CN110478484A - Inhibit the substance application in preparation of anti-tumor drugs of JDP2 expression - Google Patents
Inhibit the substance application in preparation of anti-tumor drugs of JDP2 expression Download PDFInfo
- Publication number
- CN110478484A CN110478484A CN201910730699.XA CN201910730699A CN110478484A CN 110478484 A CN110478484 A CN 110478484A CN 201910730699 A CN201910730699 A CN 201910730699A CN 110478484 A CN110478484 A CN 110478484A
- Authority
- CN
- China
- Prior art keywords
- jdp2
- expression
- tumor
- drug
- reagent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Reproductive Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gynecology & Obstetrics (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Pregnancy & Childbirth (AREA)
- Endocrinology (AREA)
- Inorganic Chemistry (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
Abstract
A kind of application of the reagent of the application for inhibiting the reagent of JDP2 expression in the sensitive drug that preparation improves cells against neoplastic drug, anti-tumor drug, and detection JDP2 expression quantity in reagent preparation box is disclosed in the present invention.To prevent and treat oophoroma, new thinking is provided.
Description
Technical field
The present invention relates to biological fields, and in particular to inhibit the application of the reagent of JDP2 expression in medicine preparation, it is a kind of
Anti-tumor drug, and detect application of the reagent of JDP2 expression quantity in reagent preparation box.
Background technique
Oophoroma is the highest gynecologic malignant tumor of lethality in world wide, seriously threatens the life and health of women.According to
Global cancer latest data in 2018 shows that oophoroma death toll is 184,799, and compared with toward annual data, the death rate is in fast
Fast ascendant trend.Since the early symptom of oophoroma is unobvious and lacks effective diagnosis screening technology, 70% oophoroma is suffered from
Person has been advanced stage (FIGO by stages III and IV phase) in initial diagnosis.Currently, the primary treatment scheme of advanced ovarian cancer is tumour
Cytoreductive art combines the postoperative chemotherapy regimen based on platinum class.In recent years, although clinical operation technical ability and tumour cell subtract
Art of going out satisfaction is continuously improved, but Patients with Advanced Ovarian Carcinoma survival region is not fundamentally improved still, and 5 years survival rates are only
Have 30%.It is counted according to latest data, Ovarian cancer is the main reason for ovarian cancer patients is dead, and about 75% oophoroma is suffered from
Person dies of tumor recurrence, wherein 60% patient is because of tumor recurrence caused by tumor drug resistance.Therefore, ovarian cancer drug-resistant is illustrated
Molecular mechanism new scientific basis is provided for tumor recurrence；Have to overcoming drug resistance of tumor and reducing ovarian cancer patients death
Significant clinical meaning.
The anticancer mechanism of most of clinical chemotherapy drugs is to lead to death of neoplastic cells by induced DNA damage.It grinds
Study carefully and show adriamycin, cis-platinum, the DNA damages drug such as Hycamtin passes through induction ROS (Reactive Oxygen Species)
It steeply rises, leads to irreversible oxidative damage and cell ageing, even result in cell death.However, more and more researchs
Show that tumour cell can resist the DNA damage effect that chemotherapeutics induces it.The cancer survived under induced by chemotherapeutic agents is thin
Born of the same parents by maintaining overactive anti-oxidant reduced level, not only activate ROS to remove system with cope with oxidative stress but also
Inhibit Apoptosis, and then generates oxidative stress tolerance.Wherein, glutathione (GSH) is anti-oxidant as important non-enzyme
Agent resists induced by chemotherapeutic agents DNA damage in tumour cell and plays an important role.GSH can not only be used for antioxidant and directly remove
ROS also can be used as the electron donor of other oxidation-reduction systems, with glutathione reductase (GP) and glutathione S-transferase
(GST) composition glutathione system removes peroxide correlation product.Report shows that GSH is in high level in kinds of tumors,
And by inhibiting Apoptosis, cell being promoted to shift and be resistant to chemicotherapy, to promote the malignant progression of tumour, show
GSH reduction system plays an important role in malignancy of tumor conversion.Currently, tumour cell is tieed up by which kind of mechanism regulating GSH
It holds unclear in overactive level.Therefore, the specific molecular mechanism and deep spy of cancer cell regulation GSH synthesis are disclosed
It begs for GSH and resists the DNA damage effect that chemotherapeutics induces it, have to exploring with the oncotherapy that the GSH that contends with synthesizes target spot
There is important clinical directive significance.
Summary of the invention
The purpose of the present invention is to provide inhibit the application of the reagent of JDP2 expression in medicine preparation.
Another object of the present invention is to provide a kind of anti-tumor drugs.
A further object of the present invention is to provide application of the reagent of detection JDP2 expression quantity in reagent preparation box.
The technical solution used in the present invention is:
The application of reagent in medicine preparation, the reagent for inhibiting JDP2 to express, the drug for it is following extremely
It is one of few:
Improve the sensibility of cells against neoplastic drug；
Inhibit the expression of intracellular GSH synthesis related gene；
Inhibit intracellular GSH horizontal；
Promote intracellular ROS level；
The methylation modification for inhibiting PRMT5 to mediate.
Further, the anti-tumor drug is chemotherapeutics.
Further, the chemotherapeutics at least one of includes and is not limited to Hycamtin, cis-platinum.
Further, the synthesis related gene includes at least one of SLC7A11, GCLM, GSS.
Further, the reagent inhibits JDP2 expression by silencing JDP2.
Further, the cell is ovarian cancer cell.
Further, the ovarian cancer cell is epithelial ovarian cancer cell.
Further, the drug is for treating oophoroma.
Further, the oophoroma is ovarian epithelial carcinoma.
A kind of anti-tumor drug, it is characterised in that: contain the reagent and chemotherapeutics for inhibiting JDP2 to express.
Further, the chemotherapeutics at least one of includes and is not limited to Hycamtin, cis-platinum.
Application of the reagent of JDP2 expression quantity in reagent preparation box is detected, the kit is used for following at least one
Kind:
Detect drug resistance of tumor；
Tumor prognosis；
Predict tumor recurrence.
Further, the drug resistance refers to tumour cell to the drug resistance of chemotherapeutics.
Further, chemotherapeutics at least one of includes and is not limited to Hycamtin, cis-platinum.
Further, the tumour is oophoroma.
Further, the oophoroma is ovarian epithelial carcinoma.
The beneficial effects of the present invention are:
The present invention mainly passes through analysis public database and clinical sample data, finds JDP2 obvious high table in oophoroma
It reaches, and negatively correlated with the overall survival prognosis of tumor patient and recurrence prognosis.Pass through internal experiment in vitro, it was demonstrated that JDP2 promotees
Into the drug resistance of oophoroma DNA damage agent such as cis-platinum and Hycamtin.Further, by JDP2ChIPseq data analysis found that
DNA damage pressure inducement JDP2 is integrated to GSH and synthesizes in the promoter of relevant key factor SLC7A11, GCLM, GSS.JDP2
It activates GSH to synthesize relevant key factor transcriptional level, and then improves the level of intracellular GSH, maintain ROS stable state and resist
Damage of the chemotherapeutics to cell.Further detection discovery, JDP2 recruits PRMT5, and then raises WDR5/MLL transmethylase
Compound promotes GSH to synthesize relevant key factor promoter H3K4me3 methylation modification.It is interacted with WDR5-MLL small
The methylation modification of molecule antagonist OICR-9429 antagonism, combined chemotherapy drug therapy, it is suppressed that JDP2/PRMT5 compound pair
The anabolic regulation of GSH causes ROS level in DNA damage to be increased to lethal dosage, to effectively treat cancer cell.It is comprehensive
Upper described: JDP2 can promote GSH to synthesize relevant key factor promoter H3K4me3 methylation and then in conjunction with PRMT5
Modification to maintain high-caliber GSH, and then promotes the drug resistance of tumor and tumor recurrence of oophoroma.It is expressed for JDP2 high
Ovarian cancer cell, the inhibitor OICR-9429 and cis-platinum/Hycamtin that WDR5/MLL need to be used in combination can effectively inhibit tumour
Intracellular GSH is horizontal, and then maintains high level ROS and promote death of neoplastic cells.The present invention provides for ovarian cancer drug-resistant
New diagnosis, treatment method.
Detailed description of the invention
Fig. 1 is public database NCBI/GEO/GSE38666；GSE14407；In GSE66957 oophoroma cancerous tissue with just
JDP2mRNA expression, further, passes through JDP high expression group and low table in Kaplan-Meier analysis of ovarian cancer patient in normal ovary
Up to the total survival region and recurrence survival region of group.
Fig. 2 is that Western blotting and Real-time PCR detect JDP2 in 2 plants of ovarian epithelial cells and 8 plants of people
Albumen and mRNA expression in ovarian cancer cell further detect the expression of JDP2 in 146 ovarian cancer tissues and divide
Analyse the expression of JDP2 and the 5 years Overall survivals and 5 years recurrence-free survival phases relationship of ovarian cancer patients.
Fig. 3 is the expression of JDP2 and the relationship of Apoptosis.
Fig. 4 is the expression that JDP2 participates in regulation SLC7A11, GCLM and GSS, and then promotes GSH, resists ROS and generates.
Fig. 5 is under DNA damage pressure, and JDP2 promotes GSH to generate in conjunction with PRMT5.
Fig. 6 is under DNA damage pressure, and JDP2/PRMT5 promotes the methylation modification of synthesis key factor promoter.
After Fig. 7 is Hycamtin chemotherapy, in JDP2 difference expression group peritonaeum the tumour growth situation of inoculation tumor bearing nude mice and
Mouse survival rate.
Fig. 8 is that building primary ovarian cancerous tissue OV-1 and OV-2 establishes PDX model, detection cis-platinum/Hycamtin joint
The therapeutic effect of OICR-9429.
Specific embodiment
Enumerate embodiment further below with the present invention will be described in detail.It will similarly be understood that following embodiment is served only for this
Invention is further described, and should not be understood as limiting the scope of the invention, those skilled in the art are according to the present invention
Some nonessential modifications and adaptations that the principle of elaboration is made all belong to the scope of protection of the present invention.Following specific works of example
Skill parameter etc. is also only an example in OK range, i.e. those skilled in the art can do suitable model by the explanation of this paper
Interior selection is enclosed, and does not really want to be defined in hereafter exemplary specific data.
It is sub combined with specific embodiments below, content that the present invention is furture elucidated.Actual conditions are not indicated in the following example
Experimental method, usually according to normal condition, the condition as described in " Molecular Cloning:A Laboratory guide " (third edition), or according to system
Make condition proposed by manufacturer.
Inventor by bioinformatics method to NCBI GEO datasets (http: //
Www.ncbi.nlm.nih.gov/gds/) common data (the GSE38666 of multiple ovarian cancer tissue mRNA expression chips；
GSE14407；GSE66957 it) is analyzed.The mRNA chip of GSE38666, GSE14407 and GSE66957 all include 12
Normal ovarian epithelial cell and respectively the mRNA data of 18,12 and 57 ovarian cancer tissues.Wherein, JDP2mRNA water
It puts down and significantly increases (Figure 1A) in ovarian cancer tissue, prompt JDP2 that may participate in the malignant development of oophoroma.
In order to which whether the expression up-regulation for verifying JDP2 is close with the malignant development of oophoroma and with the survival region of patient
Correlation, the present invention further pass through Keplan-Meier Plotter survival analysis website (http://kmplot.com/
Analysis/) analysis JDP2 and ovarian cancer patients Prognostic significance.Numerical value indicates that P < 0.05 is thought in statistics with mean ± SD
On have significant difference.JDP2 high expression and the totality and recurrence survival region of patient are negatively correlated, and JDP2 high is prompted to express
Patient's prognosis it is poor (Figure 1B).Oophoroma, including first-line drug cis-platinum and carboplatin are clinically treated using DNA damage agent, with
And Second line Drug Hycamtin, Doxorubicin and gemcitabine.These drug-induced different types of DNA damages, to kill
Tumour cell.JDP2 high expression is found by KM Plotter analysis and receives the patient of platinum class or Hycamtin drug therapy
In significant related (Figure 1B), this has prompted the expression of JDP2 and patients with ovarian tumor to resist DNA damage for total survival region and recurrence
It is closely related to hurt agent.
Based on the above content, inventor has found inventor from many levels such as molecule, cell, animal and clinical samples
JDP2 carry out system and in-depth study, with molecule mechanism of the clear JDP2 in ovarian cancer progression.
It is closely related with the poor prognosis of ovarian cancer patients that JDP2 high expression is detected in 1 clinical tissue of embodiment
The present embodiment 2 plants of oophoroma epithelial cells (IOSE80, HOSEpic) and 8 plants of ovarian cancer cells (OV90, OV56,
COV644, OVCAR3, COV362, TOV112D, A2780 and SKOV3) in detection JDP2 albumen and mRNA expression.In order to
Further whether verifying JDP2 expression is related to the malignant progression of oophoroma and survival of patients prognosis, and the present embodiment is in 146 ovum
The protein expression of JDP2 is detected in nest cancer patient tissue paraffin section and analyzes expression and the oophoroma clinical pathologic characteristic of JDP2
Correlation.Primer of the above-mentioned detection JDP2 gene used in rna level are as follows:
JDP2-F:5 '-TGGGCTGTCTCTGTCTGTTG-3 ' (SEQ ID NO:1)；
JDP2-R:5 '-GCTCTGTCATCACTCAGGCA-3 ' (SEQ ID NO:2).
As a result: JDP2 albumen and mRNA expression are apparently higher than 2 plants of oophoroma epithelial cells in 8 plants of ovarian cancer cells
(Fig. 2A, B) prompts JDP2 high expression in oophoroma, may be used as carcinogenic factor.JDP2 drug resistance tissue of patient after chemotherapy
In expression be apparently higher than non-drug resistant tissue of patient.Immunohistochemistry (IHC) statistical analysis display JDP2 level and chemotherapy
Drug resistance (P < 0.001；R=0.37) significant correlation recurs (P=0.002；) and FIGO stage (P=0.009 r=0.25；R=
0.21), but negatively correlated with shorter totality/recurrence-free survival phase, prompt the patient relative to JDP2 low expression, JDP2 high table
The ovarian cancer patients prognosis mala reached, life span are shorter (Fig. 2 C, D, E).
Conclusion:
1) JDP2 high expression in ovarian cancer cell and tissue；
2) JDP2 high expression in oophoroma, expression up-regulation with patient's drug resistance resistance, FIGO by stages and Patients on Recurrence is close
Cut phase is closed.
Embodiment 2: experiment in vitro proves that JDP2 participates in regulation chemotherapy in ovarian cancer tolerance
In order to study JDP2 in the biological function of oophoroma, the present embodiment is in two plants of ovarian cancer cells of OV90 and OVCAR3
In system, the ovarian cancer cell line for stablizing high expression and silencing JDP2 expression is established respectively using Retroviral vector systems.
Detect whether JDP2 has raising ovarian cancer cell to the resistivity of chemotherapeutics in OV90 and OVCAR3 ovarian cancer cell.
The processing of DNA damage agent cis-platinum is added in cell, detects shadow of the expression to ovarian cellular apoptosis and survival of JDP2 respectively
It rings.
As a result: Western Blotting testing result show this research be successfully established exogenous high expression JDP2 and
The oophoroma stable cell lines (Fig. 3 A) of silencing endogenous JDP2 expression.Ovarian cancer cell can be improved to chemotherapy in height expression JDP2
The anti-apoptotic ability (Fig. 3 B) of drug.The anti-of ovarian tumor cell can be improved in the high expression JDP2 of colony formation discovery simultaneously
Apoptosis promotes proliferation function (Fig. 3 C)；The above result shows that JDP2 high expression promotes the cell of ovarian cancer cell resistance chemotherapeutics
Toxicity.
Conclusion: JDP2, which has, improves ovarian cancer cell to the resistivity of chemotherapeutics.
Embodiment 3JDP2 high expression regulation glutathione reducing system
The present embodiment uses DNA damage agent (CPT) inducing cell DNA damage, and JDP2 knot is enriched in the cell of processing
The DNA fragmentation of conjunction, and the DNA of purifying is subjected to deep sequencing (ChIP sequencing, ChIP-seq).ChIP process uses
Millipore chromatin is co-precipitated kit, the DNA sample Song Jinwei intelligence company of purifying is carried out to build library sequencing, and use
HiSeq 2000 is sequenced instrument and carries out deep sequencing.Data after sequencing are fixed by reading using bowtie2 (2.2.9 version) algorithm
Human genomic sequence (hg19) is arrived in position.ChIP peak calling is fitted using MACS (1.401 version) algorithm.Work as ChIP
It is fixed when peak falls in transcription initiation site (transcriptional start site, TSS) surrounding 4kb (± 2kb) of gene
Justice is TSS.As 6kb (± 3kb) around ChIP peak falls in gene TSS, it is defined as promoter.It uses
Network2Canvas carries out Gene ontology (GO cluster) and KEGG path analysis.ChIP-qPCR, Q-PCR, WB and
GSH, ROS test experience further detect JDP2 to the regulation situation of these three genes of SLC7A11, GCLM and GSS.Wherein, Q-
The primer of PCR detection tri- gene expression amounts of SLC7A11, GCLM and GSS is respectively as follows:
SLC7A11-F:5 '-TCTCCAAAGGAGGTTACCTGC-3 ' (SEQ ID NO:3),
SLC7A11-R:5 '-AGACTCCCCTCAGTAAAGTGAC-3 ' (SEQ ID NO:4)；
GCLM-F:5 '-ATGGTTTAAACAAGGCGCTCCTGGCG-3 ' (SEQ ID NO:5),
GCLM-R:5 '-GTACCTGCAGGGATTACAGGCATGAGG-3 ' (SEQ ID NO:6)；
GSS-F:5 '-CCTAGCCGGTTTGTGCTAAAG-3 ' (SEQ ID NO:7),
GSS-R:5 '-TTTCAGGGCCTGTACCATTTC-3 ' (SEQ ID NO:8).
As a result: finding that JDP2 has apparent richness in the promoter region of genome by the ChIP-seq data analysis of JDP2
Collect (24%) (Fig. 4 A).In addition, Gene Onology (GO) clustering of the ChIP-seq data of JDP2 obtains, JDP2 with
" Response to drug ", " glutathione metabolic process ", " Glutathione biosynthetic
Process " and " Glutathione metabolism " signal path are obviously related, and JDP2 is prompted to participate in the metabolism of regulation GSH
Process, and (such as Fig. 4 B) related to drug response.Consistent with the hypothesis, this research passes through the KEGG signal of ChIP-seq data
Path analysis, JDP2 is related to " Glutathione metabolism " " Platinum drug resistance ", further
Prompt JDP2 participates in the metabolic process of GSH and promotes drug tolerance (Fig. 4 B).It is analyzed by enrichment degree, JDP2 is closed in GSH
There is obvious enrichment (Fig. 4 C) in promoter at related gene SLC7A11, GCLM, GSS, prompts JDP2 that may regulate and control this 3 bases
The transcriptional level of cause, and then influence the level of GSH.Further JDP2 is verified by ChIP qPCR to open in GSH synthesis related gene
The enrichment degree of mover.Under DNA damage, ChIP qPCR shows that JDP2 is obviously enriched in synthesis related gene promoter, and sinks
The expression of silent JDP2, hence it is evident that inhibit JDP2 in GSH synthesis related gene promoter enrichment degree (Fig. 4 D).It is consistent with the result,
Under DNA damage, JDP2 has been obviously promoted the mRNA and protein expression of SLC7A11, GCLM, GSS, and the expression of silencing JDP2,
Obviously inhibit the mRNA and protein expression (Fig. 4 E, F) of SLC7A11, GCLM, GSS.Importantly, JDP2 high expression is obvious
The level of intracellular GSH is increased, but inhibits the level (Fig. 4 G) of intracellular ROS.However the expression of silencing JDP2, hence it is evident that suppression
The level of intracellular GSH has been made, has promoted the level of intracellular ROS instead.Above the experimental results showed that, under DNA damage,
JDP2 participates in the transcriptional control of regulation GSH related gene, and promotes GSH to generate and then resist oxidative stress.
Conclusion: JDP2 resists oxidative stress and cytotoxicity caused by chemotherapeutics by regulation GSH also original system.
Embodiment 4:JDP2/PRMT5 mediates epigenetic modification and then promotes the transcriptional activity of JDP2
(1) PRMT5/JDP2 transcriptional activity
In order to further explore under DNA damage, the transcription regulation mechanism that JDP2 is mediated, the present embodiment passes through co-immunoprecipitation
Albumen situation of the experimental analysis JDP2 in conjunction with PRMT5.Firstly, camptothecine (the inhibition of DNA topoisomerase I is first used in the experiment
Agent) cell is handled, further, the antibody for passing through RPMT5 carries out co-immunoprecipitation experiment.Meanwhile constructing the JDP2 of truncated-type
Plasmid detects structural domain of the RPMT5 in conjunction with truncated-type JDP2.Strike the expression of low PRMT5, detection PRMT5 to GSH, ROS and
The influence of SLC7A11, GCLM, GSS.
As a result: in the case where DNA damage, hence it is evident that promote the combination (Fig. 5 A) of PRMT5 and JDP2.By PRMT5 and cut
Short JDP2 co-immunoprecipitation experiment proves, PRMT5 (Fig. 5 B) in conjunction with the leucine zipper motif of JDP2.There is document in the past
Report that JDP2 by recruiting DNA methylase inhibitor albumen HDAC3, starts the deacetylation of histone and leads to Transcription inhibition.That
The DNA damage the case where, PRMT5 whether with the emulative combination JDP2 of HDAC3, and participate in the transcriptional control of JDP2 pass through
Co-immunoprecipitation experiment proves, obviously inhibits the binding affinity (Fig. 5 C) of JDP2 and HDAC3 in DNA damage.Strike low PRMT5's
Expression, hence it is evident that inhibit the transcriptional level and protein level of JDP2 regulation GSH metabolism related gene, and JDP2 is inhibited to induce
The generation of GSH results in the raising (Fig. 5 D, E, F) of the ROS level of DNA damage induction.Under DNA damage pressure, PRMT5 with
HDAC3 competitive binding JDP2, and the generation of GSH is promoted to resist oxidative stress.
Conclusion:
1) PRMT5 passes through antagonism JDP2 in conjunction with HDAC3, to promote JDP2 transcriptional activation.
2) PRMT5/JDP2 participates in GSH anabolic process, resists ROS and generates.
(2) the methylation modification that PRMT5 is mediated promotes the transcription of synthesis GSH related gene
Previous literature report, PRMT5 are modified by the methylation of H3R2me1 and H3R2me2s to histone, Jin Ertong
It crosses the methylation for recruiting WDR5/MLL compound and then the H3K4me3 of promotion histone and promotes transcriptional control.Histone
The modification of H3K4me3 is also further recruited rna plymerase ii and is participated in the transcriptional control of gene.In the case where DNA damage,
The enrichment degree of H3R2me1, H3R2me2s, WDR5/MLL, H3K4me3, TFIID, POll in SLC7A11 promoter is detected,
And it detects and strikes low WDR5 to the regulating and controlling effect of SLC7A11, GCLM, GSS and GSH and ROS.
As a result: DNA damage has significantly raised the enrichment degree of H3R2me1 and H3R2me2s in SLC7A11 promoter, and
And when striking low β-catenin, JDP2, PRMT5, H3R2me1 and H3R2me2s are significantly suppressed in SLC7A11 promoter
On enrichment degree (Fig. 6 A), disclose PRMT5 by histone methylation modification promote JDP2 activate GSH process.
Consistent in this result to be, the JDP2/PRMT5 of DNA damage induction significantly promotes WDR5, MLL1-3, TFIID and Pol II
(Fig. 6 B, C) is enriched in the promoter of SLC7A11.In addition, the expression for striking low WDR5 obviously inhibits turning for GSH related gene
Record is horizontal, and then inhibits the level of GSH, but promotes the level (Fig. 6 D, E) of ROS.The micromolecular inhibitor OICR- of WDR5
9429 combined chemotherapy drugs are obviously promoted tumour cell to the sensibility of chemotherapeutics, promote death of neoplastic cells (Fig. 6 F).With
Upper the results show PRMT5 promotes JDP2 to activate the process of GSH by the methylation modification of regulation histone, and then resists
The lethal effect of chemotherapeutics.
Conclusion:
1) JDP2/PRMT5 forms compound, and then recruits the methylation that WDR5, MLL promote H3K4, to promote GSH phase
Methylation modification and transcriptional upregulation in correlation gene promoter.
2) epigenetic caused by the obvious antagonism JDP2/PRMT5 compound of WDR5 micromolecular inhibitor OICR-9429 is repaired
Decorations promote apoptosis of tumor cells with chemotherapy drugs in combination using the level of obvious up-regulation ROS.
5 micromolecular inhibitor combined chemotherapy drug of embodiment helps to treat chemotherapy in ovarian cancer tolerance
(1) it confirms in vivo experiment, JDP2 promotes oophoroma to the drug resistance of Hycamtin
Hycamtin clinically be optimize CPT after clinical medicine, be widely used in treatment oophoroma, lung cancer and other
Cancer.The present embodiment uses intraperitoneal implantation tumor as model, by 5 × 106Tumour cell note with luciferase expression
It is mapped in the abdominal cavity of BALB/C nude mice, each experimental group 6.Fluorescence using internal luciferase imaging system record tumour is strong
Degree.When the intracorporal tumour fluorescence value of mouse reaches 2 × 107p/sec/cm2/ sr begins to use Hycamtin (10mg/kg weight),
It handles weekly 3 times, handles 6 weeks always.When mouse is raised to experimental endpoints, all mouse are put to death using cervical dislocation.It takes
Tumor tissues carry out subsequent experiment out, and tumor tissues are fixed with formaldehyde and are used for destination protein with specimens paraffin embedding slices
Immunohistochemical staining.
As a result: the influence that JDP2 lacks of proper care to DNA damage pressure is checked by oophoroma mouse model in peritonaeum.Such as Fig. 7-A-
Shown in D, in the mouse of Hycamtin treatment, the cell of up-regulation JDP2 expression forms tumour and maintains higher growth rate,
As TUNEL+ cell it is less shown in, and show higher GSH concentration, but lower ROS is horizontal, and it is poor to result in mouse
Survival region.In contrast, by short interfering rna (siRNA) silencing JDP2, Dioleoyl Phosphatidylcholine (DOPC) nanometer is mixed
Liposome, the antitumor action of significant enhancing Hycamtin, the GSH for causing tumor growth rate lower and reducing in tumor are horizontal
, but ROS level and Apoptosis are higher (Fig. 7 A, B, C, D).Therefore, result above further supports JDP2 and participates in regulation
GSH is metabolized and inhibits the Apoptosis of ROS induction and treats the viewpoint for generating resistance to DNA damage.
Conclusion: in vivo experiment, chemoresistance of the JDP2 high expression enhancing ovarian cancer cell to Hycamtin.
(2) it confirms in vivo experiment, JDP2 promotes oophoroma to the drug resistance of Hycamtin
The present invention uses the heterograft of patient source (patient-derived xenografts (PDX)).PDX model is
By the fresh oophoroma tumor tissue removed of just performing the operation, the tumor mass of 1~3mm is cut into, is inoculated into the NOD-SCID of female
IL-2rγ-/-(NSG) mouse is subcutaneous.The length and width of tumour is measured weekly and is made a record, and gross tumor volume (L* is calculated
W2/ 2) growth curve for and according to gross tumor volume and record time drawing tumour, when the volume of tumour is in~0.2cm3When,
Mouse is divided into 6 groups, individually receives solvent, cis-platinum (5mg/kg weight), Hycamtin (10mg/kg weight), or
At OICR-9429 (3mg/kg weight) or OICR-9429 (3mg/kg weight) and Hycamtin (10mg/kg weight) joint
Reason or OICR-9429 (3mg/kg) and cis-platinum (5mg/kg weight) Combined Treatment are handled weekly 3 times, are handled 6 weeks always.To
When mouse is raised to experimental endpoints, all mouse are put to death using cervical dislocation.It takes out tumor tissues and carries out subsequent experiment, with
And tumor tissues are fixed to the immunohistochemical staining that destination protein is used for specimens paraffin embedding slices with formaldehyde.
As a result: the present invention further detects joint OICR-9429 using the PDX model that two clinical ovarian cancer tissues establish
With DNA damage chemotherapeutant to the therapeutic effect (Fig. 8-A) of cancer.As shown in Fig. 8-A-C, OICR-9429 is used alone simultaneously
Without significantly inhibiting tumour growth, but the significant oophoroma that enhances of OICR-9429 joint Hycamtin treatment replaces support pool
The sensibility of health leads to high-caliber ROS and inhibits tumour growth.In addition, OICR-9429 joint Hycamtin uses significant increasing
Strong sensibility of the oophoroma to Hycamtin, leads to high-caliber ROS and inhibits tumour growth.Consistent, the OICR- in this result
9429 combination with cisplatin, to the sensibility of cis-platinum, are led to high-caliber ROS and inhibit tumour growth using the significant oophoroma that enhances.
Therefore, these results indicate that being used in combination for classical DNA damage chemotherapeutant and OICR-9429 is high-caliber by promoting
ROS and then killing tumor cell provide new therapeutic strategy for drug resistance of tumor treatment.
Conclusion: in PDX model OICR-9429 inhibit JDP2/PRMT5 mediate epigenetic modification, with Hycamtin/
Cisplatin combined use has stronger chemoresistance.
In conclusion JDP2 can be as tumor drug resistance detection target spot, tumor prognosis detection target spot, prediction tumor recurrence inspection
Survey target spot.Corresponding tumor drug resistance detection kit, tumor prognosis detection kit, prediction tumor recurrence detection examination can be prepared accordingly
Agent box, the reagent of the rna transcription level containing detection JDP2 in those kits, the protein expression level of quantitative detection JDP2
Reagent etc..By the RNA and protein expression level of JDP2 in detection specimens to judge whether the patient moors support
There is chemoresistance for health/plus cisplatin in treatment.By the expression of JDP2 in detection specimens, to judge that the patient is
No tendency of recurrence with higher.By the expression of JDP2 in detection specimens, thus to the highly expressed patient of JDP2
WDR5 inhibitor and Hycamtin/Combined with Cisplatin for The Treatment scheme are formulated, to realize the effect for the treatment of.
Those skilled in the art are readily appreciated that the above description is only a preferred embodiment of the patent of the present invention, not
To limit the present invention, all modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention, etc are all fallen within
The present invention claims protection scope within.
SEQUENCE LISTING
<110>Zhongshan University
Guangzhou Jie Erke Bioisystech Co., Ltd
<120>inhibit the substance application in preparation of anti-tumor drugs of JDP2 expression
<130>
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial synthesized
<400> 1
tgggctgtct ctgtctgttg 20
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
gctctgtcat cactcaggca 20
<210> 3
<211> 21
<212> DNA
<213>artificial synthesized
<400> 3
tctccaaagg aggttacctg c 21
<210> 4
<211> 22
<212> DNA
<213>artificial synthesized
<400> 4
agactcccct cagtaaagtg ac 22
<210> 5
<211> 26
<212> DNA
<213>artificial synthesized
<400> 5
atggtttaaa caaggcgctc ctggcg 26
<210> 6
<211> 27
<212> DNA
<213>artificial synthesized
<400> 6
gtacctgcag ggattacagg catgagg 27
<210> 7
<211> 21
<212> DNA
<213>artificial synthesized
<400> 7
cctagccggt ttgtgctaaa g 21
<210> 8
<211> 21
<212> DNA
<213>artificial synthesized
<400> 8
tttcagggcc tgtaccattt c 21
Claims (10)
1. the application of reagent in medicine preparation, the reagent for inhibiting JDP2 to express, the drug for it is following at least
One of:
Improve the sensibility of cells against neoplastic drug；
Inhibit the expression of intracellular GSH synthesis related gene；
Inhibit intracellular GSH horizontal；
Promote intracellular ROS level；
The methylation modification for inhibiting PRMT5 to mediate.
2. application according to claim 1, it is characterised in that: the anti-tumor drug is chemotherapeutics.
3. application according to claim 1, it is characterised in that: the synthesis related gene includes SLC7A11, GCLM, GSS
At least one of.
4. the application according to requiring 1, it is characterised in that: the reagent inhibits JDP2 expression by silencing JDP2.
5. application according to any one of claims 1 to 4, it is characterised in that: the cell is ovarian cancer cell.
6. application according to claim 5, it is characterised in that: the drug is for treating oophoroma.
7. a kind of anti-tumor drug, it is characterised in that: contain the reagent and chemotherapeutics for inhibiting JDP2 to express.
8. detecting application of the reagent of JDP2 expression quantity in reagent preparation box, the kit is used for following at least one:
Detect drug resistance of tumor；
Tumor prognosis；
Predict tumor recurrence.
9. application according to claim 8, it is characterised in that: the drug resistance refers to tumour cell to the resistance to of chemotherapeutics
Pharmacological property.
10. application according to claim 8, it is characterised in that: the tumour is oophoroma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910730699.XA CN110478484B (en) | 2019-08-08 | 2019-08-08 | Application of substance for inhibiting JDP2 expression in preparation of antitumor drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910730699.XA CN110478484B (en) | 2019-08-08 | 2019-08-08 | Application of substance for inhibiting JDP2 expression in preparation of antitumor drugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110478484A true CN110478484A (en) | 2019-11-22 |
| CN110478484B CN110478484B (en) | 2021-10-26 |
Family
ID=68550172
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910730699.XA Active CN110478484B (en) | 2019-08-08 | 2019-08-08 | Application of substance for inhibiting JDP2 expression in preparation of antitumor drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110478484B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002247990A (en) * | 2001-02-26 | 2002-09-03 | Inst Of Physical & Chemical Res | Mouse ap1 repressor transcription factor jdp-2 |
| CN1769459A (en) * | 2004-11-03 | 2006-05-10 | 中国医学科学院基础医学研究所 | Transcription factor JDP2 cloning, expression and preparation of its multiple-clone antibody |
| CN101023349A (en) * | 2004-03-31 | 2007-08-22 | 约翰·霍普金斯大学 | Biomarkers for ovarian cancer |
| WO2015121737A2 (en) * | 2014-02-12 | 2015-08-20 | Instytut Biochemii I Biofizyki Polskiej Akademii Nauk | Transcriptomic biomarkers, method for determination thereof and use of trnascriptomic biomarkers for individual risk assessment of developing post-infraction heart failure |
-
2019
- 2019-08-08 CN CN201910730699.XA patent/CN110478484B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002247990A (en) * | 2001-02-26 | 2002-09-03 | Inst Of Physical & Chemical Res | Mouse ap1 repressor transcription factor jdp-2 |
| CN101023349A (en) * | 2004-03-31 | 2007-08-22 | 约翰·霍普金斯大学 | Biomarkers for ovarian cancer |
| CN1769459A (en) * | 2004-11-03 | 2006-05-10 | 中国医学科学院基础医学研究所 | Transcription factor JDP2 cloning, expression and preparation of its multiple-clone antibody |
| WO2015121737A2 (en) * | 2014-02-12 | 2015-08-20 | Instytut Biochemii I Biofizyki Polskiej Akademii Nauk | Transcriptomic biomarkers, method for determination thereof and use of trnascriptomic biomarkers for individual risk assessment of developing post-infraction heart failure |
Non-Patent Citations (7)
Also Published As
| Publication number | Publication date |
|---|---|
| CN110478484B (en) | 2021-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yu et al. | DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine | |
| Vernier et al. | Estrogen-related receptors are targetable ROS sensors | |
| Che et al. | Pathogenetic, prognostic, and therapeutic role of fatty acid synthase in human hepatocellular carcinoma | |
| CN104363913A (en) | CDK8/CDK19 selective inhibitors and their use in anti-metastatic and chemopreventative methods for cancer | |
| Shen et al. | PARPi treatment enhances radiotherapy-induced ferroptosis and antitumor immune responses via the cGAS signaling pathway in colorectal cancer | |
| Boudny et al. | ATR-CHK1 pathway as a therapeutic target for acute and chronic leukemias | |
| Filipits et al. | Predictive markers in the adjuvant therapy of non-small cell lung cancer | |
| Schwab et al. | Neratinib shows efficacy in the treatment of HER2 amplified carcinosarcoma in vitro and in vivo | |
| Han et al. | Docetaxel enhances apoptosis and G2/M cell cycle arrest by suppressing mitogen-activated protein kinase signaling in human renal clear cell carcinoma | |
| Song et al. | Magnolin targeting of ERK1/2 inhibits cell proliferation and colony growth by induction of cellular senescence in ovarian cancer cells | |
| Tiong et al. | CSNK1E/CTNNB1 are synthetic lethal to TP53 in colorectal cancer and are markers for prognosis | |
| Wu et al. | Up-regulation of CHAF1A, a poor prognostic factor, facilitates cell proliferation of colon cancer | |
| Bianco et al. | Chemosensitization by antisense oligonucleotides targeting MDM2 | |
| Sun et al. | Combined inhibition of KIF11 and KIF15 as an effective therapeutic strategy for gastric cancer | |
| He et al. | CDC20: a novel therapeutic target in cancer | |
| Li et al. | miR-26a reverses multidrug resistance in osteosarcoma by targeting MCL1 | |
| Hao et al. | Targeting overexpressed activating transcription factor 1 (ATF1) inhibits proliferation and migration and enhances sensitivity to paclitaxel in esophageal cancer cells | |
| CN110478484A (en) | Inhibit the substance application in preparation of anti-tumor drugs of JDP2 expression | |
| Gao et al. | Probing synergistic targets by natural compounds for hepatocellular carcinoma | |
| Hu et al. | Inhibition of Apoc1 reverses resistance of sorafenib by promoting ferroptosis in esophageal cancers | |
| US20200101070A1 (en) | Methods of treating cancer having an active wnt/beta-catenin pathway | |
| Bras et al. | Shifting KRAS hotspot mutations inhibition paradigm in colorectal cancer | |
| Oxe et al. | Treacle is upregulated in cancer and correlates with poor prognosis | |
| Wu et al. | Regulation of Hippo-YAP/CTGF signaling by combining an HDAC inhibitor and 5-fluorouracil in gastric cancer cells | |
| Wang | Combining DNA-PK inhibitors with DNA-damaging therapies expands the targeted cell population to kill more cancer cells but also elevates toxicities in normal cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |