CN107158387A - UBE2M mediations Neddylation is blocked to modify the application in renal cell carcinoma treatment - Google Patents
UBE2M mediations Neddylation is blocked to modify the application in renal cell carcinoma treatment Download PDFInfo
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- CN107158387A CN107158387A CN201710426399.3A CN201710426399A CN107158387A CN 107158387 A CN107158387 A CN 107158387A CN 201710426399 A CN201710426399 A CN 201710426399A CN 107158387 A CN107158387 A CN 107158387A
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- ube2m
- neddylation
- mediations
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- kidney cancer
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
Abstract
The application in renal cell carcinoma treatment is modified the present invention relates to a kind of blocking UBE2M mediations Neddylation, belongs to field of biomedicine technology.The present invention devises the RNA such as sh control, sh UBE2F and sh UBE2M interference related plasmids, builds the stable renal carcinoma cell line for striking low UBE2M or UBE2F protein expression levels, is found through experiments that and strikes the propagation that low UBE2M significantly inhibits kidney cancer cell.Further by notable lethal effects of the Neddylation modified specificity micromolecular inhibitor MLN4924 to kidney cancer cell, demonstrate and block the Neddylation modifications of UBE2M mediations to play key effect on kidney cancer cell proliferation activity is suppressed.Therefore, the Neddylation modification signal paths for blocking UBE2M mediations are a kind of new target spots of potential treatment clear-cell carcinoma, with very strong clinical value.
Description
Technical field
The present invention relates to one new renal cell carcinoma treatment target spot of field of biomedicine technology, and in particular to blocks
Application of the Neddylation modification signal paths of UBE2M mediations in renal cell carcinoma treatment target spot.
Background technology
Clear-cell carcinoma（Renal cell carcinoma, RCC）It is initiated by the evil of the substantive uriniferous tubule epithelial systems of kidney
Property tumour, accounts for the 80%-90% of kidney malignant cancer, accounts for the 2% to 3% of adult malignancies.At present, surgical operation（Radical cure
Property kidney surgery and Nephron -sparing surgery）It is the preferred treatment method of Limitable renal cell carcinoma, complete healing can be reached.For
Local advancement kidney is then based on operative treatment, the complex treatment of postoperative accessory cytokine or molecular targeted therapy.And turn
Shifting property kidney there is no unified standard therapeutic scheme, and research shows that molecular targeted agents can significantly extend the life cycle of patient.
From 2006, NCCN and EAU are by molecular targeted agents（Sorafenib, Sutent, for Sirolimus etc.）It is used as metastatic kidney
Cancer treatment one, two wires clinical treatment medication.Most belongs to kinase inhibitor, by suppressing tumor vascular life
It is long, antineoplastic action is reached, extends the life cycle of patient.But the toxic side effect of molecular targeted agents（Hand-foot skin reaction, it is weary
Power, Neuroleptic Leukocytopenia, hypertension, anaemia, stomatoglossitis etc.）Its extensive use in clinical treatment is limited, some patientss are therefore
Need to adjust drug dose, or even stopped treatment.Therefore, find and find new renal cell carcinoma treatment target spot and research and develop accordingly new
The molecular targeted agents of type, have important clinical value for the treatment of clear-cell carcinoma.
Ubiquitin-likeization modification Neddylation is to modify mode after a kind of new protein translation, passes through ubiquitin-like sample egg
White NEDD8（neural precursor cell-expressed developmentally downregulated 8）Specifically
Property with substrate protein covalent bond, regulate and control multiple protein complex 26S Proteasome Structure and Function, in many of cell normal physiological activity
There is important regulating and controlling effect in individual key link.However, the abnormal activation and Several Kinds of Malignancy of Neddylation modifications
Occurrence and development are closely related.So targeting is expected to turn into new anti-tumor target for Neddylation modification signal paths
Point.The key enzyme of Neddylation modification signal paths mainly includes NEDD8 activating enzymes E1, NEDD8 desmoenzymes E2 and NEDD8
Ligase E3.NEDD8 desmoenzymes E2 is highly conserved protein, and research at present only finds there are 2 kinds, is UBE2M respectively
(Ubiquitin-binding enzyme E2M, UBC12) and UBE2F (ubiquitin-binding enzyme E2F).So
And, whether Neddylation modifications and NEDD8 desmoenzymes E2 can be as renal cell carcinoma treatment target spot and mechanism of action not yet
Know.
The content of the invention
UBE2M mediation Neddylation modifications are blocked in renal cell carcinoma treatment it is an object of the invention to provide one kind
Application, to research and develop new molecular targeted therapy, there is provided the Neddylation modification works that a kind of blockings UBE2M mediate
To treat the novel targets of clear-cell carcinoma.It is related that the present invention devises the RNA interference such as sh-control, sh-UBE2F and sh-UBE2M
Plasmid, builds the stable renal carcinoma cell line for striking low UBE2M or UBE2F protein expression levels, is found through experiments that and strikes low UBE2M
Significantly inhibit the propagation of kidney cancer cell.Further by Neddylation modified specificity micromolecular inhibitors MLN4924 to kidney
The notable lethal effect of cancer cell, demonstrates and blocks the Neddylation modifications of UBE2M mediations to be lived in suppression kidney cancer cell propagation
Key effect is played in power.Therefore, the Neddylation modification signal paths for blocking UBE2M mediations are a kind of potential treatments
The new target spot of clear-cell carcinoma, with very strong clinical value.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
Block the Neddylation modification signal paths that UBE2M is mediated as drug target in anti-clear-cell carcinoma medicine is prepared
Application.
Block the Neddylation of UBE2M mediations to modify signal path as drug target and prepare suppression clear-cell carcinoma
Application in antiproliferative agent.
Expression or function by targeted inhibition UBE2M, realize the growth of targeted inhibition clear-cell carcinoma.
Targeted inhibition UBE2M expression method be：It is transferred to by lentivirus mediated shRNA in kidney cancer cell, strikes low kidney
Intracellular UBE2M expression.
Inhibitor is selected from and targets UBE2M albumen coded sequences, and can suppress UBE2M genetic transcriptions and protein expression
shRNA。
Described shRNA sequences are GCGGATCCAGAAGGACATAAA.
The beneficial effects of the present invention are：The present invention is experiments prove that mediated by blocking UBE2M
Neddylation modification signal paths can suppress the propagation of kidney cancer cell.It is further small by Neddylation modified specificities
Notable lethal effects of the molecule inhibitor MLN4924 to kidney cancer cell, it was demonstrated that block the Neddylation of UBE2M mediations to repair
Decorations play key effect on kidney cancer cell proliferation activity is suppressed.Therefore, the Neddylation modification letters of UBE2M mediations are blocked
Number path is a kind of new target spot of potential treatment clear-cell carcinoma, with very strong clinical value.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the application, this hair
Bright illustrative example and its illustrate to be used to explain the present invention, do not constitute inappropriate limitation of the present invention.
Fig. 1 can significantly inhibit kidney cancer cell 786-0's for the Neddylation modifications of the blocking UBE2M mediations of the present invention
Propagation.(A) build stabilization to strike after the kidney cancer cell 786-0 of low UBE2M or UBE2F protein expression levels, pass through cell count
Method detection kidney cancer cell propagation (P<0.001).(B) detection is analyzed by Western blot and strikes low UBE2M or UBE2F feelings
Condition.
Fig. 2 for the present invention MLN4924 significantly inhibit kidney cancer cell 786-0 propagation (P<0 .001)。
Embodiment
Further illustrate below in conjunction with the accompanying drawings the present invention detailed content and its embodiment, by specific experiment come
Further checking is of the invention.
Referring to shown in Fig. 1 and Fig. 2, the purpose of the present invention is that there is provided one kind to research and develop new molecular targeted therapy
The Neddylation modifications of UBE2M mediations are blocked as the novel targets for the treatment of clear-cell carcinoma.
In a first aspect, the invention provides block the Neddylation modification signal paths of UBE2M mediations to be used as medicine target
Application of the point in anti-clear-cell carcinoma medicine is prepared.
Second aspect, medicine target is used as the invention provides the Neddylation modification signal paths of UBE2M mediations are blocked
Application of the point in the antiproliferative agent for suppressing clear-cell carcinoma is prepared.
Described inhibitor is selected from and targets UBE2M albumen coded sequences, and can suppress UBE2M genetic transcriptions and albumen
The shRNA of expression.It is transferred to by lentivirus mediated shRNA in kidney cancer cell, strikes the expression of UBE2M in low kidney cancer cell, is realized
The growth of targeted inhibition clear-cell carcinoma.Described shRNA sequences are GCGGATCCAGAAGGACATAAA.
Embodiment 1：
Kidney cancer cell 786-0 propagation can be significantly inhibited by blocking the Neddylation modifications of UBE2M mediations
Plasmid (sh-control, sh-UBE2F and sh-UBE2M) is added in DH5 α competence respectively, placed 20 minutes on ice
After be placed in 42 DEG C of heating modules, heat shock 90 seconds.Competence after heat shock is placed in 5 minutes on ice.500 are added into competence
μ L LB liquid mediums, 200rpm shakes 40 minutes at 37 DEG C.Collect bacterium solution 300rcf to centrifuge 5 minutes, discard 400 μ L of supernatant.With
Remaining bacterium solution will be precipitated and is resuspended, and is uniformly coated in solid LB media（The μ g/ml of the antibiotic of benzyl containing ammonia 100）, it is inverted into
In 37 DEG C of constant incubators.Picking clearly single bacterium colony after 24 hours, is added into LB liquid medium（The antibiotic of benzyl containing ammonia
100μg/ml）, 200rpm shakes 24 hours at 37 DEG C.Bacterium solution is collected after 24 hours, plasmid is extracted.By the plasmid and pRSV- of acquisition
Rev, pMDLg/pRRE and pCMV-VSV-G are with 3:3:3:1 ratio is transfected into 293T cells, is placed in incubator and is cultivated（Training
Foster condition is：5%CO2；Temperature is 37 DEG C）, nutrient solution is changed after 6 hours, continues to cultivate in incubator.Harvest contains after 48 hours
The supernatant of pseudovirion, 1000rpm is centrifuged 5 minutes, by the supernatant of harvest with 0.45 μm of membrane filtration.Under the conditions of 4 DEG C,
30000rpm ultracentrifugations 2 hours in 20% sucrose.The viral pellet from after will be surpassed to be resuspended with sterile PBS.By 4 × 105It is individual
786-0 cells are seeded in 6 orifice plates, are placed in incubator and are cultivated（Condition of culture is：5%CO2；Temperature is 37 DEG C）.24 hours
Cell supernatant is discarded afterwards, the serum free medium for being mixed with corresponding virus is added, is placed in incubator and cultivates（Condition of culture
For：5%CO2；Temperature is 37 DEG C）, the culture medium containing 20% hyclone is added after 6 hours.After infection 48 hours, it is changed to and contains
There is puromycin (4ug/mL) culture medium.Cell death situation is observed, the cell of survival is passed on.
To accordingly stablize respectively kidney cancer cell 786-0 (sh-control, sh-UBE2F, sh-UBE2M and sh-UBE2F+
Sh-UBE2M) with every hole 2 × 104Individual cell is seeded in 24 orifice plates, is placed in incubator and is cultivated（Condition of culture is：5%CO2；
Temperature is 37 DEG C）, digested in different time sections application trypsase, collect cell suspension and application cell tally is carried out
Cell count.
Analyzed by Western blot.Use the Primary antibodies for target protein, including ACTIN
（Ab8227, Abcam）, UBE2M（Ab109507,Abcam）And UBE2F（Ab185234, Abcam）.Secondary antibody includes anti-rabbit
IgG and anti-mouse IgG（Jackson ImmunoResearch Laboratories）.
Embodiment 2：
MLN4924 significantly inhibits kidney cancer cell 786-0 propagation
By kidney cancer cell 786-0 with every hole 2 × 104Individual cell is inoculated in 24 orifice plates, is placed in incubator and is cultivated（Condition of culture
For：5%CO2；Temperature is 37 DEG C）, after after cell attachment, using DMSO or MLN4924（0.5 μM and 1 μM）Handled, not
Digested with the period using trypsase, collect cell suspension and application cell tally carries out cell count.
Understand in summary, kidney cancer cell can be suppressed after the Neddylation modification signal paths for blocking UBE2M mediations
Propagation.And by notable lethal effects of the MLN4924 to kidney cancer cell, the Neddylation of UBE2M mediations is demonstrated from side
Modify the key effect in kidney cancer cell proliferation activity.Therefore, the Neddylation modification signals of UBE2M mediations are blocked to lead to
Road is a kind of new target spot of potential treatment clear-cell carcinoma, with very strong clinical value.
The preferred embodiment of the present invention is the foregoing is only, is not intended to limit the invention, for the technology of this area
For personnel, the present invention can have various modifications and variations.All any modification, equivalent substitution and improvements made for the present invention etc.,
It should be included in the scope of the protection.
Claims (6)
1. blocking the Neddylation of UBE2M mediations to modify signal path as drug target is preparing anti-clear-cell carcinoma medicine
In application.
2. block the Neddylation modification signal paths of UBE2M mediations to suppress clear-cell carcinoma in preparation as drug target to increase
Grow the application in reagent.
3. application according to claim 1 or 2, expression or function by targeted inhibition UBE2M, realizes targeted inhibition kidney
The growth of cell cancer.
4. application according to claim 3, the method for targeted inhibition UBE2M expression is：Turned by lentivirus mediated shRNA
Enter in kidney cancer cell, strike the expression of UBE2M in low kidney cancer cell.
5. application according to claim 4, inhibitor is selected from and targets UBE2M albumen coded sequences, and can suppress
UBE2M genetic transcriptions and the shRNA of protein expression.
6. application according to claim 5, described shRNA sequences are GCGGATCCAGAAGGACATAAA.
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| CN201710426399.3A CN107158387A (en) | 2017-06-08 | 2017-06-08 | UBE2M mediations Neddylation is blocked to modify the application in renal cell carcinoma treatment |
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| CN201710426399.3A CN107158387A (en) | 2017-06-08 | 2017-06-08 | UBE2M mediations Neddylation is blocked to modify the application in renal cell carcinoma treatment |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101583622A (en) * | 2006-11-02 | 2009-11-18 | 黄岚 | Inhibitors for disrupting the interaction of ubiquitination related enzymes and uses thereof |
| US20150079590A1 (en) * | 2013-09-18 | 2015-03-19 | Beth Israel Deaconess Medical Center, Inc. | Characterization and analysis of the composition and dynamics of the mammalian riboproteome |
-
2017
- 2017-06-08 CN CN201710426399.3A patent/CN107158387A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101583622A (en) * | 2006-11-02 | 2009-11-18 | 黄岚 | Inhibitors for disrupting the interaction of ubiquitination related enzymes and uses thereof |
| US20150079590A1 (en) * | 2013-09-18 | 2015-03-19 | Beth Israel Deaconess Medical Center, Inc. | Characterization and analysis of the composition and dynamics of the mammalian riboproteome |
Non-Patent Citations (6)
| Title |
|---|
| DANNY T. HUANG，等: "E2-RING Expansion of the NEDD8 Cascade Confers Specificity to Cullin Modification", 《MOLECULAR CELL》 * |
| JI-GEN PING，等: "The expression of Cullin1 is increased in renal cell carcinoma", 《TUMOR BIOLOGY》 * |
| SCOTT CUKRAS，等: "Inactivating UBE2M Impacts the DNA Damage Response and Genome Integrity Involving Multiple Cullin Ligases", 《PLOS ONE》 * |
| T HOSONO，等: "NUB1, an interferon-inducible protein, mediates anti-proliferative actions and apoptosis in renal cell carcinoma cells through cell-cycle regulation", 《BRITISH JOURNAL OF CANCER》 * |
| TERESA A. SOUCY，等: "An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer", 《NATURE》 * |
| 张爱联: "《生物化学与分子生物学实验教程》", 31 January 2009, 中国农业大学出版社 * |
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Application publication date: 20170915 |