg Glu His Gly Pro Glu Ser Gly Lys Ala

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Leu Ala Ala Met Val Glu Asp Leu Thr Glu Arg Ala Leu Glu Ala Lys

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Ala Val Pro Cys His Arg Gln Gly Glu Gln Glu Glu Thr Ala Leu Cys

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Arg Gly Ala Trp Glu Pro Gly Ile Gln Leu Gly Pro Val Glu Val Lys

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Pro Glu Trp Gly Met Pro Pro Gly Glu Gly Val Gln Gly Pro Asp Pro

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Gly Thr Glu Glu Gln Leu Ser Gln Asp Pro Gly Asp Glu Thr Arg Ala

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Phe Gln Glu Gln Ala Leu Pro Val Leu Gln Ala Gly Pro Gly Leu Pro

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Ala Val Asn Pro Arg Asp Gln Glu Met Ala Ala Gly Phe Phe Thr Ala

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Gly Ser Gln Gly Leu Gly Pro Phe Lys Asp Met Ala Leu Ala Phe Pro

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Glu Glu Glu Trp Arg His Val Thr Pro Ala Gln Ile Asp Cys Phe Gly

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Glu Tyr Val Glu Pro Gln Asp Cys Arg Val Ser Pro Gly Gly Gly Ser

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Lys Glu Lys Glu Ala Lys Pro Pro Gln Glu Asp Leu Lys Gly Ala Leu

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Val Ala Leu Thr Ser Glu Arg Phe Gly Glu Ala Ser Leu Gln Gly Pro

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Gly Leu Gly Arg Val Cys Glu Gln Glu Pro Gly Gly Pro Ala Gly Ser

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Ala Pro Gly Leu Pro Pro Pro Gln His Gly Ala Ile Pro Leu Pro Asp

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Glu Val Lys Thr His Ser Ser Phe Trp Lys Pro Phe Gln Cys Pro Glu

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Cys Gly Lys Gly Phe Ser Arg Ser Ser Asn Leu Val Arg His Gln Arg

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Thr His Glu Glu Lys Ser Tyr Gly Cys Val Glu Cys Gly Lys Gly Phe

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Thr Leu Arg Glu Tyr Leu Met Lys His Gln Arg Thr His Leu Gly Lys

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Arg Pro Tyr Val Cys Ser Glu Cys Trp Lys Thr Phe Ser Gln Arg His

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His Leu Glu Val His Gln Arg Ser His Thr Gly Glu Lys Pro Tyr Lys

420 425 430

Cys Gly Asp Cys Trp Lys Ser Phe Ser Arg Arg Gln His Leu Gln Val

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His Arg Arg Thr His Thr Gly Glu Lys Pro Tyr Thr Cys Glu Cys Gly

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Lys Ser Phe Ser Arg Asn Ala Asn Leu Ala Val His Arg Arg Ala His

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Thr Gly Glu Lys Pro Tyr Gly Cys Gln Val Cys Gly Lys Arg Phe Ser

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

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Leu Asn Arg His Gln Lys Thr Gln His Arg Gln Glu Pro Leu Val Gln

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tttcggctga ggtttcggca gttccgctac caggaggcag ctggacccca ggaagctctt 180

agggagctcc aggagctctg tcgtcggtgg ctgaggcccg agttgcacac caaggagcag 240

atcctggagc tgctggtgct ggagcagttc ctcactatcc tgccccgcga gttctacgcc 300

tggatccggg agcatggccc agagagtggc aaggccctgg ccgccatggt ggaggacctg 360

acagaaagag cactggaggc caaggcggtt ccatgccaca ggcagggaga gcaggaggaa 420

acagcacttt gcagaggcgc ttgggagcca ggcatccagc tggggccagt ggaggtcaag 480

cctgaatggg ggatgccccc tggggaagga gttcaaggtc cagacccagg taccgaggag 540

cagctcagtc aggaccctgg agatgagaca cgggccttcc aggagcaagc actacctgtt 600

ctgcaggcgg gtcctggcct ccccgcagtg aatcccagag accaagagat ggcagctggg 660

ttctttactg ctggatcgca ggggttgggg ccatttaaag atatggccct ggccttccct 720

gaggaggagt ggaggcatgt gaccccagcc cagatagact gctttgggga gtatgtggaa 780

ccgcaggact gcagggtctc tccaggcggt gggagcaagg aaaaggaggc aaaaccccca 840

caggaagacc tgaaaggggc gctggtggca ctgacatcag agaggtttgg ggaagcctct 900

ctccagggcc ctgggctcgg aagggtctgt gagcaggagc ctggtggccc tgcaggcagt 960

gcgcctgggc ttcctcctcc ccagcacggt gccatccccc tgcctgacga agtcaaaacc 1020

cacagctcct tctggaagcc tttccagtgc cctgagtgtg ggaaaggatt cagtcggagc 1080

tccaatctcg tcaggcacca gcgaacccac gaagagaagt cttatggctg tgtggagtgt 1140

gggaagggct ttaccctgag agaatacctg atgaagcacc agagaaccca cctgggaaag 1200

aggccctacg tgtgcagcga gtgctggaaa accttcagcc agagacacca cctggaggtg 1260

caccagcgca gccacactgg ggagaagccc tacaagtgcg gggactgctg gaagagcttc 1320

agccgcaggc agcacctgca ggtgcaccgg aggacgcaca ccggggagaa gccctacacc 1380

tgcgagtgtg gcaagagctt cagcaggaat gccaatctgg cggtgcaccg gcgtgcccac 1440

actggcgaga agccatatgg gtgccaggtg tgcgggaagc ggttcagcaa aggggagcgg 1500

ctggtccgac accagagaat ccatacaggg gagaagccct accactgtcc tgcctgcggg 1560

cgaagcttca accagaggtc catcctcaac cggcaccaga agacccagca ccgccaggag 1620

ccgctggtgc agtga 1635

<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

agcgcaccau cacaucuaat t 21

<210> 6

<211> 21

<212> DNA/RNA

<213>Artificial sequence

<220>

<223>

<400> 6

uuagauguga uggugcgcut t 21

<210> 7

<211> 21

<212> DNA/RNA

<213>Artificial sequence

<220>

<223>

<400> 7

cccacgaaga gaagucuuat t 21

<210> 8

<211> 21

<212> DNA/RNA

<213>Artificial sequence

<220>

<223>

<400> 8

uaagacuucu cuucgugggt t 21

Claims

1. application of the material of suppression ZNF498 protein actives and/or expression quantity in product is prepared；The function of the product is At least one of following C1) to C12)：

C1) pre- anti-cancer；C2) treating cancer；C3 the propagation of tumour cell) is suppressed；C4 the growth of tumour) is suppressed；C5) promote swollen The apoptosis of oncocyte；C6 the apoptosis of p53 positive tumor cells) is promoted；C7 it is horizontal) to increase p53 protein phosphorylations；C8 p53) is increased The phosphorylation level of the 46th serine of albumen；C9 the expression of Puma albumen) is increased；C10 the table of Gadd45 albumen) is increased Up to level；C11 the expression quantity of puma genes) is raised；C12 the activity of p53 albumen) is increased.

At least one of it is following C1 2. suppressing the application of the material of ZNF498 protein actives and/or expression quantity) to C12)：

3. application as claimed in claim 1 or 2, it is characterised in that：It is described " to suppress ZNF498 protein actives and/or expression quantity Material " be z1) z2) or z3) z4) or z5) or z6)：

Z1) nucleic acid oligomer siRNAa；The nucleic acid oligomer siRNAa single stranded nucleic acid molecule and sequence as shown in the sequence 5 in sequence table Single stranded nucleic acid molecule composition shown in the sequence 6 of list；

Z2) nucleic acid oligomer siRNAb；The nucleic acid oligomer siRNAb single stranded nucleic acid molecule and sequence as shown in the sequence 7 in sequence table Single stranded nucleic acid molecule shown in the sequence 8 of list forms；

Application of the 4.ZNF498 albumen as drug target in product is prepared；The function of the product is following C1) to C12) At least one of：

5. the application as described in Claims 1-4 is any, it is characterised in that：

The C1) or the C2) in, the cancer is liver cancer, lung cancer or colon cancer；

The C3) in, the tumour cell is human liver cancer cell；

The C4) in, the tumour is the tumour that human liver cancer cell is formed in Mice Body；

The C5) in, the tumour cell is human liver cancer cell or human colon cancer cell；

The C6) in, the tumour cell is human liver cancer cell or human colon cancer cell；

The C7) in, " increase p53 protein phosphorylations are horizontal " is horizontal for p53 protein phosphorylations in increase cell；It is described thin Born of the same parents are human liver cancer cell；

The C8) in, the phosphorylation level of p53 the 46th serine of albumen " increase " is p53 albumen in increase cell the The phosphorylation level of 46 serines；The cell is human liver cancer cell；

The C9) in, " expression of increase Puma albumen " is the expression of Puma albumen in increase cell；It is described Cell is human liver cancer cell；

The C10) in, " expression of increase Gadd45 albumen " is the expression water of Gadd45 albumen in increase cell It is flat；The cell is human liver cancer cell；

The C11) in, " expression quantity of up-regulation puma genes " is the expression quantity of puma genes in up-regulation cell；It is described thin Born of the same parents are human liver cancer cell；

The C12) in, " activity of increase p53 albumen " is the activity of p53 albumen in increase cell；The cell is behaved Liver cancer cells.

6. the material of any the suppression ZNF498 protein actives and/or expression quantity in claim 1,2,3 or 5.

7.X1) or X2)：

X1) application of the ZNF498 albumen in product is prepared；At least one of the function of the product is A1) to A11)；

At least one of X2) the application of ZNF498 albumen, it is A1) to A11)；

A1 the activity of p53 albumen) is suppressed；A2 the expression of Puma albumen) is suppressed；A3 the expression quantity of puma genes) is lowered； A4) with p53 protein bindings；A5 the propagation of tumour cell) is promoted；A6 the growth of tumour) is promoted；A7 withering for tumour cell) is suppressed Die；A8 the apoptosis of p53 positive tumor cells) is suppressed；A9 it is horizontal) to suppress p53 protein phosphorylations；A10 p53 albumen the 46th) is suppressed The phosphorylation level of position serine；A11 the expression of Gadd45 albumen) is suppressed.

8. application as claimed in claim 7, it is characterised in that：

The A1) in, " activity for suppressing p53 albumen " is the activity for suppressing p53 albumen in cell；The cell behaviour liver Cancer cell；

The A2) in, " expression for suppressing Puma albumen " is the expression for suppressing Puma albumen in cell；It is described Cell is human liver cancer cell；

The A3) in, " expression quantity for lowering puma genes " is the expression quantity for lowering puma genes in cell；The cell For human liver cancer cell；

The A4) in, " with p53 protein bindings " be in cell with p53 protein bindings；The cell is that human lung cancer is thin Born of the same parents；

The A7) in, " apoptosis for suppressing tumour cell " is the apoptosis for suppressing tumour cell in cell；The cell is behaved Colon cancer cell.

9. product first or product second；

The product first contains the thing of any the suppression ZNF498 protein actives and/or expression quantity in claim 1,2,3 or 5 Matter；At least one of the function of the product first is following C1) to C12)：

C1) pre- anti-cancer；C2) treating cancer；C3 the propagation of tumour cell) is suppressed；C4 the growth of tumour) is suppressed；C5) promote swollen The apoptosis of oncocyte；C6 the apoptosis of p53 positive tumor cells) is promoted；C7 it is horizontal) to increase p53 protein phosphorylations；C8 p53) is increased The phosphorylation level of the 46th serine of albumen；C9 the expression of Puma albumen) is increased；C10 the table of Gadd45 albumen) is increased Up to level；C11 the expression quantity of puma genes) is raised；C12 the activity of p53 albumen) is increased；

The product second contains ZNF498 albumen；At least one of the function of the product second is following A1) to A11)：

10. application of the material of suppression ZNF498 protein actives and/or expression quantity in exploitation or screening reagent；The reagent At least one of purposes is following C1) to C12)：