CN110302382A - A kind of drug of targets neoplastic cells - Google Patents
A kind of drug of targets neoplastic cells Download PDFInfo
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Abstract
The present invention relates to a kind of drugs of targets neoplastic cells.The present invention provides a kind of ZER6 inhibitor, is used to prevent and/or treat tumour, wherein the inhibitor is preferably the expression that specificity inhibits p52-ZER6 gene and/or albumen, the expression without inhibiting p71-ZER6 gene and/or albumen.The present invention also provides a kind of methods for preparing ZER6 inhibitor, prevent and/or treat the purposes of tumour by the inhibitor.
Description
Technical field
The present invention relates to the prevention of disorders such as cancers and/or therapy fields.Specifically, the present invention relates to act on ZER6
The inhibitor of gene and/or albumen is for preventing and/or treating disorders such as cancers.
Background technique
According to the statistical estimate of the World Health Organization (World Health Organization): cancer is 91 countries 70
Year the pervious big cause of death of the first or second.Cancer is in rising trend always in the morbidity and mortality of China, and cancer is certainly
Have become the most important cause of death over 2010, cancer mortality accounts for the 23.91% of China resident whole cause of the death, and becomes mesh
The preceding most important public health problem of China.During the nearly last ten years, Chinese cancer morbidity, the death rate rise every year, cancer hair
The sick every average annual growth rate of rate about 3.9%, the every average annual growth rate 2.5% of the death rate, therefore malignant tumour are to endanger people's life to be good for
The research and development of the major disease of health, anti-tumor drug shoulder heavy responsibilities.In recent years, with the rapid development of medicine and pharmacy, people
The essence for gradually recognizing cell carcinogenesis is cell infinite multiplication caused by the imbalance of intracellular signal transduction pathway, following
It is the great change of anti-tumor drug research and development theory.The focus of research and development is being transferred to from conventional cell cytotoxic drug thin for tumour
The specificity of abnormal signal system target site intracellular anti-tumor drug of new generation.Different from conventional cell cytotoxic drug poor selectivity, poison
Side effect is strong, is also easy to produce the features such as drug resistance, and target spot specificity antineoplastic medicine is directed between normal cell and tumour cell
Difference has reached highly selective, hypotoxicity therapeutic effect, including targeting tyrosine kinase, angiogenesis, tumour cell cycle
Correlation factor, inhibitors of histone deacetylase, tumor microenvironment, tumor stem cell, tumor metabolic exception etc..Although in recent years
The relevant molecular mechanism of tumor development is gradually illustrated, but still does not have to find most effective inhibition cancer at present
The suitable molecule target spot of development.
ZER6 (alias zinc finger protein 39 8, zinc finger protein 398, ZNF398) gene was in quilt in 2002
The discovery such as Conroy.ZER6 gene generates two kinds of spliced bodies, i.e. p52-ZER6 and p71-ZER6.However be found from 2002 with
Come, biological function and their molecular regulation mechanism in relation to ZER6 and its spliced body p52-ZER6 and p71-ZER6 are not yet
By reporting, the state of complete blank is currently in the understanding of their function and molecular mechanism.
Summary of the invention
Inventor has screened the shRNA expression plasmid library comprising 3,354 shRNA expression plasmids, explores regulation p21
The new regulatory factor of transcriptional activity, and before the selection result 10% p21 candidate inhibiting factor is analyzed and studied.Wherein,
Zinc finger protein ZER6 (alias ZNF398) is found in 10% candidate inhibiting factor before the selection result.ZER6 includes p52-ZER6
With two kinds of hypotypes of p71-ZER6 (two kinds are respectively referred to caused by the mRNA that No. Refseq is NM_020781.3 and NM_170686.2
Albumen, protein Refseq is respectively each mRNA of NP_065832.1 and NP_733787.1 or corresponding and protein
No. Refseq more new version), as shown in Figure 1.P52-ZER6 and p71-ZER6 protein C-terminal having the same, however their N
It holds different.The zinc finger protein structure of p52-ZER6 and p71-ZER6 is their C-terminal.The N-terminal of p71-ZER6
Comprising a KRAB structural domain (59 amino acid) and a HUB-1 structural domain, and the N-terminal of p52-ZER6 only include one not
(30 amino acid of the C-terminal of KRAB structural domain, inventor are named as truncated KRAB to complete KRAB structural domain
Or tKRAB structural domain).Inventor has made intensive studies ZER6 albumen (especially p52-ZER6 albumen), it has been found that its
Important regulative is played in tumour regulation, to provide completely new anti-tumor target and drug.The present invention is also for the first time
It was found that the function of tKRAB structural domain, and the region tKRAB is found in ZER6, especially p52-ZER6 albumen is to tumor suppressor
Crucial effect is played in the negative regulation that p53 plays the role of.
In some embodiments, the present invention relates to following one or more aspects:
1. a kind of ZER6 inhibitor, is used to prevent and/or treat tumour.
2. inhibitor described in project 1, wherein the Inhibitor specificity inhibits the table of p52-ZER6 gene and/or albumen
It reaches, the expression without inhibiting p71-ZER6 gene and/or albumen.
3. inhibitor described in project 1 or 2, wherein the inhibitor includes the reagent for inhibiting p52-ZER6 gene expression,
Such as reduce the inhibitor of p52-ZER6 gene expression, including such as nucleic acid drug, such as siRNA, shRNA, antisense RNA, RNA
Aptamer, such as using the reagent of homologous recombination progress gene knockout, such as utilize the examination of CRISPR/Cas9 progress gene editing
Agent.
4. the described in any item inhibitor of project 1-3, wherein the inhibitor targets p52-ZER6 gene specific area, such as
Target p52-ZER6 upstream region of gene expression regulation area, such as 5 ' non-translational regions of p52-ZER6 gene, such as targeting coding p52-
The nucleic acid sequence of the N-terminal of ZER6 albumen, such as targeting encode the nucleic acid of the tKRAB structural domain of the N-terminal of p52-ZER6 albumen
Sequence, such as the polynucleotide sequence or its segment of targeting SEQ ID NO:1.
5. the described in any item inhibitor of project 1-4, wherein the inhibitor includes p52-ZER6 protein inhibitor, such as
Including small molecule compound, the antibody of p52-ZER6 albumen such as monoclonal antibody, such as the protease of degradation p52-ZER6 albumen,
Such as the inhibitor of the N-terminal for p52-ZER6 albumen, such as the tKRAB structural domain of the N-terminal for p52-ZER6 albumen
Inhibitor, such as antibody, such as monoclonal antibody and/or polyclonal antibody.
6. a kind of method for preparing p52-ZER6 inhibitor, the method includes making candidate agent and p52-ZER6 gene
And/or albumen contact, measure the amount of p52-ZER6 gene and/or albumen, wherein reduce compared with the control p52-ZER6 gene and/
Or the reagent of protein content is prepared as p52-ZER6 inhibitor.
7. method described in project 6, wherein further including following one or more steps: 1) amount of p53 and/or p21 is measured,
Amount including gene and/or albumen, wherein the reagent for increasing the amount of p53 and/or p21 compared with the control is prepared as p52-ZER6 suppression
Preparation；2) measurement p53 protein is in conjunction with MDM2 protein, wherein reducing p53 protein and MDM2 protein compared with the control
In conjunction with reagent be prepared as p52-ZER6 inhibitor；3) cell cycle activity and/or ability of cell proliferation are measured, wherein causing thin
Born of the same parents' period stops and/or the reagent of ability of cell proliferation decline is prepared as p52-ZER6 inhibitor；4) anticancer of measurement targeting p53
The effect of drug, wherein the reagent of the anticancer drug effect of enhancing targeting p53 is prepared as p52-ZER6 inhibitor；And/or it 5) surveys
The one-tenth knurl ability of cancer cell is determined, wherein the reagent for reducing cancer cell one-tenth knurl ability is prepared as p52-ZER6 inhibitor.
8. method described in project 6 or 7, wherein candidate agent is following one or more: 1) inhibiting p52-ZER6 gene
The reagent of expression, such as the inhibitor of p52-ZER6 gene expression, including such as nucleic acid drug, such as siRNA, shRNA are reduced,
Antisense RNA, RNA aptamer, such as using the reagent of homologous recombination progress gene knockout, such as CRISPR/Cas9 is utilized to carry out base
Because of the reagent of editor, the preferably described reagent targets p52-ZER6 gene specific area, such as targets the expression of p52-ZER6 upstream region of gene
Control region, such as 5 ' non-translational regions of p52-ZER6 gene；And/or 2) p52-ZER6 protein inhibitor, for example including small molecule
Compound, the antibody of p52-ZER6 albumen such as monoclonal antibody, such as the protease of degradation p52-ZER6 albumen.
9. the described in any item inhibitor of project 1-5 or the inhibitor of the described in any item method preparations of project 6-8 are being made
It is ready for use on prevention and/or the drug for treating tumour and/or the purposes in kit.
10. purposes described in project 9, wherein the inhibitor and another or the use of a variety of anti-cancer agent in combination, example
The anticarcinogen of p53 is such as targeted, such as promotes the anticarcinogen of tumor suppressor p53 and cycle regulating factor p21 effect, such as press down
The anticarcinogen of the combination of p53 processed and its negative regulatory factor MDM2, such as NSC 207895, MI-773 (SAR405838), NSC
207895、MX69、NVP-CGM097、RITA(NSC 652287)、Tenovin-1、MRT59、YH239-EE、HDM201、
RG7112(RO5045337)、Inauhzin、MDM2-p53-IN-1b、Nutlin-1、Nutlin-2、Nutlin-3、Nutlin-
3a, Nutlin-3b, Caylin-1, Caylin-2, MI-219, MI-319, Idasanutlin (RG-7388) etc., such as
Nutlin anticarcinogen and its derivative Nutlin-1, Nutlin-2, Nutlin-3, Nutlin-3a, Nutlin-3b, Caylin-
1, Caylin-2, MI-219, MI-319, Idasanutlin (RG-7388) etc..
11. the purposes of any one of project 9-10, wherein the tumour includes such as solid tumor or neoplastic hematologic disorder, early stage is swollen
Tumor, mid-term tumour, late tumor, refractory neoplasm, the tumour including for example expressing ZER6, such as expression p52-ZER6's swells
Tumor, tumour of the preferably specific expressed p52-ZER6 without expressing p71-ZER6, including such as leukaemia, for example, it is acute and chronic
Leukaemia, such as AML, ALL, CML, intractable CLL/SCLL, myelodysplastic syndrome, piastrenemia, including example
Such as colon cancer, breast cancer, kidney, the cancer of the brain, bladder cancer, cervical carcinoma, prostate cancer, gastric cancer, carcinoma of testis, oophoroma, cancer of pancreas, hang down
Body tumor, adrenal, thyroid cancer, salivary-gland carcinoma, liver cancer, intestinal cancer, thymic carcinoma, leukaemia, myelosarcoma, lung cancer and tracheae
Cancer.
In some embodiments, p52-ZER6 gene specific area include following SEQ ID NO:1 sequence or
Its segment:
(SEQ ID NO:1).
In some embodiments, the sequence or its segment of the Inhibitor specificity targeting SEQ ID NO:1, or with
Sequence described in SEQ ID NO:1 or its segment are prepared for specific target.Needle can be prepared by methods known in the art
To the specific inhibitor of particular sequence, such as the siRNA of the sequence, shRNA, antisense RNA, RNA aptamer, such as benefit
The reagent of gene knockout is carried out with homologous recombination, such as the reagent of gene editing, small molecule chemical combination are carried out using CRISPR/Cas9
Object, for the antibody such as monoclonal antibody of the target, such as protease etc..
In some embodiments, the inhibitor includes the shRNA target sequence of p52-ZER6:
Shp52-1AS:GAGAGGACGTAGTCTTGCT (SEQ ID NO:2)
Shp52-2AS:GACTGAGAGGCAGCGAGAA (SEQ ID No:3).
In some embodiments, the patient can be any appropriate tumor patient, for example, any appropriate solid tumor or
Patients with hematological tumor, such as late tumor or refractory neoplasm patient.In some embodiments, the patient may include example
Such as it is suitble to the tumour of the anticancer drug therapy using targeting p53, such as the drug for being suitble to targeting to prevent MDM2-P53 conjugate target spot
The tumour treated.In some embodiments, the tumour may include such as leukaemia, such as acute and chronic white blood
Disease.In some embodiments, the tumour is comprehensive including such as AML, ALL, CML, intractable CLL/SCLL, myelodysplasia
Simulator sickness, piastrenemia etc..In some embodiments, the tumour may include the tumour of such as expression ZER6, such as
Express the tumour of p52-ZER6, tumour of the preferably specific expressed p52-ZER6 without expressing p71-ZER6, such as the tumour
It may include colon cancer, breast cancer, kidney, the cancer of the brain, bladder cancer, cervical carcinoma, prostate cancer, gastric cancer, carcinoma of testis, oophoroma, pancreas
Gland cancer, hypophysoma, adrenal, thyroid cancer, salivary-gland carcinoma, liver cancer, intestinal cancer, thymic carcinoma, leukaemia, myelosarcoma, lung cancer
And tracheocarcinoma.
Detailed description of the invention
Fig. 1 is the gene and protein structure ideograph of two spliced bodies of ZER6.A.mRNA sequential patterns graph；B. egg
White matter tactic pattern figure.
Fig. 2 shows transcriptional activity, mRNA and the protein of p52-ZER6 specific effect p21, and p71-ZER6 cannot.
It is struck in a HCT116 colon cancer cell and subtracts p52-ZER6 p71-ZER6mRNA expression quantity is not significantly affected；And it strikes and subtracts p71-
ZER6 is not also significantly affected to the expression quantity of p52-ZER6mRNA and (is struck and subtract specificity).It is struck in b HCT116 colon cancer cell
Subtract p52-ZER6 not significantly affect p71-ZER6 protein expression quantity；And it strikes and subtracts p71-ZER6 to p52-ZER6mRNA's
Expression quantity does not also significantly affect and (strikes and subtract specificity).It is struck in c HCT116 colon cancer cell and subtracts p52-ZER6 and cause
P21mRNA significantly rises；And it strikes and subtracts p71-ZER6 and do not cause this effect.It is struck in d HCT116 colon cancer cell and subtracts p52-ZER6
Luciferase reporter gene (1uciferase reporter vector) activity with p21 promoter is caused to significantly rise；
And it strikes and subtracts p71-ZER6 and do not cause this effect.It is struck in e HCT116 colon cancer cell and subtracts p52-ZER6 and lead to p21 protein water
It is flat to significantly rise；And it strikes and subtracts p71-ZER6 and do not cause this effect.P52-ZER6 is overexpressed in fHCT116 colon cancer cell to be caused
P21 mRNA expression is remarkably decreased；And it is overexpressed p71-ZER6 and does not cause this effect.In g HCT116 colon cancer cell
Being overexpressed p52-ZER6 causes p21 protein level to be remarkably decreased；And it is overexpressed p71-ZER6 and does not cause this effect.
Fig. 3, which shows to strike, to be subtracted p52-ZER6 the cell cycle is caused to stop, ability of cell proliferation decline；Being overexpressed p21 can save
Above-mentioned phenotype.A strike subtract p52-ZER6 cause HCT116 colon cancer cell sum reduce；B, which strikes, to be subtracted p52-ZER6 HCT116 is caused to tie
The decline of colon-cancer cell proliferative capacity；C, which strikes, to be subtracted p52-ZER6 the cell cycle is caused to stop at G0-G1Phase；D is thin in HCT116 colon cancer
It is struck simultaneously in born of the same parents and subtracts p52-ZER6 and p21, lead to have significant rise due to striking the decline of total number of cells caused by subtracting p52-ZER6；e
It is struck simultaneously in HCT116 colon cancer cell and subtracts p52-ZER6 and p21, caused due to striking cell Proliferation caused by subtracting p52-ZER6
Ability decline has significant rise.
Fig. 4 shows the one-tenth knurl ability struck and subtract p52-ZER6 significant decrease colon cancer cell HCT116.A, which strikes, subtracts p52-ZER6's
(p52-ZER6, which strikes to subtract respectively to strike, to be subtracted in group, in 6 mouse for one-tenth knurl ability significant decrease after HCT116 colon cancer cell is transplanted to nude mice
Only a mouse forms very small tumour after 28 days), the control group and strike the appearance of tumors and tumour for subtracting group that a is 14 days
Volume measurements (in this time point, p52-ZER6, which strikes, to be subtracted group and can not detect apparent tumour)；It b control group (14 days) and strikes
Subtract p53 and p21 expression quantity (ImmunohistochemistryResults Results) in the tumor tissues of group (28 days), strikes the expression quantity for subtracting p53 and p21 in group
It significantly rises.
Fig. 5 shows that p52-ZER6 promotes p53 protein in conjunction with MDM2 protein, induces p53 protein ubiquitination, inhibits
The accumulation of p53 protein.(a) FLAG-p52-ZER6, FLAG-p52-ZER6 and FLAG-p71-ZER6 intermediate (FLAG-p52K
And FLAG-p52KH) and FLAG-p71-ZER6 overexpression Plasmid pattern figure；(b) p52-ZER6 and FLAG-p52KIt is overexpressed aobvious
MDM2 albumen quality of the work promotion in conjunction with p53 protein, and FLAG-p53KHAnd FLAG-p71 cannot；(c) p52-ZER6 and
FLAG-p52KIt is overexpressed and remarkably promotes the ubiquitination of p53 protein, and FLAG-p53KHAnd FLAG-p71 cannot；(d)p52-
ZER6 and FLAG-p52KThe accumulation of overexpression significant decrease p53 protein, and FLAG-p53KHAnd FLAG-p71 cannot.
Fig. 6 shows that p52-ZER6 hinders the work of Nutlins kind anti-cancer drugs object induction tumor suppressor p53 protein accumulation
Use mechanism.(a) amount that Nutlin-3 reduces the MDM2 protein in HCT116 colon cancer cell in conjunction with p53 protein (is exempted from
Epidemic disease co-precipitation experiment, both Nutlin caused the reduction of the MDM2 protein expression quantity in conjunction with p53 protein that p53 protein is caused to drop
Xie Yue is few, so that the accumulation of p53 protein be induced to cause apoptosis of tumor cells, plays antitumous effect), however be overexpressed
This effect is eliminated in the cell of p52-ZER6；(b) after Nutlin-3 processing, the p53 egg in HCT116 colon cancer cell
White matter accumulation is significant to be increased, and this function and effect significantly reduces after being overexpressed p52-ZER6.
The setting of Fig. 7 display example qPCR response procedures.
Fig. 8 shows influence of the amount of p52-ZER6 in HCT116 colon cancer cell to Nutlin-3 drug effect.(a) it transfects in figure
The p52-ZER6 of amount shown, which is overexpressed plasmid pair Nutlin-3, reduces cell number energy after processing 24 hours, 48 hours and 72 hours
The influence of power；(b) p52-ZER6 for transfecting 2 μ g is overexpressed plasmid amount to the IC of the Nutlin-3 cell handled50。
Fig. 9 shows influence of the amount of p52-ZER6 in MCF-7 breast cancer cell to Nutlin-3 drug effect.(a) it transfects in figure
The p52-ZER6 of amount shown, which is overexpressed plasmid pair Nutlin-3, reduces cell number energy after processing 24 hours, 48 hours and 72 hours
The influence of power；(b) p52-ZER6 for transfecting 2 μ g is overexpressed plasmid amount to the IC of the Nutlin-3 cell handled50。
Figure 10 shows influence of the amount of p52-ZER6 in HepG2 breast cancer cell to Nutlin-3 drug effect.(a) it transfects in figure
The p52-ZER6 of amount shown, which is overexpressed plasmid pair Nutlin-3, reduces cell number energy after processing 24 hours, 48 hours and 72 hours
The influence of power；(b) p52-ZER6 for transfecting 2 μ g is overexpressed plasmid amount to the IC of the Nutlin-3 cell handled50。
Specific embodiment
Inventor has found that one of two spliced bodies of ZER6, p52-ZER6 can inhibit tumor suppressor p53 and period tune
Control factor p21 (p53 can promote p21 transcription in conjunction with the promoter of p21)；And another transcription factor p71-ZER6 cannot.
Experimental data shows that ZER6 strikes and subtracts rear p21 rising, and p52-ZER6, which strikes, subtracts rear p21 rising (mRNA, protein, transcription
Activity)；It strikes to subtract and be carried out with shRNA (RNA interference).Also show that p52-ZER6 leads to G0-G1Stop, cell Proliferation decline.Referring to thin
Born of the same parents' cycle analysis and cell proliferation experiment.Have shown that the reason of p52-ZER6 regulates and controls p53 is that p52-ZER6 promotes p53 and its
The combination of negative regulatory factor MDM2 causes the ubiquitination of p53 to rise, and promoting p53 protein degradation, (MDM2 is p53 ubiquitination
E3 ligase is the negative regulatory factor of p53).Simultaneously, it has proved that strike and subtract p52 one-tenth knurl ability is caused to decline very significantly.Ginseng
See zoopery (Tumor formation).The experimental data that inventor provides sufficiently proves important function of the ZER6 in tumour adjusting.
Material and reagent
Instrument
Key instrument used herein is as listed by table 1.1:
1.1 main experimental device information of table
Reagent consumptive material
1.2 experiment reagent of table
1.3 kit of table
1.4 antibody information of table
1.5 real-time quantitative PCR primer of table
The shRNA target sequence of 1.6 p52-ZER6 of table
1.7 fusion protein of table
1. plasmid construction
1. the building of shRNA expression plasmid
Synthesize have hairpin structure sequence, cantilever sequence, termination sequence three parts oligonucleotide sequence and it
Complementary series is inserted into the BspM I site of the pcPUR+U6i carrier containing U6 promoter (ginseng after two complementary sequence anneals
See Huang et al., ScienceAdvances, 2017；3 (10): e1701383).
2. being overexpressed the building of plasmid
1) the overexpression plasmid construction of FLAG label is had
The nucleotide sequence insertion of FLAG with NheI and HindIII restriction enzyme site cohesive end had into CMV starting
Between the site NheI and HindIII of the pcDNA3.1+ plasmid (U.S., Invitrogen Life Technologies) of son,
A successfully expression plasmid with FLAG label is constructed after being sequenced successfully.Corresponding encoded is obtained by template amplification of cDNA respectively
Area's segment is inserted between the site BamHI and NotI of pcFLAG carrier, after single colonie PCR and double digestion verifying
Sequencing, sequencing result is compared with target fragment with GENETYX software, sequence alignment success after identification plasmid construction at
Function.With the method building FLAG-p52-ZER6 is overexpressed plasmid (FLAG-p52), FLAG-p71-ZER6 is overexpressed plasmid
(FLAG-p71), the FLAG-p52-ZER6 with the region overall length KRAB is overexpressed plasmid (FLAG-p52K) and with overall length
The FLAG-p52-ZER6 in the region KRAB and the region HUB-1 is overexpressed plasmid (FLAG-p52KH)。
3. reporter plasmid constructs
Cell genomic dna is extracted, PCR amplification reporter sequences, PCR product carries out gel electrophoresis, to purpose band
Glue recycling is carried out, target fragment and pGL4.13 plasmid carry out double digestion with HindIII and BglII respectively, and target fragment carries out
DNA purifying, glue recycling, target fragment and carrier are attached, and Escherichia coli are converted, with small amount plasmid extraction kit upgrading
HindIII and BglII carry out double digestion after grain, agarose gel electrophoresis verifying, sequencing, with GENETYX software by sequencing result
It is compared with target fragment, plasmid construction success is assert after sequence alignment success.
2. cell experiment
1. cell culture
Wild type HCT116 colon carcinoma cell line uses McCoy ' s 5A culture medium, contains 10% fetal calf serum and 1%
Pen .- Strep (Penicillin-Streptomycin Solution) it is dual anti-, contain 5%CO under the conditions of 37 DEG C2
Incubator culture.
Wild type MCF-7 breast cancer cell and HepG2 liver cancer cell lines use DMEM high glucose medium, contain 10%
Fetal calf serum and 1% Pen .- Strep (Penicillin-Streptomycin Solution) it is dual anti-, in 37 DEG C of conditions
Under contain 5%CO2Incubator culture.
3. clpp gene subtracts experiment
Six orifice plates inoculation 5 × 105A cell is simultaneously cultivated for 24 hours, and every hole transfects 2 μ g shRNA expression plasmids.After transfection for 24 hours,
Puromycin screening (final concentration: 1.2 μ g/ml) is carried out to cell.After screening 36h, after negative control group complete cell death, receive
Collection cell does subsequent experimental.
4. gene overexpression is tested
Six orifice plates inoculation 5 × 105A cell is simultaneously cultivated for 24 hours, and every hole transfects the overexpression plasmid of the amount shown on 2 μ g figures.
After transfection for 24 hours, puromycin screening (final concentration: 1.2 μ g/ml) is carried out to cell.After screening 36h, negative control group cell is complete
After portion is dead, collects cell and do subsequent experimental.
5.RNA is extracted and real-time quantitative PCR (qPCR)
1. RNA is extracted and reverse transcription
PBS cleans cell, and Trizol lysate is added, and 100 μ l chloroforms are added, are stored at room temperature 2-3min, and 12,000rpm, 4
DEG C centrifugation 15min, be added isopropanol, be stored at room temperature 10min, 12,000rpm 4 DEG C of centrifugation 10min.75% ethyl alcohol of 1ml is added,
12000rpm, is centrifuged 5min, adds DEPC water by 4 DEG C.Nanodrop 2000 measures RNA concentration, and 1 μ g RNA is taken to carry out reverse transcription.
Reverse transcription step is carried out entirely by reference to the specification of kit PrimeScript Reagent Kit with gDNAEraser.
2. real-time quantitative PCR (qPCR)
1) sample for obtaining above-mentioned reverse transcription dilutes 10 times, the template as subsequent quantitation PCR.
The reaction system of PCR is prepared according to following table:
2) qPCR reaction is carried out
According to setting qPCR response procedures shown in Fig. 5.
6. Protein Extraction and western blot analysis
RIPA lysis buffer be added protease inhibitors PMSF and inhibitors of phosphatases Cocktail (RIPA, PMSF and
Cocktail is according to 100: 1: 2).Add lysate, is placed in and cracks 10min on ice, 12,000rpm, 4 DEG C of centrifugation 10min, in collection
Clearly, protein quantification is carried out according to the specification that green skies BCA protein quantification kit provides.The protein of equivalent is configured
Protein band is transferred on pvdf membrane after electrophoresis by electrophoresis on PAGE gel, after transferring film with 5% defatted milk
Powder room temperature closes 1h, and 4 DEG C of deposited primary antibodies are stayed overnight, and TBS-T washes film 2-3 times, and room temperature applies corresponding secondary antibody 90min, and TBS-T is washed three times.
It is exposed and is taken pictures using SuperSignal West Femto Maximum Sensitivity Substrate detection system.
7. luciferase reporter gene is tested
Inoculation 8 × 104A cell is in 24 orifice plates, the specified shRNA expression plasmid of cotransfection, p21 firefly luciferase
Reporter gene vector and renilla luciferase expression plasmid (pRL-SV40, Promega) as internal reference remove after transfecting 18h
Culture medium, is added lysate, 12,000rpm, centrifugation 5min, and absorption supernatant takes lysate to be transferred in white 96 orifice plates, sample
After adding, Dual-Luciferase luciferase buffer, detection firefly luciferase chemistry hair is added with 8 pipettors
Light value, then Stop-GLO is added in the every hole detected, the values of chemiluminescence of renilla luciferase is detected, is sent out by chemistry twice
Light radiometer calculates relative light unit.
8.MTS measures cell quantity
5 × 10 are inoculated in six orifice plates5Cell, culture for 24 hours, transfect shRNA plasmid, for 24 hours after, with contain 1.2 μ g/ml purine
The Screening of Media cell of mycin, after screening 36h, by the cell renewed vaccination collected again into 96 orifice plates, 5000, every hole
Cell makes the kind of collection according to the cell number gradient in every hole 0/5000/10000/20000/40000/80000 in 96 orifice plates
Make standard curve.The MTS that 10 μ l are added in every hole in standard curve plate in the medium, after being put into 37 DEG C of culture 1.5h,
Under 490nm wavelength, the absorbance in every hole is detected.Standard curve is fabricated to absorbance with corresponding cell number.Sample detection: point
Not for 24 hours, 48h, 72h, cell culture medium is changed to the culture medium for being mixed with MTS reagent into, same method detects the extinction in each hole
Degree, then calculates each hole in the cell number of various time points by standard curve.
9.EdU marks cell proliferation experiment and Colony forming experiment
According to Cell-LightTM EdU488 In Vitro Imaging Kit specifications carry out EdU dyeing.
Every hole inoculation 5 × 10 in six orifice plates5Cell transfects corresponding shRNA plasmid after culture for 24 hours respectively.After transfection for 24 hours, with containing
The Screening of Media cell of 1.2 μ g/ml puromycins, after screening 36h, by the cell renewed vaccination collected again to 48 orifice plates
In, every hole 8 × 104A cell, culture to cell for 24 hours it is adherent after start to do EdU dyeing.With culture medium by EdU reagent according to 1:
1,000 dilution proportion, final concentration of 50 μM.After culture medium culture cell 1.5h containing EdU, culture medium is removed.Cell is used
PBS is gently cleaned 2 times, each 5min.It is added 4% paraformaldehyde fixer in each hole the cells are fixed 30min, removes fixation
Liquid.Every hole is gently added glycine solution (concentration 2mg/ml), is incubated for 5min, sucks glycine solution.PBS is gently cleaned.
Bleeding agent (0.5%TritonX-100) is added and is incubated for 10min, PBS cleaning.Use Microsystems LAS AF-TCS MP5
(Leica) quantifying for the EdU positive and DAPI positive cell is carried out.
10. cell cycle analysis
5 × 10 are inoculated in six orifice plates5Cell, culture for 24 hours, transfect shRNA plasmid.Transfect the culture of puromycin for 24 hours
Base screening, cell sufficiently suspend, and every hole is divided into two holes.After screening 36h, with trypsin digestion and cell, renewed vaccination 3 × 105
A cells/well is into six orifice plates.After adherent, change the culture of serum free medium starvation for 24 hours, be further cultured under normal operation for 24 hours.Disappear
Change cell, and adjusting concentration is 1 × 106A/ml.70% ethyl alcohol of 9 times of volumes, -20 DEG C of fixed 12h are added.Centrifuge cell,
Cell removal ethyl alcohol residual is washed with PBS, cell is resuspended in Buffer A, RNase A (final concentration 0.25mg/ is added
Ml), it is protected from light 30min for 37 DEG C.Propidium iodide PI is added, is incubated at room temperature 30min.With flow cytometer excitation wavelength 488 into
Row detection, determines the percentage of cell in each cell cycle phase, obtains each cycling cells distributed number figure of cell.
11. zoopery
Zoopery is carried out in medical officer army medical university, ground force great Ping hospital animal house.BALB/c-nu/nu mouse (male, body
Weight: 18-22g, 6 week old) it is purchased from army medical university, ground force (Chongqing in China；Licensing number SYXK-PLA-20120031), and through land
Army medical university, army Laboratory Animal Welfare and Ethics Committee ratify.All zooperies meet Third Military Medical University's animal welfare
Committee member may require that.BALB/c-nu/nu mouse is divided into three groups, every group of subcutaneous injection 5 × 106A cell.Pass through vernier within every two days
Tumor size of calliper to measure, with reference to following equation: V=a x b2/ 2, wherein a and b is the long axis of tumour respectively and short
Axis.It is measured after mouse inoculation in total the 21st day.After the data for measuring last day, tumor mass is peeled, is taken pictures.Then tumour is cut
It opens, a part is immersed directly in 4% paraformaldehyde fixer, and for doing subsequent immunohistochemical experiment, two pieces directly add
Liquid nitrogen grinding, respectively plus protein cleavage liquid RIPA and Trizol lysate, for extract protein and RNA carry out it is further
Detection, one piece saves for a long time in liquid nitrogen.
12. immunohistochemical analysis
Slice is put into the ethyl alcohol for being then placed in gradient dilution in the dimethylbenzene of gradient dilution, is rinsed with water；It will slice
It immerses in antigen retrieval buffers (pH9.0), slide is placed in PBS (PH7.4), 5min is washed；Slice is put into 3% hydrogen peroxide solution
(H2O2∶H2O=1: 9), it is incubated for 25min；3%BSA is added dropwise, room temperature closes 30min；Primary antibody, 4 DEG C of overnight incubations are added dropwise；It is added dropwise two
It is anti-, it is incubated at room temperature 50min；DAB developing solution is added dropwise, observes and control developing time under the microscope, positive display brown color is multiple
Dye nucleus: redying 3min for tissue with haematoxylin, and tap water rinses, and is broken up several seconds with 1% hydrochloride alcohol, then again with certainly
Water is rinsed, and is returned indigo plant using ammonium hydroxide, is finally rinsed with flowing water；Slice is sequentially placed into -85% alcohol of 75% alcohol-in sequence
Dehydrated alcohol twice-dimethylbenzene twice, dehydration is to transparent, with neutral gum mounting；Use Pannoramic Midi
(3DHistech, Budapest, Hungary) shoots image (scanning).
13. co-immunoprecipitation
5 × 10 are inoculated in 10em culture plate6A cell, transfection is overexpressed plasmid (pcMDM2, pcp53) afterwards for 24 hours, culture
After cell cleaned 2 times with PBS, the dedicated RIPA lysate of IP of Fresh, ice bath 30min is added.12,000rpm, 4 DEG C
It is centrifuged 10min, collects supernatant, takes out 100 μ L as Input.Draw 40 μ L of proteinA+G pearl.Be added PBS, 4 DEG C, 2,
500rpm is centrifuged 5min.The above-mentioned dedicated lysate of configured IP is added, IgG is added in negative control group, and MDM2 is added in experimental group
Primary antibody, 4 DEG C of rotation 2h, 2,500rpm centrifugation 5min remove supernatant, and PBS is cleaned twice, and the above-mentioned thin of equivalent is added after removing supernatant
Cellular lysate liquid.4 DEG C of rotations 4h, 2,500rpm centrifugation 5min remove supernatant, and PBS is cleaned twice, and lysate cleans twice, and SDS is added
PAGE loading buffer boils 5min.Then, western blotting experiment is carried out to immunoprecipitate as described above
Analysis.
14.Nutlin processing
5 × 10 are inoculated in six orifice plates5A cell after culture for 24 hours, transfects the specified plasmid of 2 μ g, changes after 6h final concentration of
The culture medium of the Nutlin-3 of 5 μm of ol/L collects cell extraction protein afterwards for 24 hours.
15. ubiquitination detects
By cell inoculation in 10em culture dish (5 × 106A cell/ware) in, transfection ubiquitin is overexpressed plasmid (pc- afterwards for 24 hours
Ubi), the overexpression plasmid (pcp53) of p53 and shRNA expression plasmid or overexpression plasmid.It is (dense eventually with MG132 after transfection for 24 hours
20 μM of degree) processing cell 8h, then uses the dedicated lysate lytic cell of IP, collects lysate.ProteinA+G pearl is drawn, is added
Enter PBS, be gently mixed by inversion, 4 DEG C of 2,500rpm are centrifuged 5min.Supernatant carefully is sopped up, repeated washing is primary.Above-mentioned configuration is added
The primary antibody of p53,4 DEG C of rotation 2h on rotary shaker is added in the good dedicated lysate of IP, every pipe, and 2,500rpm centrifugation 5min are gone
Clearly, PBS cleaning twice, removes the cell pyrolysis liquid of addition equivalent after supernatant.4 DEG C of rotation 4h, 2,500rpm centrifugations on rotary shaker
5min removes supernatant, then is cleaned twice with PBS, and the liquid in pearl is blotted, and SDS PAGE loading buffer is added and boils
5min.Then western blotting points are carried out to the protein of immunoprecipitation using anti-ubiquitin protein antibody and other antibody
Analysis.
16.MTT method measures cell survival rate and IC50
The attached tumor cells for selecting logarithmic growth phase after being digested with pancreatin, are matched with the culture medium containing 10% fetal calf serum
At 500,000 kinds of every hole in 6 orifice plates；Control plasmid (pcCon) is used afterwards for 24 hours, p52 plasmid pair cell is overexpressed and is transfected；It prepares
Transfection cocktail takes 1.5ml EP to manage, is divided into plasmid group and 2000 groups of liposome lipo, is separately added into 200 μ lopti-MEM,
It is tried according to plasmid (μ g) and transfection reagent (μ l) plasmid that corresponding amount is added by 1: 1,1: 1.5,1: 2,1: 2.5,1: 3 and transfection
Agent.It is incubated at room temperature 5min, allows plasmid and lipo2000 to be uniformly dispersed in respective pipe, then mixes the two, room temperature
Stationary incubation 20min；Waste liquid is sopped up with waste drains pump, 6 orifice plates replace fresh culture, and then transfection mixture is added dropwise in every hole respectively
Neoblast culture solution is replaced after 400 μ l, 6h；After for 24 hours, 4~5 × 10 are made into the culture medium containing 10% fetal calf serum4/ ml's is thin
Born of the same parents' suspension is seeded in 96 well culture plates, 100 μ l of every hole, and 37 DEG C, 5%CO2Culture is for 24 hours.What experimental group more renewed contains
Nutlin3 concentration is 40 μM of drug, and control group then replaces the culture solution containing isometric solvent, and every group sets 3 parallel holes, 37
DEG C, 5%CO2Culture for 24 hours, 48h, 72h.The mixed liquor for preparing MTS and culture medium (presses every 10 μ l MTS of hole and 100 μ l culture mediums
Ratio prepared).96 orifice plate of a plate is taken out, culture medium is carefully discarded, the 110 prepared mixed liquors of μ l are added in every hole.37
It DEG C is protected from light after being incubated for 90min, the light absorption value for measuring each hole is protected from light at microplate reader 490nm.The experimental method of table 1 removes plasmid (μ
G) with transfection reagent (μ l) by 1: 2 transfection, the concentration containing Nutlin3 that experimental group more renews is 0.1,1,10,20,40,60 μM
It is other to be same as above other than drug.
Claims (11)
1. a kind of ZER6 inhibitor, is used to prevent and/or treat tumour.
2. inhibitor described in claim 1, wherein the Inhibitor specificity inhibits the table of p52-ZER6 gene and/or albumen
It reaches, the expression without inhibiting p71-ZER6 gene and/or albumen.
3. inhibitor of any of claims 1 or 2, wherein the inhibitor includes the reagent for inhibiting p52-ZER6 gene expression,
Such as reduce the inhibitor of p52-ZER6 gene expression, including such as nucleic acid drug, such as siRNA, shRNA, antisense RNA, RNA
Aptamer, such as using the reagent of homologous recombination progress gene knockout, such as utilize the examination of CRISPR/Cas9 progress gene editing
Agent.
4. the described in any item inhibitor of claim 1-3, wherein the inhibitor targets p52-ZER6 gene specific area, such as
Target p52-ZER6 upstream region of gene expression regulation area, such as 5 ' non-translational regions of p52-ZER6 gene, such as targeting coding p52-
The nucleic acid sequence of the N-terminal of ZER6 albumen, such as targeting encode the nucleic acid of the tKRAB structural domain of the N-terminal of p52-ZER6 albumen
Sequence, such as the polynucleotide sequence or its segment of targeting SEQ ID NO:1.
5. the described in any item inhibitor of claim 1-4, wherein the inhibitor includes p52-ZER6 protein inhibitor, such as
Including small molecule compound, the antibody of p52-ZER6 albumen such as monoclonal antibody, such as the protease of degradation p52-ZER6 albumen,
Such as the inhibitor of the N-terminal for p52-ZER6 albumen, such as the tKRAB structural domain of the N-terminal for p52-ZER6 albumen
Inhibitor, such as antibody, such as monoclonal antibody and/or polyclonal antibody.
6. a kind of method for preparing p52-ZER6 inhibitor, the method includes make candidate agent and p52-ZER6 gene and/or
Albumen contact, measures the amount of p52-ZER6 gene and/or albumen, wherein reducing p52-ZER6 gene and/or egg compared with the control
The reagent of white amount is prepared as p52-ZER6 inhibitor.
7. method of claim 6, wherein further including following one or more steps: 1) amount of p53 and/or p21 is measured,
Amount including gene and/or albumen, wherein the reagent for increasing the amount of p53 and/or p21 compared with the control is prepared as p52-ZER6 suppression
Preparation；2) measurement p53 protein is in conjunction with MDM2 protein, wherein reducing p53 protein and MDM2 protein compared with the control
In conjunction with reagent be prepared as p52-ZER6 inhibitor；3) cell cycle activity and/or ability of cell proliferation are measured, wherein causing thin
Born of the same parents' period stops and/or the reagent of ability of cell proliferation decline is prepared as p52-ZER6 inhibitor；4) Nutlins anticarcinogen is measured
The effect of object, wherein the reagent of enhancing Nutlins anticancer drug effect is prepared as p52-ZER6 inhibitor；And/or 5) measure cancer
The one-tenth knurl ability of cell, wherein the reagent for reducing cancer cell one-tenth knurl ability is prepared as p52-ZER6 inhibitor.
8. method described in claim 6 or 7, wherein candidate agent is following one or more: 1) inhibiting p52-ZER6 gene
The reagent of expression, such as the inhibitor of p52-ZER6 gene expression, including such as nucleic acid drug, such as siRNA, shRNA are reduced,
Antisense RNA, RNA aptamer, such as using the reagent of homologous recombination progress gene knockout, such as CRISPR/Cas9 is utilized to carry out base
Because of the reagent of editor, the preferably described reagent targets p52-ZER6 gene specific area, such as targets the expression of p52-ZER6 upstream region of gene
Control region, such as 5 ' non-translational regions of p52-ZER6 gene；And/or 2) p52-ZER6 protein inhibitor, for example including small molecule
Compound, the antibody of p52-ZER6 albumen such as monoclonal antibody, such as the protease of degradation p52-ZER6 albumen.
9. the described in any item inhibitor of claim 1-5 or the inhibitor of the described in any item method preparations of claim 6-8
Preparing the purposes in drug and/or kit for preventing and/or treating tumour.
10. purposes as claimed in claim 9, wherein the inhibitor and another or the use of a variety of anti-cancer agent in combination, example
The anticarcinogen of p53 is such as targeted, such as promotes the anticarcinogen of tumor suppressor p53 and cycle regulating factor p21 effect, such as press down
The anticarcinogen of the combination of p53 processed and its negative regulatory factor MDM2, such as NSC 207895, MI-773 (SAR405838), NSC
207895、MX69、NVP-CGM097、RITA(NSC 652287)、Tenovin-1、MRT59、YH239-EE、HDM201、
RG7112(RO5045337)、Inauhzin、MDM2-p53-IN-1b、Nutlin-1、Nutlin-2、Nutlin-3、Nutlin-
3a, Nutlin-3b, Caylin-1, Caylin-2, MI-219, MI-319, Idasanutlin (RG-7388) etc., such as
Nutlin anticarcinogen and its derivative Nutlin-1, Nutlin-2, Nutlin-3, Nutlin-3a, Nutlin-3b, Caylin-
1, Caylin-2, MI-219, MI-319, Idasanutlin (RG-7388) etc..
11. the purposes of any one of claim 9-10, wherein the tumour includes such as solid tumor or neoplastic hematologic disorder, early stage is swollen
Tumor, mid-term tumour, late tumor, refractory neoplasm, the tumour including for example expressing ZER6, such as expression p52-ZER6's swells
Tumor, tumour of the preferably specific expressed p52-ZER6 without expressing p71-ZER6, including such as leukaemia, for example, it is acute and chronic
Leukaemia, such as AML, ALL, CML, intractable CLL/SCLL, myelodysplastic syndrome, piastrenemia, including example
Such as colon cancer, breast cancer, kidney, the cancer of the brain, bladder cancer, cervical carcinoma, prostate cancer, gastric cancer, carcinoma of testis, oophoroma, cancer of pancreas, hang down
Body tumor, adrenal, thyroid cancer, salivary-gland carcinoma, liver cancer, intestinal cancer, thymic carcinoma, leukaemia, myelosarcoma, lung cancer and tracheae
Cancer.
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