CN109420169A - Novel tumor associated target point Zscan4 and its application in inhibition tumour - Google Patents

Abstract

The present invention relates to novel tumor associated target point Zscan4 and its inhibiting the application in tumour.Important biological action may be played during SASP phenotype occurrence and development by disclosing Zscan4, and it is also closely related with the development of tumour.Zscan4 can be used as the research target spot of SASP phenotype regulation, can be used as diagnosis, the prognosis evaluation marker of tumour, be alternatively arranged as the drug that target spot exploitation inhibits tumour.

Description

Novel tumor associated target point Zscan4 and its application in inhibition tumour

Technical field

The invention belongs to field of pharmacology, more particularly it relates to novel tumor associated target point Zscan4 and its Inhibiting the application in tumour.

Background technique

Diseases associated with senescence is the direct factor for leading to human death, mainly includes malignant tumour and a variety of organ degenerations Disease such as heart failure, osteoporosis, arthritis, Alzheimer disease, parkinsonism etc..Wherein malignant tumour is in recent years Surmount various other diseases, becomes the number one killer for endangering human health.With the cell ageing of human diseases close association, Initially found and proposed the sixties in 19th century by American scientist Hayflick, be obstructed with normal cell proliferation, cell week The biological phenomena that phase stagnates as main feature.50 for many years, numerous studies be dedicated to probing into the triggering of cell ageing because Element, signal path, phenotypic characteristic and the influencing mechanism for a variety of diseases.In multicellular organism, cell ageing declines with body There are different meanings, but close association between the two always.It is unbalance and repair in period homeostasis of degenerating that body aging is organism A kind of general performance of reactivation power decline, cell ageing are then that cell recurring structure under the influence of internal and external environment many factors damages Wound and dysfunction and a kind of passive state for entering.With metabolism, cell ageing is objective reality, belongs to cell The basic law of vital movement.Under normal physiological conditions, cell ageing can be shortened by telomere to be caused.However, a series of reasons Cell ageing, such as ionising radiation, chemicals, oxidative stress and specific oncogene can also be caused by changing stimulation or bio signal Expression etc., but influence of these factors to cell relates generally to the generation of the stress event such as DNA damage.

It is even more important, significant changes also have occurred in protein synthesis and the metabolism of chronic senile cell, i.e. cell enters A kind of strong and lasting secretor state, educational circles are referred to as aging correlation secretion phenotype (senescence-associated Secretory phenotype, SASP), the Acute stress for being different from previously reported mistake stress phenomenon (acute stress- Associated phenotype, ASAP).The occurrence and development of SASP are secreted program (DNA damage by DNA damage Secretory program, DDSP) driving, the latter has conspicuousness and uniqueness at several aspects.Firstly, DDSP program Be it is highly complex, several hundred kinds of albumen are induced to different degree simultaneously；Secondly, reaction be it is violent, many transcriptons and They drive albumen, be above transferred to as many as 10 times it is even higher；Third, there is obvious between different cell types for this program Consistency and otherness；4th, DDSP program up-regulation a large amount of protein moleculars be it is currently known to stimulate tumour growth and The factor of evolution, including protease (MMPs), growth factor (AREG, EREG), preceding angiogenesis factor (ANGPTL4, ANGPT1), interleukins (IL-6, IL-8) and Bones morphology occurrence factor (BMP6) etc..

Although the effector of SASP secreting type cell release can excite the immune response of internal local organization, promote to decline The removing of old cell, but can more significantly change the structure and function of periphery microenvironment, accelerate the diseases associated with senescence such as tumour into Exhibition.2016, the researcher of the U.S. clinic Mayo confirmed that senile cell influences the series of negative that health generates, Neng Gourang The lost of life of normal mouse is up to 35%.It is formed on the contrary, removing senile cell and can postpone tumour, keeps tissue and organ normal State function, extending mouse average life span is more than 20%.It is high using selectively targeted drug since senile cell is unable to fast breeding Effect, rapidly remove in vivo with the age increase and accumulate senile cell will to health and the service life generate profound impact, thus It plays a positive role in clinic.

It is the research of core development using biological phenomena as target, clinical sample, can reveal that cell ageing is micro- for organizing The pathophysilogical significance of environment.The present inventor has found in recent years, because of disease caused by the chemical factors such as drug side-effect during anticancer Tumor-related cell plastid DNA damage and while there is aging feature in stove, with WNT16B, SFRP2 and SPINK1 etc. The height synthesis of soluble protein and a large amount of releases (typical SASP feature), can cause intralesional cancer cell Twist, Snail It is final to accelerate cancer cell that epithelial-mesenchymal occurs to convert (epithelial- with the expression up-regulation of the transcription factors such as Smad Mesenchymal transition, EMT).So far, more and more data verifications this development of pre-clinical setting tumor Rule is to target the contemporary clinicals routine hand such as the chemotherapy of target, radiotherapy or the targeted therapy for interfering DDR related pathways with DNA The risen effect of missing the target can activate tumor microenvironment, cause stroma cell aging and form SASP to secrete section over the course for the treatment of Phenotype, the malignant characteristics such as proliferation rate, mobility, invasion and heterogeneity that the latter shows cancer cell in each stage Occurrence and development there is very important pathological effect, objectively impart cancer cell shifted with acquired resistance and height it is latent Power.Therefore, senile cell not only interferes the physiology of normal body homeostasis to safeguard, non-autonomous with cell more under therapeutic damage The paracrine mode of property pushes a series of development processes of the diseases such as cancer.

Regulation of the expression of SASP albumen by Cellular stress induction albumen p38MAPK and several transcription factors, it is main to wrap Include nuclear factor NF- κ B, CCAAT enhancer binding protein β (CCAAT enhancer binding protein β, C/EBP- β) and Activated protein-1 (activator protein-1, AP-1).These transactivator multidigits are in control immunity cell factor table The downstream of the signal cascade reached.For example, normal cell for aging induction sexual stimulus early reaction first is that interleukin I L-1 α Up-regulation, and this cell membrane relevant cell factor can be in conjunction with its membrane receptor IL-1R, and the latter transfer to activate a series of signal logical Road simultaneously finally activates NF- κ B.Then, the NF- κ B of activation raises a large amount of eggs including immune-mediated factor IL-6 and IL-8 White expression causes the synthesis intracellular of the SASP factor of wide spectrum and extracellular release.The present inventor has found in the recent period, mainly swashs as cytoplasm The mTOR of one of enzyme plays crucial mediation in fibroblast IL-1 α/NF- κ B signal access that DNA damage induces, and SASP secretion phenotype can substantially be weakened for the drug such as rapamycin of mTOR, to block cancer cell from periphery microenvironment Middle acquisition survival signaling.In addition, mTOR also realizes the translation of Differential regulation MK2 by 4EBP1, MK2 transfers in senile cell Middle phosphorylation rna binding protein ZFP36L1, to inhibit the activity of its countless SASP component of degrading.

To sum up, the mechanism that cell ageing and tumour occur is extremely complex, and there is an urgent need in the art to therefrom find rule Rule, finds new regulatory mechanism, obtains useful clinical medicine on this basis.

Summary of the invention

The purpose of the present invention is to provide novel tumor associated target point Zscan4 and its inhibiting the application in tumour.

In the first aspect of the present invention, a kind of purposes of the lower adjustment of Zscan4 gene or albumen is provided, suppression is used to prepare The pharmaceutical composition of tumour processed.

In a preferred embodiment, the tumour is selected from: after gene cytotoxic drug or DNA damage drug-treated or being passed through Cross the tumour of DNA damage treatment, or the tumour of expression Zscan4.

In another preferred example, the gene cytotoxic drug or DNA damage drug include but is not limited to: bleomycin, Mitoxantrone, cis-platinum, carboplatin, camptothecine, adriamycin；Or the DNA damage treatment includes but is not limited to: ionising radiation Treatment.

The pharmaceutical composition and gene cytotoxic drug or DNA damage Drug combination are in suppression in another preferred example Tumour processed；Or the pharmaceutical composition and DNA damage treatment are united and applied in inhibition tumour.

The lower adjustment is selected from another preferred example: the disturbing molecule of specificity interference Zscan4 gene expression；Or The gene editing reagent (such as targeting the sgRNA of Zscan4 gene) of specific knockdown Zscan4 gene；Or specificity with The protein bound antibody or ligand of Zscan4 gene coding.

The lower adjustment is shRNA in another preferred example；Preferably, it is with SEQ ID NO:1 and SEQ ID Nucleotide sequence shown in NO:2.

In another aspect of this invention, the purposes of a kind of Zscan4 gene or albumen is provided, regulation aging phase is used to prepare Close the composition of secretion phenotype (SASP)；Or it is used to prepare the pharmaceutical composition for inhibiting diseases associated with senescence.

In a preferred embodiment, the diseases associated with senescence includes: atherosclerosis, osteoarthritis, osteoporosis, And other organ degenerative diseases.

In another aspect of this invention, a kind of method of the potential substance of screening inhibition tumour is provided, which comprises

(1) system of expression Zscan4 gene is handled with candidate substances；With

(2) expression or activity of Zscan4 gene in the system are detected；

Wherein, if the candidate substances can reduce the expression or activity of Zscan4 gene, show that the candidate substances are suppressions The potential substance of tumour processed.

In a preferred embodiment, step (1) includes: that candidate substances are added to the body of expression Zscan4 in test group In system；And/or

Step (2) includes: the expression or activity of Zscan4 in the system for detect test group, and compared with the control group, wherein The control group is the system for not adding the expression Zscan4 of the candidate substances；

If the expression of Zscan4 or activity are statistically lower than control group in test group, indicate that the candidate is suppression The potential substance of tumour processed.

In another preferred example, the system is selected from: cell system (cell or cell culture such as expression Zscan4 Object), subcellular system, solution system, organizational framework, organ systems or animal system.

In another preferred example, described statistically lower than preferably significantly lower than, such as low 20% or more, it is preferably low 50% or more；More preferably low 80% or more.

In another preferred example, the candidate substances include but is not limited to: being directed to Zscan4 gene or protein design Small molecule compound, the signal path participated in for Zscan4 gene or albumen or its upstream or downstream protein design it is dry Disturb molecule, nucleic acid inhibitor, binding molecule (such as antibody or ligand).

In another preferred example, the method further include: further cell experiment is carried out to the potential substance of acquisition And/or animal experiment, further to select and determine the substance for inhibiting tumour useful from candidate substances.

In another aspect of this invention, it provides a kind of for inhibiting the pharmaceutical composition of tumour, the pharmaceutical composition In include: Zscan4 gene or albumen lower adjustment；With gene cytotoxic drug, DNA damage drug or DNA damage ionising radiation Therapeutic agent.

In another aspect of this invention, it provides a kind of for inhibiting the medicine box of tumour, includes: container in the medicine box, And it is placed in the lower adjustment of the Zscan4 gene or albumen of the container；With

Container, and it is placed in the gene cytotoxic drug of the container, DNA damage drug or DNA damage ionizing radiation treatment Drug.

In a preferred embodiment, the lower adjustment includes: the disturbing molecule of specificity interference Zscan4 gene expression；Or The gene editing reagent (such as targeting the sgRNA of Zscan4 gene) of specific knockdown Zscan4 gene；Or specificity with The protein bound antibody or ligand of Zscan4 gene coding.

In another preferred example, the gene cytotoxic drug or DNA damage drug include but is not limited to: bleomycin, Mitoxantrone, cis-platinum, carboplatin, camptothecine, adriamycin.

In another aspect of this invention, the reagent of a kind of specific recognition Zscan4 gene or the albumen of its coding is provided Purposes is used to prepare the reagent or kit for carrying out tumor prognosis evaluation.

In a preferred embodiment, the reagent of the albumen of the specific recognition Zscan4 gene or its coding is selected from: special The primer of specific amplification Zscan4 gene；The probe of specific recognition Zscan4 gene；Or specific binding Zscan4 gene is compiled The antibody or ligand of the albumen of code.

In another aspect of this invention, a kind of kit for tumor prognosis evaluation is provided, is contained in the kit Have: the reagent of the albumen of specific recognition Zscan4 gene or its coding.

Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.

Detailed description of the invention

The cell of Fig. 1, source of people Normal primary Prostate Stromal Cells after two groups of clinically used anti-cancer therapies processing Morphological feature.Upper layer, the bright field picture after SA- β-Gal dyeing；Lower layer differs (phase contrast) picture.Arrow, Typical vacuole structure in cytoplasm.

Fig. 2, stroma cell are handling the statistical analysis after SA- β-Gal dyeing again through various anti-cancer modalities.

BdrU Embedding efficiency after Fig. 3, stroma cell are damaged statisticallys analyze.

Fig. 4, DNA damage focus through immunofluorescence dyeing determine after, according to 4 classification used in the past carry out sort out and Compare.

Fig. 5, according to microarray data, by PSC27 cell respectively after different classes of drug-treated, carry out GO function It can analyze, CC (cellular component) statistics shows different generics.Left side, DT (DNA damaging treatment) are right Side NDT (non-DNA damaging treatment).The corresponding item name of each color is with reference in comment field in pie chart Mark.

Fig. 6, histogram mode show the statistical result in Fig. 5.Left side, DT (DNA damaging treatment) are right Side NDT (non-DNA damaging treatment).Each color in Fig. 5 the same as corresponding to each other in histogram.

Fig. 7, pie chart show microarray data in GO-BP statistic of classification as a result, the correspondence class name of each category color Title is shown in intermediate mark.

Fig. 8, pie chart show microarray data in GO-MF statistic of classification as a result, the correspondence class name of each category color Title is shown in intermediate mark.

Fig. 9, Venn diagram figure, which are shown, reconciles down-regulated gene after two groups of drug-treateds on stroma cell.The up-regulation of DT group There are change > 3 fold of 271 genes in list, and there are 71 change > 3 gene fold to appear in up-regulation column in NDT group In table.

Figure 10, the upper synthesis Venn diagram figure lowered that reconciles.It removes outside the up-regulation gene in figure (9), Shang You 407 Gene appears in DT downward list and change > 3 fold, and 147 genes appear in the respective list of NDT.

Upper highest preceding 30 genes of modulation in stroma cell after Figure 11, Heatmap display DT group drug-treated, Corresponding expression value after the processing of NDT group is shown simultaneously.The expression value of each gene is respectively DT damage group compared to control The result of group (unprocessed group).

The result of Heatmap in Figure 12, Figure 11 after hierarchical agglomerate (drug and gene be two parameters) analysis.

The influence of Figure 13, Whole genome analysis DT group and NDT group for stroma cell express spectra, the fold of 3107 genes change>1.To compare visualization, hierarchical agglomerate is not done.

Figure 14, stroma cell were by the 7th day after bleomycin (50 μ g/ml) processing, by fixed and immunofluorescence dyeing Result.γ H2AX and Zscan4 antibody is primary antibody, and DAPI is to redye.Control group, the primary stroma cell without drug-treated. Scale, 20 μm.

The expression that Figure 15, Western blot analyze Zscan4 in the 7th day stroma cell after bleomycin is handled becomes Change and subcellular localization.After cytoplasm and Nuclear extract are separated through kit, ATM activation, NF- κ B compound subunit The expression of the nuclear translocation and Zscan4 of (p50, p65) detects simultaneously.GAPDH and Histone H3 is respectively cytoplasm and cell Core loading control.

Figure 16, Western blot analyze Zscan4 albumen, p38 and its substrate HSP27, p16, p21 and typical case SASP because Expression of the son in stroma cell.* P<0.05, * * P<0.01, ^P>0.05.

It includes cis-platinum (Cisplatin) that Figure 17, PSC27 cell pass through chemotherapeutics respectively, carboplatin (Carboplatin), Camptothecine (Camptothecin), Tarceva (Erlotinib) receive Wu Dankang (Nivolumab) and adriamycin (Doxorubicin) after extracorporeal treatment, with expression of the Western blot analysis Zscan4 in cell.

The expression of Zscan4 in Figure 18, non-small cell lung cancer (NSCLC) patient's primary tumo(u)r.Patient remembers according to pathology Record is divided into without chemotherapy group and experience chemotherapy group, and histotomy is passed through to be dyed by the IHC of primary antibody dyeing and HE of anti-Zscan4. Scale, 50 μm.

Figure 19, the pathology assessment based on Zscan4 coloration result in NSCLC tissue.Patient according to Zscan4 in the tissue Staining power be divided into 4 classifications, including 1 (feminine gender), 2 (weak), 3 (medium) and 4 (strong).P<0.05.

Figure 20, with laser capture microdissection (LCM) technology by tumor tissues epithelial cell and stroma cell carry out After specific isolation, qRT-PCR analyzes expression of the Zscan4 in two kinds of cell types.Box-shaped figure shows that Zscan4 exists Expression under the conditions of various.

Figure 21, Kaplan-Meier analyze Zscan4 expression with the pass between NSCLC patient's DFS phase (DFS) Connection.DFS is defined as going out from patient clinical diagnoses the number of days after the NSCLC time to treatment the palindromia moment.

Zscan4 protein expression is the same as the relationship between p38 and mTOR intensity of activation in Figure 22, NSCLC patient matrix organization. Share 99 patient tissues by analysis, the score value of each albumen is derived from 3 different independent pathology judgements.

(Pearson analysis, r=are assessed in association in Figure 23, matrix organization between Zscan4 and p38 pathology score value 0.82；P<0.0001).

(Pearson analysis, r are assessed in association in Figure 24, matrix organization between Zscan4 and mTOR pathology score value =0.87；P<0.0001).

The expression of Zscan4 in Figure 25, breast cancer (BCa) patient's primary tumo(u)r.Patient is divided into not according to recording pathological mechanism Through chemotherapy group and experience chemotherapy group, histotomy is passed through to be dyed by the IHC of primary antibody dyeing and HE of anti-Zscan4, scale, 50 μ m。

Figure 26, the pathology assessment based on Zscan4 coloration result in NSCLC tissue.Patient according to Zscan4 in the tissue Staining power be divided into 4 classifications, including 1 (feminine gender), 2 (weak), 3 (medium) and 4 (strong).P<0.05.

Figure 27, with laser capture microdissection (LCM) technology by tumor tissues epithelial cell and stroma cell carry out After specific isolation, qRT-PCR analyzes expression of the Zscan4 in two kinds of cell types.Box-shaped figure shows that Zscan4 exists Expression under the conditions of various.

Figure 28, Kaplan-Meier analyze Zscan4 expression with the pass between BCa patient's DFS phase (DFS) Connection.DFS is defined as going out from patient clinical diagnoses the number of days after the BCa time to treatment the palindromia moment.

Zscan4 protein expression is the same as the relationship between p38 and mTOR intensity of activation in Figure 29, BCa patient matrix organization.Altogether There are 62 patients by fabric analysis, the score value of each albumen is derived from 3 different independent pathology judgements.

(Pearson analysis, r=are assessed in association in Figure 30, matrix organization between Zscan4 and p38 pathology score value 0.81, P < 0.0001).

(Pearson analysis, r=are assessed in association in Figure 31, matrix organization between Zscan4 and p38 pathology score value 0.67；P<0.0001).

Figure 32, the PSC27 subbreed including Zscan4 overexpression group are after bleomycin (50ug/ml) processing DDR foci statistical result.

BrdU incorporation situation statistics in Figure 33, Figure 31 after each group stroma cell DNA damage.

The cell ageing assessment that group of cells is dyed based on SA- β-Gal in Figure 34, Figure 31.

Figure 35, the PSC27 subbreed including Zscan4 knockout group are after bleomycin (50ug/ml) processing DDR foci statistical result.

BrdU incorporation situation statistics in Figure 36, Figure 34 after each group stroma cell DNA damage.

The cell ageing assessment that each group stroma cell is dyed based on SA- β-Gal in Figure 37, Figure 34.

Clonality detection under Figure 38, conditions in vitro after various Cell subline experience bleomycin processing.

The statistical analysis of group of cells clone colony number in Figure 39, Figure 38.

Expression analysis of each group stroma cell typical case SASP factor in transcriptional level in Figure 40, Figure 31.

Expression analysis of each group stroma cell typical case SASP factor in transcriptional level in Figure 41, Figure 34.

Figure 42, Western blot detection Zscan4 protein expression and the DDR factor include H2AX, ATM, Kap1, Chk2, and Activation situation including p53.Right side, control group (SCR-shRNA) and Zscan4 knockout group (Zscan4-shRNA) are by rich next Various factor expressions or Activation after mycin extracorporeal treatment.The different time nodes that cell indicates in figure are collected It is analyzed later for immune-blotting method.* P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001, ^P>0.05.

Figure 43, PSC27 are handled via bleomycin (50ug/ml) and then with ATM micromolecular inhibitor KU55933 (KU, 10 μM) effect.The PI product that Anti-p-ATM is mediated, the Western mediated by anti-p-ATM and anti-TRAF6 Blot analysis.

Figure 44, PSC27 control group and process TRAF6-specific shRNAs knockout group are after bleomycin damage IP the and immunoblot assays analysis mediated with anti-TRAF6.

Figure 45, PSC27 cell are aided with 5Z-7 (500nM) processing by bleomycin and then mediate through anti-TAK1 IP sedimentation, western blot analyze product in p-TAK1, TRAF6, p-ATM and ATM expression.

Figure 46, PSC27 control cell and TRAF6specific shRNA surely transfer from one department to another after handling by bleomycin, warp The IP that anti-TRAF6 is mediated, product are analyzed through western blot again.

Figure 47, stroma cell cytoplasmic protein and nucleoprotein after bleomycin processing efficiently separate the sample after extracting This, wherein TAK1 is activated and NF- κ B nuclear translocation situation for analysis.Control cell is parallel with the cell progress by 5Z-7 processing to divide Analysis.

Possible NF- κ B binding site in Figure 48, the diagram signal upstream Zscan4 proximal promoter DNA sequence dna.Promoter Product of the region after fragmentary sequence clone, is connected into pGL4.22 carrier (pGL-Zscan4-P01 to P06) respectively. TSS, transcription initiation site.

293 cell of HEK of Figure 49, in advance transfection Zscan4, are handled in the medium with TNF-α (20ng/ml).It is unloaded As negative control, the expression vector NAT11-Luc2CP for encoding multiple NF- κ B binding sites and IL-2 minimal promoter is body Positive control.

Figure 50, the PSC27 transfected using plasmid involved in Figure 48 or carrier, through 50 μ g/ml bleomycins or 1uM meters After holding in the palm anthraquinone processing, its respective firefly luciferase activity is detected.

Figure 51, co-immunoprecipitation chromatin immunoprecipitation (ChIP) are for analyzing and identifying Zscan4 The NF- κ B binding site of upstream promoter region.The Three Represents position of Zscan4-p1/p2/p3 expression Zscan4 promoter region Point.

Figure 52, NF- κ B-, which are not intended to be mutated PSC27 subbreed, passes through bleomycin, after mitoxantrone and radioactive ray process, warp It crosses qRT-PCR and analyzes its signal strength or weakness for expressing Zscan4.

Figure 53, PSC27 stroma cell are after bleomycin and 5Z-7 processing, the table of various typical case's SASP effector molecules Change up to level.BLEO, bleomycin.5Z-7,5Z-7-oxozeaenol.*P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P<0.0001, ^P>0.05.

Figure 54, stroma cell pass through 50 μ g/ml bleomycins, various typical case SASP after 5 μM of Bay 11-7082 processing The expression of the factor.

Figure 55, the stroma cell established after SCR-shRNA and Zscan4-shRNA transfection surely transfer from one department to another, and undergo to win and Its corresponding CM is collected after mycin processing, is used for a variety of cancerous prostate epithelial cell systems of in vitro culture.Proliferation rate is with cell number Amount is evaluated.

The external mobility test that various epithelial cell lines in Figure 56, Figure 54 are mediated by Transwell.

The vitro invasion rate test that various epithelial cell lines in Figure 57, Figure 54 are mediated by Transwell.

Figure 58, immunodeficient mouse (SCID) are inoculated with the thin of the various subbreed compositions of PC3 and/or PSC27 into subcutaneous location is crossed After born of the same parents' recombinant, by a pre- clinical trial in 8 weeks by a definite date, detect the gross tumor volume of mouse and carry out pathological analysis when Between be oriented to schematic diagram.

The each group mouse tumor volume detected at the end of Figure 59, pre- clinical trial, the independent inoculation group of PC3 cell are to compile Number 1-6；The group for having stroma cell to participate in is combined as number 7-12.Between 9th group and the 10th group, between the 11st group and the 12nd group Statistical difference is indicated with the latter compared to the former variation ratio.Every group of animal is 10.Zscan4 knockout, which uses, is shRNA#1。

The each group mouse tumor volume detected at the end of Figure 60, pre- clinical trial, the independent inoculation group of PC3 cell are to compile Number 1-6；The group for having stroma cell to participate in is combined as number 7-12.Between 9th group and the 10th group, between the 11st group and the 12nd group Statistical difference is indicated with the latter compared to the former variation ratio.Every group of animal is 10.Zscan4 knockout, which uses, is shRNA#2。

Specific embodiment

The present inventor after extensive and in-depth study, discloses Zscan4 in the process of SASP phenotype occurrence and development for the first time The important biological action of middle possible performance, and it is also closely related with the development of tumour.Therefore, Zscan4 can be used as SASP The research target spot of phenotype regulation, can be used as diagnosis, the prognosis evaluation marker of tumour, is alternatively arranged as target spot exploitation and inhibits tumour Drug.

Zscan4

Mankind Zscan4 (zinc finger and SCAN domain containing 4) gene cluster include 6 can be with There is the sequence similarity of height, collectively referred to as Zscan4 (also known as ZNF494) each other in the paralog gene of transcription.Wherein The Zscan4d mainly expression in two cell stages (two-cell embryo), and then primary expression is dry thin in embryo by Zscan4c Born of the same parents (embryonic stem cell, ES cell) simultaneously regulate and control self-renewing.Zscan4 actively participates in the telomere maintenance of ES cell, And ensure that this cell has long-term Genome stability.At any one time, only about 5% ES cell expresses Zscan4, But almost all of ES cell at least expresses a Zscan4 in continuous succeeding generations.On biological function, Zscan4 wink When expression it is related with the homologous recombination gene upregulation of rapid extension and the meiosis specificity of telomere, the latter then encode with Zscan4 common location is in the albumen of Telomere regions.However, the missing of Zscan4 but causes telomere to shorten, karyotype is abnormal, Spontaneous sister chromatid exchange, cellular morphology is flat, and growth rate slows down, and apoptosis activity rises, until cell enters crisis (crisis) period, be finally stopped division and it is dead.

Understanding in relation to Zscan4 mostly extends with telomere related with Genome stability so far.Under DNA damage stress, Zscan4 can not only reduce DDR response intensity, but also can significantly improve and induce multi-potent stem cell (induced Pluripotent stem cell, iPSC) generation efficiency；Its express by PI3K catalytic subunit p110 α regulation and With being parallel to each other for DNA double chain signs of failure object γ H2AX.It is generated in a manner of increasing Zscan4 expression The iPSC of iPSC ratio under normal circumstances has longer telomere, so maintain Genome stability that can mention during reprogramming It rises reprogramming efficiency, the iPSC of high quality is promoted to be formed.In addition, Zscan4 can be played in non-dry cell telomere extend and Genome stablizes relevant function.Telomerase positive cancer cells system HeLa, MCF7 and telomerase negative cancer cells system SaOS2, In U2OS, Zscan4 can with telomere repeatability binding factor 1/2 (telomere repeat binding factor1/2, TRF1/2) and repressor Activating protein-1 (repressor activator protein 1, Rap1) is fixed altogether in nucleus Simultaneously direct interaction occurs for position.In this case, Zscan4 is by way of telomerase activation dependent/non-dependent, in cancer cell Middle maintenance telomere and genomic integrity.

Using Protocols in Molecular Biology, the inventors discovered that, human fibroblast DNA caused by chemicotherapy processing is damaged Under the conditions of wound, Zscan4 expression quantity obviously rises, and gradually develops from the up-regulation in acute senescent stage as chronic aging in cell The feature of lasting, the constant expression in period.Compared to non-genomic poison anticancer drug, those establish change on the basis of targeting DNA Reagent and ionising radiation are treated, more can induce the expression of Zscan4.It is worth noting that, after gene knockout Zscan4, because of DNA The cell of chronic aging is damaged and enters, the expression of SASP component protein is remarkably decreased, and ectopic expression Zscan4 makes At result on the contrary, implying that Zscan4 may play important biology during SASP phenotype occurrence and development and make With.In recent years it has been reported that the cell ageing of DNA damage induction reacts related with duration DDR, and end caused by extrinsic factor Just unrepairable once occurs for grain DNA damage, to excite permanent DDR reaction and cell ageing.

In this regard, present inventors believe that cell acute Stress responses DDR caused by DNA damage passes through the transcriptions such as activation NF- κ B The factor raises expression of the Zscan4 in stroma cell, and the latter raises telomeric dna affected area in core immediately, with the eggs such as Rap1 The white DDR related complex for being assembled into collaboration and repairing telomeric dna damage；This duration DNA damage signal forces cell with non- Independence mode enters aging state, and further activates the transcription such as NF- κ B by the signal cascade effect of DDSP driven by program The factor eventually leads to the occurrence and development of wide spectrum SASP secretion phenotype to form the positive feedback mechanism that Zscan4 is mediated.

It is adjusted under Zscan4

Above-mentioned new discovery based on the present inventor, the present invention provides a kind of use of the lower adjustment of Zscan4 gene or albumen On the way, it is used to prepare the pharmaceutical composition for inhibiting tumour.The tumour is selected from: passing through gene cytotoxic drug or DNA damage drug Tumour after processing or by DNA damage treatment, or the tumour of expression Zscan4.The gene cytotoxic drug or DNA damage Drug can be but not limited to: bleomycin, mitoxantrone, cis-platinum, carboplatin, camptothecine and adriamycin etc..The DNA damage Property treatment can be but not limited to: ionizing radiation treatment.

The embodiment of the present invention is the results show that missing of the Zscan4 in stroma cell can be significantly reduced under conditions in vitro Drug resistance of the various acquired shapes and tumor-bearing mice in-vivo tumour of cancer cell under chemotherapy regimes；Traditional gene poison therapy Joint microenvironment (TME) targeted therapy, can significantly inhibit the drug resistance that tumour is obtained from microenvironment, and therapeutic effect obviously changes It is kind.It therefore, can be by the lower adjustment of Zscan4 gene or albumen of the invention and gene poison as preferred embodiment of the invention Object or DNA damage Drug combination are in inhibition tumour；Or the pharmaceutical composition and DNA damage treat use in conjunction In inhibition tumour.It generally, can be after using traditional gene cytotoxic drug or DNA damage drug therapy, using the present invention Zscan4 gene or albumen lower adjustment treatment.

As used herein, the lower adjustment of the Zscan4 gene or albumen includes inhibitor, antagonist agent, retardance Agent, blocking agent etc..

The lower adjustment of the Zscan4 gene or albumen refers to any activity for reducing Zscan4 albumen, reduces The stability of Zscan4 gene or albumen, the expression for lowering Zscan4 albumen reduce Zscan4 albumen effective acting time or suppression The substance of the transcription and translation of Zscan4 gene processed, these substances are used equally for the present invention, as useful for lowering Zscan4 Substance, so as to for inhibiting tumour.For example, the lower adjustment is: the interference of specificity interference Zscan4 gene expression RNA molecule or GEM 132；Or specificity and the protein bound antibody of Zscan4 gene coding or ligand, etc..

As a kind of preferred embodiment of the invention, the lower adjustment is a kind of disturbance RNA molecule of Zscan4 specificity (shRNA), the inventor have observed that, using disturbance RNA molecule of the invention, Zscan4 is lowered with can dramatically, for tumour Inhibiting effect highly significant.

The present invention is not particularly limited the preparation method of disturbance RNA molecule, including but not limited to: chemical synthesis, In-vitro transcription method etc..The RNA interfering can be transported into the cell by using transfection reagent appropriate, or this also can be used Multiple technologies known to field are transported into the cell.

As another kind selection of the invention, the gene editing that CRISPR/Cas9 system is targeted can be used, thus The region of targeting disease knocks out Zscan4 gene.The method of common knockout Zscan4 gene includes: that by sgRNA or can form institute It states the nucleic acid of sgRNA, Cas9 mRNA or the nucleic acid corotation of the Cas9 mRNA can be formed into target area or targeting cell. After target site has been determined, it can adopt and come by known method so that sgRNA and Cas9 are introduced in into the cell.The energy The nucleic acid for forming the sgRNA is nucleic acid construct or expression vector or the nucleic acid that can form the Cas9 mRNA is These expression vectors are imported into the cell by nucleic acid construct or expression vector, to form active sgRNA in the cell And Cas9 mRNA.

The reagent or kit of tumor prognosis evaluation

Above-mentioned new discovery based on the present inventor, can be using Zscan4 as the marker of tumor prognosis evaluation: (i) is carried out Parting, antidiastole, and/or the susceptibility analysis of tumour；(ii) assess the tumor therapeutic agent of correlated crowd, curative effect of medication, Prognosis, and the suitable treatment method of selection.For example, may separate out by the crowd of Zscan4 abnormal gene expression, so as into Row is more targetedly treated.

The new discovery of people according to the present invention, can be by judging the expression of Zscan4 or active feelings in sample to be assessed Condition, come predict to provide the sample to be assessed subject tumor prognosis situation, select suitable drug to implement treatment.In general, The threshold value that can specify that a Zscan4 is considered as inhibiting when the expression of Zscan4 is higher than the threshold value of defined The scheme of Zscan4 is treated.The threshold value is easy to determining to those skilled in the art, such as can lead to It crosses the expression of the Zscan4 in the expression of the Zscan4 in health adult tissue's microenvironment and tumor patient microenvironment After being compared, the threshold value of Zscan4 abnormal expression is obtained.

Therefore, the present invention provides the purposes of Zscan4 gene or albumen, be used to prepare tumor prognosis evaluation reagent or Kit.

Various techniques known in the art can be used to detect presence or absence and the expression of Zscan4 gene, these Technology is included in the present invention.Such as existing technology such as Southern blotting, western blot method, DNA sequence dna can be used Analysis, PCR etc., these methods may be used in combination.

The present invention also provides the examinations of presence or absence and expression for detecting Zscan4 gene in analyte Agent.Preferably, when carrying out the detection of gene level, the primer of specific amplification Zscan4 can be used；Or specific recognition The probe of Zscan4 determines the presence or absence of Zscan4 gene；It, can be using specificity when carrying out the detection of protein level The expression of Zscan4 albumen is determined in conjunction with the antibody or ligand of the Zscan4 albumen encoded.

Design for the specific probe of Zscan4 gene is technology well known in the art, for example, preparing one kind Probe can be specifically bound with specific site on Zscan4 gene, without with other genes other than Zscan4 gene Specific binding, and the probe has detectable signal.

The method for testing and analyzing Zscan4 protein expression situation in object using the antibody of specific binding Zscan4 albumen It is also technology well known in the art.

The present invention also provides the examinations of presence or absence and expression for detecting Zscan4 gene in analyte Agent box, the kit include: the primer of specific amplification Zscan4 gene；The probe of specific recognition Zscan4 gene；Or it is special The opposite sex combines the antibody or ligand of the albumen of Zscan4 gene coding.

In addition, may also include in the kit for various reagents needed for extracting DNA, PCR, hybridization, colour developing etc., Including but not limited to: extract, amplification liquid, hybridization solution, enzyme, comparison liquid, developing solution, washing lotion etc..

In addition, may also include operation instructions and/or Nucleotide Sequence Analysis Software etc. in the kit.

Drug screening

It can promote the growth of tumour cell knowing overexpression of the Zscan4 in stroma cell, and knock out stroma cell In Zscan4 can inhibit this feature of the growth of tumour cell after, can be screened based on this feature inhibit Zscan4 expression Or active substance.The drug for inhibiting tumour actually useful can be found from the substance.

Therefore, the present invention provides a kind of method of the potential substance of screening inhibition tumour, and the method includes: with candidate The system of substance processing expression Zscan4；With the expression or activity for detecting Zscan4 in the system；If the candidate substances can The expression or activity for inhibiting Zscan4 then show that the candidate substances are the potential substances for inhibiting tumour.The expression Zscan4 System be preferably cell (or cell culture) system, the cell can be the cell of endogenous expression Zscan4； Or it can be the cell of recombinant expression Zscan4.

In a preferred embodiment of the present invention, when being screened, in order to be more easily observable the expression or activity of Zscan4 Change, also settable control group, the control group can be do not add the candidate substances expression Zscan4 system.

As preferred embodiment of the invention, the method further include: the potential substance of acquisition is carried out further thin Born of the same parents' experiment and/or animal experiment, further to select and determine the substance for inhibiting tumour actually useful.

On the other hand, the present invention also provides the potential substances of the inhibition tumour obtained using the screening technique.These Preliminary screening go out substance may make up a screening library, in order to people may finally be screened out from it can for inhibition The expression and activity of Zscan4, and then the substance for inhibiting tumour useful.

Pharmaceutical composition

The present invention also provides a kind of pharmaceutical compositions, it contains effective quantity (such as 0.000001-50wt%；Preferably 0.00001-20wt%；More preferably, 0.0001-10wt%) the Zscan4 gene or albumen lower adjustment and pharmacy Upper acceptable carrier.The lower adjustment of any Zscan4 gene above-mentioned or albumen is used equally for the preparation of composition.

As a kind of preferred embodiment of the invention, provide a kind of for inhibiting the composition of tumour, the composition Contain a effective amount of disturbance RNA molecule of the present invention and pharmaceutically acceptable carrier.

As a kind of preferred embodiment of the invention, provide a kind of for inhibiting the composition of tumour, the composition Lower adjustment containing a effective amount of Zscan4 gene or albumen and a effective amount of other preparations, other preparations are for example It is gene cytotoxic drug or DNA damage drug, ionizing radiation treatment drug.

As used herein, described " effective quantity " refer to people and/or animal can be generated function or it is active and can by people and/ Or the amount that animal is received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various figurations Agent and diluent.The term refers to medicament carriers some in this way: themselves not being necessary active constituent, and does not have after applying Excessive toxicity.Suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable load in the composition Body can contain liquid, such as water, salt water, buffer.In addition, there is likely to be complementary substance in these carriers, as filler, Lubricant, glidant, wetting agent or emulsifier, pH buffer substance etc..Lipofectamine can also be contained in the carrier.

After knowing the purposes of lower adjustment of the Zscan4 gene or albumen, it can use well known in the art a variety of The lower adjustment or its encoding gene or its pharmaceutical composition are delivered medicine to mammal by method.Including but not limited to: skin Lower injection, intramuscular injection, for percutaneous administration of, administer locally to, be implanted into, be sustained and give；Preferably, the administration mode is non-bowel It gives.

Preferably, the means that gene therapy can be used carry out.For example, can be directly by the lower adjustment of Zscan4 by such as infusing It the methods of penetrates and to deliver medicine to subject；Alternatively, can will be carried by certain approach the lower adjustment of Zscan4 ceneme (such as Expression vector or virus etc. or siRNA) it is delivered on target spot, and be allowed to adjust under the Zscan4 of expression activity, concrete condition needs Depending on the type of the lower adjustment, these are well-known to those skilled in the art.

The effective quantity of the lower adjustment of Zscan4 gene or albumen of the present invention can be with the mode of administration and to be treated The severity of disease etc. and change.Preferred a effective amount of selection can be by those of ordinary skill in the art depending on various factors To determine (such as passing through clinical test).The factor includes but is not limited to: the downward of the Zscan4 gene or albumen Pharmacokinetic parameter of agent such as bioavailability, metabolism, half-life period etc.；Patient institute the severity of disease to be treated, The weight of patient, the immune state of patient, approach of administration etc..

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.

I, material and method

1. cell culture

(1) cell line maintains

The normal primary stromal cell lines PSC27 of source of people prostate (be obtained from U.S.'s Fred and breathe out green gloomy Cancer Research Center) in It is proliferated and passes in PSCC complete culture solution.Prostate Benign Epithelial cell line BPH1, carcinoma of prostate epithelial cell line M12, DU145, PC3, LNCaP and VCaP (be purchased from ATCC) in the RPMI-1640 complete culture solution of 5%FBS, in 37 DEG C, It is cultivated in the incubator of 5%CO2 condition.

(2) cell cryopreservation and recovery

A cell cryopreservation

Logarithmic growth phase cell is collected with 0.25% trypsase, 1000rpm is centrifuged 2min, discards supernatant, suspend again Cell is in the frozen stock solution of fresh configuration.Cell is dispensed in the sterile cryopreservation tube indicated.Then through (4 DEG C of gradient cooling 10min, -20 DEG C of 30min, -80 DEG C of 16-18h), finally it is transferred to liquid nitrogen stored for extended periods.

B cell recovery

The cell frozen in liquid nitrogen is taken out, 37 DEG C of water-baths is immediately placed in, makes its fast melt.It is directly added into the training of 2ml cell Nutrient solution makes cell even suspension.After cell is adherent, the culture solution that more renews.

(3) experiment in vitro is handled

To cause cellular damage, 100nM is added in culture solution when PSC27 cell grows to 80% (abbreviation PSC27-Pre) Taxotere (docetaxel, DTX), 100nM taxol (paclitaxel, PTX), 200nM vincristine (vincristine, VCR), 50 μ g/ml bleomycin (bleomycin, BLEO), 1 μM of mitoxantrone (mitoxantrone, MIT) or 10Gy¹³⁷Cs ionising radiation (γ-radiation is in 743rad/min, RAD).In addition, 100 μM of cis-platinums (cisplatin, CIS), 100 μ M carboplatin (carboplatin, CARB), 50nM camptothecine (camptothecin, CAM) and 20 μM of adriamycins (doxorubicin, DOX) after drug-treated 6 hours, cell is simply washed 3 times by PBS, then indwelling carries out subsequent reality 7~10 days in culture solution It tests.

2. plasmid preparation and slow-virus transfection

Overall length source of people Zscan4 is cloned in Lentiviral pLenti-CMV/To-Puro-DEST2 (Invitrogen) between restriction enzyme site EcoRI and XbaI.Packaging is that 293FT is used for cell transfecting and slow virus manufacture.With It is respectively as follows: in small hairpin RNAs (shRNAs) sequence for knocking out Zscan4

5'-CAAATAGTTTCCCTAATCATC-3'(SEQ ID NO:1)；With

5’-CTACGGGTGCATCAGATAATT-3’(SEQ ID NO:2)。

3. immunofluorescence and histochemical analysis

Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser139) (clone JBW301, ) and rabbit polyclonal antibody anti-Zscan4 (Cat#A12015, Abclonal) and secondary antibody Alexa Millipore488 (or 594)-conjugated F (ab ') 2 are sequentially added on the glass slide for being covered with fixed cell.Cell Core is redyed with the DAPI of 2 μ g/ml.A most representative image is chosen from 3 field of view carries out data analysis It is shown with result.FV1000 laser scanning co-focusing microscope (Olympus) is for obtaining cell confocal fluorescent image.

Clinical patients with lung cancer and patient with breast cancer organize IHC dyeing antibody Zscan4 used to be same as above, and are purchased from Abclonal.Tool Steps are as follows for body: then conventional dewaxing uses the citric acid of 0.01M pH6.0 with 0.6%H2O2 methanol in 37 DEG C of incubation 30min Buffer repairs 20min, the cooling 30min of room temperature.20min is closed with normal sheep serum, with Zscan4 primary antibody (1:200) at 37 DEG C It is incubated for 1h, moves to 4 DEG C of refrigerator overnights.It is washed three times with TBS within second day, is incubated for secondary antibody (the goat-anti rabbit of HRP coupling) at 37 DEG C 45min, then washed 3 times with TBS, finally developed the color with DAB.

4. matrix-epithelium co-cultures and experiment in vitro

With culture solution culture PSC27 cell 3 days of DMEM+0.5%FBS, the cell of full abundance is then cleaned with 1 times of PBS Group.Supernatant is simply collected after centrifugation as Conditioned immunolresponse and stores -80 DEG C or directly uses.Prostate epithelial cell is this Carry out experiment in vitro in the time for continuously cultivating 3 days in Conditioned immunolresponse.For chemoresistant, epithelial cell ties up to low serum It is cultivated in DMEM (0.5%FBS) or in Conditioned immunolresponse, while mitoxantrone (MIT) is for handling cell 1 to 3 day, it is dense Spend the IC close to each cell line₅₀Numerical value is then observed under bright-field microscope.

5. full-length genome range expresses chip analysis (Agilent expression microarray)

Stromal cell lines PSC27 primary to normal source of people prostate carries out full-length genome range expression chip (4x 44k) point The program and method of analysis are referring to Sun, Y. etc., Nat.Med.18:1359-1368.

6. quantitative PCR (RT-PCR) measures gene expression

(1) extraction of cell total rna

Growth period cell total rna is extracted with Trizol reagent, every T25 culture bottle cell is added 1ml Trizol, uses cell Scraper transfers them in centrifuge tube after scraping cellular layer, mixes well to not sticky.Every 1ml Trizol adds 0.2ml chloroform, Acutely concussion 15 seconds are incubated at room temperature 5-10min；4 DEG C, 11,000g centrifugations 15 minutes；Colorless supernatant liquid is moved into a new centrifugation Guan Zhong adds 0.5ml isopropanol by every 1ml Trizol, is incubated at room temperature 10 minutes, 11,000g, 4 DEG C are centrifuged 10 minutes；It outwells Clearly, it is washed, 4 DEG C with 75% ethyl alcohol (every 1ml Trizol is at least with 75% ethyl alcohol of 1ml), 7,500g centrifugations 5 minutes；Room temperature is dry RNA precipitate 5-10 minutes dry (RNA cannot be dried), uses DEPC-H²O dissolution precipitating.

After spectrophotometer quantifies RNA, a small amount of total serum IgE is taken to carry out 1% agarose electrophoresis, checks RNA state and quality.

(2) reverse transcription reaction

OligodT₂₃VN(50uM) 1ul

Total RNA 1-2ug

RNase Free ddH₂O adds to 8ul

65 DEG C are heated 5 minutes, are immediately placed in and are quenched on ice, and stand 2 minutes.

Configure the first chain cDNA Synthesis liquid

2x RT Mix 10ul

HiScript II Enzyme Mix 2ul

The first chain cDNA synthesis is carried out according to the following conditions:

25℃ 5min

50℃ 45min

85℃ 5min

(3) real-time quantitative PCR reacts

Reverse transcription reaction product cDNA is diluted 50 times and is used as template.

It is loaded according to the above standard, reaction condition are as follows: 95 DEG C of initial denaturation 15s, then 95 DEG C of 5s, 60 DEG C of 31s, 40 are followed Ring；Melt curve analysis condition is 95 DEG C of 15s, 60 DEG C of 30s, 95 DEG C of 15s.Sample is reacted on ABI ViiA7 (ABI) instrument.With Internal reference is made in the expression of β-actin.After the reaction was completed, the amplification situation that each gene is checked through software analysis, exports corresponding domain It is worth recurring number and the relative expression quantity of each gene is calculated using 2- Δ Δ Ct method.To melt curve analysis (melting surve) Wave crest and waveform analyzed to determine whether obtained amplified production is specific single goal segment.

7.Western blot analysis

(1) total protein of cell extracts

After the PBS buffer solution that cell is pre-chilled through ice is simply washed, the RIPA of PMSF containing 1mM (protease inhibitors) is added Cell lysis buffer solution (Invitrogen), sets lytic cell 30min on ice, with cell scraper collect cell pyrolysis liquid, 4 DEG C 12,000rpm centrifugation 15min take supernatant, -80 DEG C of preservations.

(2) BCA method protein quantification

Reagent A and reagent B are mixed in the ratio of 1:50, working solution are made and waits for by BCA protein quantification kit (Pierce) With.Dilution standard albumen makes its concentration be followed successively by 0 μ g/ μ l, 25 μ g/ μ l, 50 μ g/ μ l, 100 μ g/ μ l, 250 μ g/ μ l, 500 μ g/ μ l, 750 μ g/ μ l, 1000 μ g/ μ l, 2000 μ g/ μ l.5 μ l standard proteins or 5 μ l samples are added in ELISA Plate, add 100 μ L BCA working solution, 37 DEG C of water-bath 30min, the absorbance value of 570nm wavelength is read with microplate reader after mixing.With absorbance Value is ordinate, and standard protein concentration is abscissa, draws standard curve.According to standard curve, the concentration of sample is calculated.

(3) SDS-PAGE electrophoresis

Preparing 12%SDS-PAGE, (5ml system includes 30% acrylamide 2ml, ddH2O 1.6ml, 1.5M 50 μ l of pH8.8Tris-HCl 1.3ml, 10%SDS, 10% ammonium persulfate, 50 2 μ l of μ l, TEMED), it is injected after mixing rapidly dry It is quiet at room temperature in net gap preset glass plate (Bio-Rad), and in top layer addition appropriate amount of deionized water to promote gel polymerisation Set 30min, after glue to be separated agglomerates completely, abandon upper layer deionized water and with filter paper exhaust residual liquid.Prepare concentration glue (2ml Include 30% 20 μ l of acrylamide 0.33ml, 1.0M pH6.8Tris-HCl0.25ml, 10%SDS, 10% persulfuric acid in system 20 2 μ l of μ l, TEMED of ammonium), separation gel upper layer is added to after mixing immediately, clean 10 stripping forks is inserted into, stands at room temperature 30min takes out comb after gelling to be concentrated is admittedly complete, uses ddH₂O washs loading slot for several times, and gel is placed in electrophoresis tank (Bio- Rad), electrophoretic buffer (pH8.0Tris containing 25mM, 0.25M Glycine, 0.1%SDS) is added.Protein sample presses 5:1 ratio 6 × sample-loading buffer (pH6.8Tris-HCl containing 300mM, 12%SDS, 600mM DTT, 60% glycerol, 0.6% bromine is added in example Phenol is blue) it mixes, boiling water bath 10min, the cooling 5min of ice bath, binding protein quantitative result, each swimming lane is added equal protein sample, uses Bio-Rad electrophoresis apparatus carries out electrophoresis, after first entering separation gel to bromophenol blue forward position within electrophoresis about 20 minutes with the progress of 80V voltage, Voltage is improved to 120V, continues to reach separation gel bottom to bromophenol blue band in electrophoresis about 1 hour, and electrophoresis terminates.

(4) albumen transferring film

After SDS-PAGE electrophoresis, excision concentration glue and no sample region buffer nitrocellulose filter with electrophoretic transfer The of short duration immersion of liquid.Bio-Rad 3mm filter paper, cellulose nitrate are successively put by anode to cathode on electrotransfer device (Bio-Rad) Plain filter membrane, gel, Bio-Rad 3mm filter paper.Electrotransfer 1.5h is carried out with 100V voltage.After transferring film, pass through pre-dyed Marker and transfer effect is judged with 0.1% Ponceaux dyeing (Ponceau Stain), and use ddH₂O decoloration 5min.

(5) antibody label and ECL detection

By nitrocellulose filter in confining liquid (TBST (0.1%Tween-20in TBS) containing 5% skimmed milk power) Room temperature is closed 1 hour.4 DEG C of overnight incubations in primary antibody hybridization solution.It is rinsed 3 times, every time 2 minutes with TBST room temperature.It is added to seal The corresponding secondary antibody hybridization solution for having HRP to be coupled for closing liquid preparation, is incubated at room temperature 0.5 hour.With PBST room temperature rinse filter membrane 3 times, often Secondary 2min.

The substrate of SuperSignal West Pico kit (Pierce) moderate proportions and reinforcing agent are mixed, uniformly It is added drop-wise on filter membrane, is incubated at room temperature 1 minute, expose X-ray, after development, fixing, scan X-ray and save to analyze.

8.NF- κ B regulation analysis

Anti-virus carrier containing two IKK phosphorylation mutational sites S32A and S34A on coding I κ B α protein sequence PBabe-Puro-I κ B α-Mut (super repressor), be used to transfect slow virus package cell line PHOENIX.Slow virus It is used subsequently to infect PSC27 stromal cell lines, and 1 μ g/ml puromycin (puromycin) is then used for screening positive clone. Alternatively method, 5 μM of micromolecular inhibitor Bay 11-7082 (being purchased from Selleck) are used for NF- kB activity control System.Stroma cell is subsequently exposed to several various forms of cell toxicants, records resulting phenotype in time, analyzes dependency basis Because of expression.By the processed cell of this mode, the conditionity culture solution generated is collected, for being directed to The various detections of epithelial cell.

9.SFRP2 promoter Analysis and chromosome immunosedimentation (ChIP) detection

It is used for people Zscan4 gene (Gene ID 201516, Genbank accession NM_152677.2) soft Part CONSITE is analyzed, to find potential core NF- κ B binding site.6 pairs of PCR primers are designed to be segmented and expand the base Because of 5 ' upstream proximal promoter subsequences:

[primer set#1 (- 1610~0):

Forward 5 '-cagtggctgtgtgtctttgccttc-3 ' (SEQ ID NO:1),

reverse 5'-gtggcagagggcaaggctaatgctg-3'(SEQ ID NO:2)；

Primer set#2 (- 1997~0)

Forward 5 '-ctacatactgaggcggattggc-3 ' (SEQ ID NO:3),

reverse 5'-gtggcagagggcaaggctaatgctg-3'(SEQ ID NO:4)；

Primer set#3 (- 2496~0)

Forward 5 '-ggggaccccagtctttgcgttca-3 ' (SEQ ID NO:5),

reverse 5'-gtggcagagggcaaggctaatgctg-3'(SEQ ID NO:6)；

Primer set#4 (- 2996~0)

Forward 5 '-ctttgcgaggcccaagtaggtttac-3 ' (SEQ ID NO:7),

reverse 5'-gtggcagagggcaaggctaatgctg-3'(SEQ ID NO:8)；

Primer set#5 (- 3730~0)

Forward 5 '-tgcactctgctaatgcaagctacag-3 ' (SEQ ID NO:9),

reverse 5'-gtggcagagggcaaggctaatgctg-3'(SEQ ID NO:10)；

Primer set#6 (- 4000~0)

Forward 5 '-agcgccccctcctcatggagaccg-3 ' (SEQ ID NO:11),

reverse 5’-gtggcagagggcaaggctaatgctg-3’(SEQ ID NO:12)。

The above PCR product corresponding sequence is verified in TNF-α and DNA damage drug-treated by molecular cloning and experiment in vitro Later, the real NF-kB binding site of Zscan4 promoter region is determined.Then, 3 pairs of primers are designed for specific amplification ChIP The gDNA section to settle down, primer are respectively as follows:

[primer set#a(-4000to-3820)

Forward 5 '-agcgccccctcctcatggagaccg-3 ' (SEQ ID NO:13),

reverse 5'-catgaggactgccaggctctcaga-3'(SEQ ID NO:14)；

[primer set#b(-3277to-3097)

Forward 5 '-cactcctgtaatcccaacac-3 ' (SEQ ID NO:15),

reverse 5'-gttcaagcaattctcctgcc-3'(SEQ ID NO:16)；

[primer set#c(-1844to-1645)

Forward 5 '-agcactacctgccttcccaa-3 ' (SEQ ID NO:17),

reverse 5’-atgagcaggaggatttatcc-3’(SEQ ID NO:18)。

Meanwhile the other 4 pairs of primers of design are used to expand the promoter sequence (being positive control) of following gene respectively.

WNT16B:

Forward 5 '-caggaaaggtcatgacacacc-3 ' (SEQ ID NO:19),

Reverse 5 '-agagcagcctggggatct-3 ' (SEQ ID NO:20),

SFRP2:

Forward 5 '-attcattacccggctcctct-3 ' (SEQ ID NO:21),

Reverse 5 '-cctgcctagagatccacgag-3 ' (SEQ ID NO:22),

IL-6:

Forward 5 '-aaatgcccaacagaggtca-3 ' (SEQ ID NO:23),

Reverse 5 '-cacggctctaggctctgaat-3 ' (SEQ ID NO:24), and

IL8:

Forward 5 '-aaaactataggagctacatt-3 ' (SEQ ID NO:25),

reverse 5’-tcgcttctgggcaagtaca-3’(SEQ ID NO:26)。

PSC27 cell (such as p8) for early stage algebra and the PSC27 cell by bleomycin (50ug/ml) processing into Row ChIP analysis.Fixed chromosome in vitro is carried out using mouse monoclonal anti-p65antibody (F-6, Santa Cruz) Settlement treatment extracts DNA to expand.By amplified production be cloned into pCR2.1-TOPO (Invitrogen) and In pGL4.22vector (Promega).The report expression vector system for being loaded with multiple NF- κ B binding site mutation passes through site- The design of directed mutagenesis (Strategene) method and generation.In addition, covering multiple NF- κ B binding sites and warp Cross report carrier NAT11- of the IL-2 minimal promoter of optimization as NF- κ B activation transgenic system (NAT system) Luc2CP-IRES-nEGFP (is obtained from Hokkaido university, Japan), is used as positive control in an experiment.Each report carrier by PRL-TK vector (Addgene) cotransfection is to carry out signal normalization processing.

The purifying of 10.WNT16B monoclonal antibody, it is multiple fold and effect determine clinical lung cancer and patient with breast cancer's tissue samples obtain and Analysis

Chemotherapeutics scheme is according to recurrence and refractory non-small cell lung patient (clinical trial registration number NCT02889666) and the pathological characteristics of permeability ductal breast cancer patient (clinical trial registration NCT02897700) refer to Fixed.Clinical stages is primary lung cancer more than I subtype A (IA) (T1a, N0, M0) but without obvious far-end transfer lesion Patient be recruited into clinical queue.Meanwhile the age 75 years old or more is NSCLC or age greater than 18 years old through clinical definite Organizationally it is proved that the patient side of permeability BCa is recruited.All patients are provided informed consent form and confirmation of signing. Related tumor size, organization type, tumour infiltration, the data of lymphatic metastasis and pathology TNM disease stage are from recording pathological mechanism system System obtains.Tumour is processed as FFPE sample and is processed into Histological section for assessment.OCT frozen section selectively divides through LCM From for gene expression analysis.Particularly, according to the method (Sun et al., 2012) being previously reported, the body of gland of chemotherapy Related stroma cell is separated through LCM.Immunocompetence scores (IRS) according to the histochemical staining colour generation depth of each tissue samples difference It ranges 0-1 (feminine gender), 1-2 (if), 2-3 (in), four class of 3-4 (strong) (Fedchenko and Reifenrath, 2014). The diagnosis of NSCLC and BCa sample is judged and is scored by pathology doctor independent of each other.Randomized controlled trial (RCT) side Case and all experimental arrangements are ratified and are authorized through Medical College, Shanghai Communication Univ. IRB, and are gradually opened according to authoritative guideline Exhibition.

11. mice transplanted tumor model and pre- clinical chemotherapy program

The experiment of all experiment mices follow strictly Shanghai Inst. of Life Science, CAS experimental animal look after and It is carried out using the related regulations of the committee (IACUC).Age 6 weeks or so immune-deficient mice ICR SCID mice (weight About 25g) it is tested for relevant animal of the present invention.Stroma cell PSC27 and epithelial cell are mixed with the ratio of 1:4, and each shifting Implant includes 1.25 × 10⁶Cell is used for reconstructed tissue.Transplantable tumor is implanted into Mice Body by subcutaneous transplantation mode, transplants hand 8 weekend animals are performed euthanasia after art terminates.Gross tumor volume is calculated according to following formula: V=(π/6) x ((l+w)/2)³ (V, volume；L, length；W, width).

In the test of pre- clinical chemotherapy, standard test recipe, implementationization after 2 weeks are supplied to by the mouse of subcutaneous transplantation Treat Drug-Mitoxantrone (0.2mg/kg dosage) and/or SASP inhibitor (500 μ l, 10mg/kg dosage) intraperitoneal administration.Time point For first day of the 3rd, 5,7 week, the entire course for the treatment of carried out 3 circulation administrations altogether, and each circulation is 2 weeks.After the course for the treatment of, Mouse Kidney It is dirty to be collected for measurement of tumor and histologic analysis.Every mouse cumulative bad receives mitoxantrone 0.6mg/kg weight altogether, SASP inhibitor 30mg/kg weight.To cause the systemic body-wide SASP factor to express under chemotherapy induction, mitoxantrone is according to above Step and sequence are administered mouse by venoclysis mode, but dosage drops to 0.1mg/kg weight/each (entire course for the treatment of The accumulative mitoxantrone dosage that receives is 0.3mg/kg weight) to mitigate drug related toxicity.Chemotherapy test proceeds to the 8th weekend knot Beam, mouse are dissected immediately after putting to death, and transplantable tumor is collected and used pathology network analysis.

12. biometrical method

The experiment in vitro of related to cell proliferation rate in the present invention, migration, invasion and viability etc. and mouse move The in vivo studies for planting tumor and chemotherapy processing is repeated 3 times above, and data are presented in the form of mean value ± standard error.Statistical analysis It establishes on the basis of initial data, passes through one-way analysis of variance or a two-tailed Student ' s t-test is calculated, and the result of P < 0.05 is taken as with significant difference.

II. embodiment

Embodiment 1, transcriptome analysis are the result shows that Zscan4 is shown in the source of people stroma cell handled by gene cytotoxic drug Write up-regulated expression

For the express spectra variation characteristic of source of people stroma cell within the scope of research full-length genome, inventors used normal originals For anti-cancer therapies common in source of people Prostate Stromal Cells system PSC27 and two kinds of clinics, that is, non-DNA damage processing and DNA Damaging processing.The former (non-DNA-damaging treatment, NDT) includes docetaxel (DTX, taxotere), Paclitaxel (PTX, taxol) and vincristine (vincristine), the latter (DNA-damaging treatment, DT) It then include bleomycin (BLEO, bleomycin) mitoxantrone (mitoxantrone, MIT) and radiation (RAD, ionization Radiation).The dosage standard of every kind of processing mode be can cause severe cellular destroy and cause Apoptosis or its The death of its form, so that expression under detection cellular damage state and exploring with closely related important point of SASP Son is possibly realized.It is interesting that the above processing mode can cause significant cell ageing, including some characteristic features are such as SA- β-Gal stained positive and apparent cellular morphology variation (Fig. 1, Fig. 2).But the result that these processing modes ultimately cause It has differences in some respects, such as the appearance of blister corpusculum in cytoplasm, DNA aggregate velocity and DNA damage degree etc. (Fig. 1, Fig. 3, Fig. 4).

Microarray results show compared to NDT group drug, DT medicine group cause extracellular matrix (ECM) GAP-associated protein GAP and The significant up-regulation of soluble protein.(Asia of usually display albumen related with a certain function is thin in GO function-CC classification for this tendency Born of the same parents positioning) in clearly (Fig. 5, Fig. 6), although GO function other two classification-BP and MF analysis result in and have no Marked difference (Fig. 7, Fig. 8).It is worth noting that, the gene that consistency raises in this two groups of drugs accounts for the up-regulation of DT medicine group The 12.5% of list, and 47.9% (expression variation multiple threshold value is at 3 times or more) (figure is then accounted in NDT medicine group respective list 9).(Figure 10, respectively 24.3% and 67.3%), whole result confirms after merging with two groups of drug induced down-regulated genes There is essential differences for the source of people stroma cell full-length genome range expression pattern that two groups of anticancer drugs cause.

In the list of the above chip results, other than the extracellular protein of various secretories, the inventors discovered that with It is that telomere extends one closely related with Genome stability in same embryonic stem cell toward the factor Z scan4 not being concerned A transcription factor.In the data of the present inventor, Zscan4 is located at the apical position of the up-regulation list of the factor intracellular, and can be with Clearly distinguish the generated Different Results (Figure 11) of two groups of drugs of DT and NDT.Rank cluster analysis result shows, compared to NDT group drug has similitude outstanding and close relevance (figure in terms of gene expression caused by each drug of DT group 12, Figure 13).More importantly once DNA damage occurs, Zscan4 can be detected in entire cytoplasm and nucleus, But the signal in core area then becomes apparent (Figure 14, Figure 15), and weight may be had in DNA damage repair action by imply Zscan4 It acts on.

In addition to this, the inventors have further noted that p38, one is used as stress-inducible mitogen in normal fibroblast Former activated protein kinase (MAPK) member regulates and controls the protein factor of SASP, also appears among up-regulation list, and p38 obviously can be with area It is divided to the different function (Figure 16) of two groups of cell toxicity medicaments.Further experimental result confirms, after the processing of gene cytotoxic drug Occur Zscan4 up-regulated expression in stroma cell, and there is no this effect (Figure 17, cisplatin/ for non-genomic cytotoxic drug Carboplatin/camptothecin/doxorubicin is gene cytotoxic drug；Erlotinib/nivolumab is non- Gene cytotoxic drug).Zscan4 and this while expression pattern of typical case's SASP effector in human stroma cell, imply Under DNA damage event and secretion phenotype development, there is potential molecule and cell biological mechanisms, the latter can be by this Functionally tight association is together for a little important molecules.

Embodiment 2, Zscan4 are expressed in the cancer patient matrix organization in stage after chemotherapy and with poor clinical prognosis phases It closes

On the basis of finding that DT drug can induce Zscan4 to express in stroma cell, the present inventor is next proceeded to Whether repeated under the conditions of studying similar phenomenon in vivo.Because SASP be one independent of organ and organization type cell it is non-from Main property process, the present inventor are assessed first against non-small cell lung cancer (NSCLC) clinical samples.Without staging tomography Patient's body tumour and matrix in the protein expression of Zscan4 can substantially ignore, because in tissue no matter epithelium or matrix it is thin The IHC dyeing of born of the same parents is negative (Figure 18).However, the inventors discovered that, it experienced the chemotherapy for relating generally to gene cytotoxic drug Occurs quite surprising Zscan4 expression in NSCLC patient tissue later, and a large amount of concentrate appears in stroma cell, with week Side dye-free or the epithelial cell of limited dyeing form sharp contrast.

To further describe expression of the Zscan4 in these patient's primary carcinoma, inventors used a set of preparatory Designed pathology assessment system, the latter can carry out qualitative interpretation according to the staining power of specific antigen in the tissue.It compares In the tumour of the patient of untreated, significant raised Zscan4 expression is shown as by the sample of the patient in chemotherapy stage (Figure 19).After separating epithelial cell and stroma cell by LCM, the present inventor observes Zscan4 Phase patient after treatment Transcript degree in stroma cell is skyrocketing, and the epithelial cell closed on changes (Figure 20) without this.More importantly The Kaplan-Meier survival analysis of the NSCLC patient being layered for Zscan4 expression the result shows that, Zscan4 high Expression presented with the progression free survival phase of patient's queue in period after treating it is significant negatively correlated, and low-level Zscan4 then support compared with Good prognosis (Figure 21).

Once there is document report p38 to play a significant role the formation of SASP in the past, it can regulate and control in senile cell NF- κ B transcriptional activity simultaneously stablizes SASP effector mRNA in post-transcriptional phase.In addition, mTOR can be turned over by improving IL-1 α The synthesis of MAPKAPK2 kinases is translated and controlled to monitor the development of SASP.However, related p38, mTOR and Zscan4 occur in SASP Biology association in development process, does not know so far.Zscan4 is being had detected after intracorporal expression, the present invention Activation levels of the people using similar analysis system for p38 and mTOR have carried out one-to-one assessment.As a result, it has been found that There is synchronize phenomenon (Figure 22) between Zscan4 expression and p38, mTOR activation.In turn, statistical data is shown in p38, MTOR and Zscan4 have significant correlation (respectively r=0.83, r=0.87, and P value under every kind of Correlation Criteria < 0.001) (Figure 23, Figure 24).

To make experimental result have generality, the present inventor is then directed to another solid tumor and has detected identical one Cover index.Zscan4 is in the expression in patient's matrix in stage after chemotherapy and the significant phase between Zscan4 and p38/mTOR Guan Xing is verified by the pathological analysis of patient with breast cancer's sample, this crowd of patient is divided extremely according to respective clinography Untreated group and through treatment group (r=0.81for correlation of Zscan4 and p38, r=0.67for Zscan4 and mTOR；Two kinds of associations are P < 0.01) (Figure 25-Figure 31).Therefore, the present inventor obtains from clinical queue Volume of data confirms that Zscan4 tends to occur high expression in cancer patient's matrix by chemotherapy processing comprehensively, and There are significant correlation between Zscan4 and SASP key regulator, the latter includes but is not limited to general in damage stroma cell The molecules such as p38 and mTOR all over activation.

Embodiment 3, Zscan4 do not change cell ageing but can significantly regulate and control SASP occurrence and development

In the Zscan4 expression found in cancer patient's sample with the hight coordinate between SASP regulating element, promote this Inventor and then the functional meaning for inquiring into Zscan4 in damage stroma cell.Firstly, the present inventor constructs high expression Zscan4 Knockout system (respectively PSC27-Zscan4 and the PSC27- for surely transfering from one department to another to lack with Zscan4 silencing as foreign gene Zscan4-KD).The result shows that no matter in cell ectopic expression Zscan4 or thoroughly removing Zscan4 from substrate level, Fail change gene poison induction cell pattern, including significantly increase DNA damage focus (DNA damage foci, i.e., DDF), the DNA aggregate velocity slowed down, is presented the cell ageing of characteristic feature, and decline to a great extent clonality (Figure 32, Figure 33, Figure 34, Figure 35, Figure 36, Figure 37, Figure 38, Figure 39).But as heterogenous expression in stromal cell lines PSC27 Zscan4 can reduce most of SASP factors substrate horizontal expression (including but not limited to IL-6, IL-8, GM-CSF, SPINK1, SFRP2, AREG, EREG, ANGPTL4, MMP3 and WNT16B), although not significant.In contrast, these excretions because The expression of son is but substantially raised in the stroma cell that Zscan4 is overexpressed and is handled by bleomycin, respective variation times Number is significantly higher than the up-regulation multiple (Figure 40) of control group (PSC27-Vector) by appearance after same dose of drug induced injury.

Zscan4 effect played in SASP phenotype development process is recognized for deep layer, and the present inventor also has evaluated Zscan4 Missing is influenced for caused by SASP factor expression.It is interesting that SASP factor substrate is presented in the stroma cell that Zscan4 is knocked out Slightly raising in level, but it is not significant (Figure 41).It is compared with control group (PSC27-SCR-shRNA), knockout is stroma cell The expression of SASP effector disappears substantially under the conditions of DNA damage.Therefore, it is overexpressed Zscan4 and seems that slight decrease matrix is thin The background expression of SASP, the expression amplitude of each factor of SASP can be but significantly expanded when Zscan4 is overexpressed in born of the same parents.

Next, the present inventor attempts to determine reaction of the effect of Zscan4 defect for DNA Damage, this is to pass through Detect with DNA damage reaction the closely related some factors of (DNA damage response, DDR) signal transduction expression and/ Or Activation is unfolded.The inventors discovered that Zscan4 expression almost occurs simultaneously with the activation of DDR molecule machine, the latter H2AX (γ-H2AX) including phosphorylation, ATM, Kap, Chk2 and p53 (Figure 42).When DNA damage occurs, Zscan4 is presented Expression rapidly, although in a manner of time dependence, the variation of protein level can go out for 6 hours after drug-treated It is existing.This is than most of DDR GAP-associated protein GAP such as p-ATM, the time of γ-H2AX, p-53BP1 and their substrate variations in cytoplasm Node usually wants late.However, Zscan4 missing under conditions of, the activation of most of main DDR albumen be it is of short duration, generally not It can exceed that 24 hours (Figure 42).Although there is so of short duration reaction time, some events in the downstream DDR includes Chk2, p53 and The up-regulation of p21, the present inventor will test the time extend to cellular damage after the 7th day when, still maintain through SASP The whole process of development.Above data shows that the cell ageing reaction of gene poison induction is substantially complete, including checkpoint resistance Stagnant and downstream reaction successively occurs in the case where substantially not adversely effected according to cascade order.

As the details having described above, Zscan4 under DNA Damage background by being synthesized in cytoplasm after, greatly Amount indexing is in nucleus, i.e. the position (Figure 14, Figure 15) that initially issues of DNA damage signal.This phenomenon shows Zscan4 pairs In being persistently necessary for maintenance typical case's DDR reaction signal chain, and have by functionally participating in the thin of damage stroma cell In karyon the activity of early stage DDR to coordinate by ASAP (acute stress associated phenotype, ASAP, i.e., it is acute Stress Relevant phenotype) transit to chronic event and the potentiality of SASP.

Embodiment 4, the acute reaction caused as DNA damage, Zscan4 are expressed by ATM-TRAF6-TAK1 signal shaft Regulation

On the basis of the discovery of Zscan4 functional sense, the present inventor has further probed into Zscan4 by gene poison Expression mechanism in the stroma cell of damage.In the past once studies have reported that DNA damage can excite the prompt activation of ATM, and after And it is promoted to carry out indexing from nucleus to cytoplasm, finally cause downstream sequence of events, swashing including NF- κ B compound It is living.Therefore, after the present inventor uses ChIP to handle by phosphorylation ATM (p-ATM) antibody analysis bleomycin first Stroma cell lysate finds there is interaction between the ATM and TRAF6 of activation, but can be by ATM inhibitor KU55933 institute It abolishes (Figure 43).Because ATM is bound on TRAF6 poly-ubiquitination (more ubiquitin that TRAF6 can be activated to mediate Change) and cause it is some including TAK1 activate including downstream reaction, the present inventor analyzes in stroma cell immediately whether there is Similar phenomenon.For this purpose, cell lysate is had detected with anti-TRAF6 using IP after bleomycin handles stroma cell, Notice the auto-ubiquitination (single ubiquitination) of the TRAF6 increased rapidly, it was confirmed that it is in damaging cells Ubiquitin ligase (ubiquitin ligase) activity (Figure 44).The present inventor then uses phosphorylated TAK1 (p- TAK1) antibody has carried out further IP analysis, it is found that between TAK1 and TRAF6 that this is in DNA there is physical interaction Occur immediately after damage, but (Figure 45) can be abolished by TAK1 inhibitor 5Z-7-oxozeaenol (being hereafter abbreviated as 5Z-7). In contrast, this interaction is then not present between TAK1 and ATM, although the two molecules equal quilt after DNA damage Soon activate.Meanwhile this data also implies the particularity (Figure 45) acted between TAK1-TRAF6.As supportive card According to TRAF6 antibody-mediated IP experiment shows that TRAF6 can interact with the ATM and TAK1 of the state of activation, implies TRAF6 can be used as middle element and transmit ATM signal and give TAK1 (Figure 46).Meanwhile the knockout of TRAF6 can be in damaged matrix TAK1 activation is abolished in cell, rather than ATM is activated, and reconfirms that TRAF6 is mediating the special work in ASAP acute reaction signal With (Figure 44).

Next, whether research is related with the activation of NF- κ B compound by the TAK1 of upstream DDR signal institute indirect activation, And the latter, which was once reported, to be situated between by the mono-ubiquitination of I κ B kinase subunit γ (IKK γ) in cytoplasm It leads.After being separated nucleus with cytoplasm protein, the inventors discovered that TAK1 phosphorylation is same NF- κ B compound The nuclear translocation of main two subunit p50and p65 is related (Figure 47).However in the presence of 5Z-7, the phosphorylation of TAK1 It is blocked with the core that enters of p50/p65, it was demonstrated that NF- κ B is the signal path downstream that TAK1 is mediated in the cytoplasm of stroma cell One event (Figure 47).

In order to determine NF- κ B compound, the SASP transcriptional machinery that wide spectrum is expressed within the scope of full-length genome, if drive The acute expression of Zscan4, the present inventor are analyzed using bioinformatics method in stroma cell after dynamic DNA damage, It was found that in the proximal promoter region Zscan4, there is 6 predictive NF- κ B binding sites (Figure 48).Pass through sequentiality molecule Clone and TNF-α combination gene cytotoxic drug based on promoter qualitative detection, disclose and surely put upstream in Zscan4 transcription initiation There is 4 real NF- κ B binding sites (Figure 49, Figure 50) in the range of 4000bp.These results are by subsequent with matrix Cell lysate confirms (Figure 51) by the IP experiment of target.The present inventor once set up ectopic expression I κ B α mutant's in the past Stromal cell lines (PSC27^IκBα), the latter can block NF- κ B compound nuclear translocation in DNA damage and its transcription that decays Activity.Compared to wild type control, the PSC27 of I κ B α mutant is expressed^IκBαCell occurs under bleomycin treatment conditions Significantly reduced Zscan4 expression (Figure 52).Therefore, the data consistency of the present inventor confirms really being damaged for Zscan4 Stroma cell in NF- κ B a real, direct binding site.

In addition, the present inventors have additionally discovered that, integration or NF- κ B little molecules in inhibiting of the either I κ B α mutant in genome The processing of agent (Bay 11-7082), can cause the expression of the typical SASP factor to be substantially reduced, although these factors are as SASP Marker generally raises (Figure 53, Figure 54) after bleomycin processing.These are as a result, in conjunction with Zscan4 after DNA damage Stroma cell in regulation SASP expression a key protein the fact that (Figure 41), once collectively show that is anti-in acute DDR It should be raised in the process by ATM-TRAF6-TAK1 signal shaft, Zscan4 can then pass through the formation for promoting a positive feedforward loop circuit And the development of signal path is covered, further expansion SASP phenotype amplitude strengthens the slow of each factor of SASP in subsequent period Property expression.

The various acquired shapes of cancer cell and lotus under conditions in vitro can be significantly reduced in embodiment 5, the missing of Zscan4 Drug resistance of the tumor mouse interior tumor under chemotherapy regimes

For the result of study for further extending Zscan4, the present inventor uses Zscan4 specific knockdown stromal cell lines, A series of phenotypes of cancer cell are had detected under condition of culture in vitro.The present inventor has chosen representative prostatic epithelium Benign cell system BPH1, epithelial cancer cell line include M12, PC3, DU145 and LNCaP.The born of the same parents collected from stromal cell lines PSC27 External solution (conditioned media, CM) significantly improves the in-vitro multiplication rate of the above various epithelial cell lines in culture bottle (Figure 55).

In order to determine cancer cell while obtaining higher multiplication potentiality from stroma cell extracellular fluid, if there are also other The malignant progression of aspect, the present inventor analyze the mobility and invasion rate of prostate gland cancer cell.It the use of Transwell is detection One group of experiment of tool, the results showed that lacked compared to control group (PSC27-CTRL and PSC27-SCR-shRNA), Zscan4 CM caused by stroma cell subbreed fails to assign the higher mobility of cancer cell and invasion rate (Figure 56, Figure 57).Rich next mould Under CM condition of culture derived from PSC27-CTRL and PSC27-SCR-shRNA after element damage, cancer cell generally occurs higher Migration and invasive ability, but the knockout of Zscan4 reverses these acquired malignant characteristics, has returned to substantially thorn Original level before swashing.

Since Zscan4 can be used as a target spot of stroma cell SASP phenotype, presence or absence can cause cancer cell Significant Traits change under conditions in vitro, the present inventor further verify this effect under the conditions of then seeking in vivo.Pre- First designed a set of lower experiment (Figure 58) carried out of pre- clinical treatment guidance, the results showed that, 8 weeks by a definite date, MIT Jie At the end of the chemotherapy treatment led, for mouse tumor terminal volume on the basis of reducing by 36% by MIT, Zscan4 is in gene level Expression silencing can cause tumour terminal volume further to reduce, and amplitude is up to about 78-79% (Figure 59, Figure 60).Therefore, in conjunction with Physiology reality under clinical condition, traditional gene poison therapy combine microenvironment (TME) targeted therapy, can significantly inhibit tumour The drug resistance obtained from microenvironment, therapeutic effect are obviously improved.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

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Claims (17)

1. a kind of purposes of the lower adjustment of Zscan4 gene or albumen is used to prepare the pharmaceutical composition for inhibiting tumour.

2. purposes as described in claim 1, which is characterized in that the tumour is selected from: passing through gene cytotoxic drug or DNA damage Property drug-treated after or by DNA damage treatment tumour, or expression Zscan4 tumour.

3. purposes as claimed in claim 2, which is characterized in that the gene cytotoxic drug or DNA damage drug include: it is rich come Mycin, mitoxantrone, cis-platinum, carboplatin, camptothecine, adriamycin；Or

The DNA damage treatment includes: ionizing radiation treatment.

4. purposes as claimed in claim 2 or claim 3, which is characterized in that the pharmaceutical composition and gene cytotoxic drug or DNA are damaged Wound property Drug combination is in inhibition tumour；Or the pharmaceutical composition and DNA damage treatment are united and applied in inhibition and swell Tumor.

5. purposes as described in claim 1, which is characterized in that the lower adjustment is selected from:

The disturbing molecule of specificity interference Zscan4 gene expression；Or

The gene editing reagent of specific knockdown Zscan4 gene；Or

The protein bound antibody or ligand of specificity and Zscan4 gene coding.

6. purposes as claimed in claim 5, which is characterized in that the lower adjustment is shRNA；Preferably, it is with SEQ Nucleotide sequence shown in ID NO:1 and SEQ ID NO:2.

7. purposes as described in claim 1, which is characterized in that the tumour includes: prostate cancer, lung cancer, breast cancer.

8. the purposes of a kind of Zscan4 gene or albumen is used to prepare the composition of regulation aging correlation secretion phenotype；Or it is used for Preparation inhibits the pharmaceutical composition of diseases associated with senescence.

9. a kind of method that screening inhibits the potential substance of tumour, which comprises

(1) system of expression Zscan4 gene is handled with candidate substances；With

(2) expression or activity of Zscan4 gene in the system are detected；

Wherein, if the candidate substances can reduce the expression or activity of Zscan4 gene, show that the candidate substances are to inhibit swollen The potential substance of tumor.

10. method as claimed in claim 9, which is characterized in that step (1) includes: that candidate substances are added in test group Into the system of expression Zscan4；And/or

Step (2) includes: the expression or activity of Zscan4 in the system for detect test group, and compared with the control group, wherein described Control group be do not add the candidate substances expression Zscan4 system；

If the expression of Zscan4 or activity are statistically lower than control group in test group, indicate that the candidate is to inhibit swollen The potential substance of tumor.

11. a kind of for inhibiting the pharmaceutical composition of tumour, which is characterized in that include: in the pharmaceutical composition

The lower adjustment of Zscan4 gene or albumen；With

Gene cytotoxic drug, DNA damage drug or DNA damage ionizing radiation treatment drug.

12. a kind of for inhibiting the medicine box of tumour, which is characterized in that include: in the medicine box

Container, and it is placed in the lower adjustment of the Zscan4 gene or albumen of the container；With

Container, and it is placed in the gene cytotoxic drug of the container, DNA damage drug or DNA damage ionizing radiation treatment drug.

13. the medicine box as described in claim 11 or 12, which is characterized in that the lower adjustment includes: specific interference The disturbing molecule of Zscan4 gene expression；Or the gene editing reagent of specific knockdown Zscan4 gene；Or specificity with The protein bound antibody or ligand of Zscan4 gene coding.

14. medicine box as claimed in claim 12, which is characterized in that the gene cytotoxic drug or DNA damage drug include: rich Bleomycin, mitoxantrone, cis-platinum, carboplatin, camptothecine, adriamycin.

15. it is pre- to be used to prepare progress tumour for a kind of purposes of the reagent of the albumen of specific recognition Zscan4 gene or its coding The reagent or kit assessed afterwards.

16. purposes as claimed in claim 15, which is characterized in that the specific recognition Zscan4 gene or its coding The reagent of albumen is selected from:

The primer of specific amplification Zscan4 gene；

The probe of specific recognition Zscan4 gene；Or

Specifically bind the antibody or ligand of the albumen of Zscan4 gene coding.

17. a kind of kit for tumor prognosis evaluation, which is characterized in that contain in the kit: specific recognition The reagent of the albumen of Zscan4 gene or its coding.