CN107907674A - Applications of the Drp1 as drug targets in the medicine that screening prepares treatment breast cancer - Google Patents
Applications of the Drp1 as drug targets in the medicine that screening prepares treatment breast cancer Download PDFInfo
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Abstract
Disclosed by the invention is applications of the Drp1 as drug targets in the medicine that screening prepares treatment breast cancer, the medicine refers to the reagent that can suppress Drp1 expression, and the reagent of the suppression Drp1 expression includes suppressing the reagent of Drp1 protein stabilities, suppresses the reagent of Drp1 protein actives, suppresses the reagent of Drp1 protein functions.The medicine includes the activator of GBP2 genes or GBP2 albumen, and the activator can promote or strengthen GBP2 or be related to expression or the activity of the material of GBP2 upstreams or downstream pathway, and the medicine includes GBP2 albumen and/or Mdivi 1.Method and a kind of screening technique of breast cancer medicines present invention also offers breast cancer of the diagnosis characterized by Drp1 overexpressions and/or up-regulation.The present invention for the treatment of breast cancer provides new medicament selection, can carry out that specific aim is stronger, reaches more effective treatment to breast cancer, Death Rate of Breast Cancer is further reduced, with very high clinical medicine value.
Description
Technical field
The present invention relates to technical field, is specifically related to Drp1 and prepares the medicine for treating breast cancer in screening as drug targets
Application in thing.
Background technology
Mitochondria is important " energy plants " of cell, and in the generation of tumour, development etc. plays an important role.Mitochondria work(
Can be normally essential for tumour cell energy production and cells survival.The energetic supersession that mitochondria passes through modulate tumor cell
The growth of tumour cell is controlled with apoptosis isoreactivity.The abnormal growth that can influence breast cancer cell of mitochondrial function and
Metabolism.Mitochondrial fusion and division play necessary adjustment effect in mitochondrial function.Moreover, the fusion and division of mitochondria
Process occurs in tumour, also plays the role of in development important.In recent years research finds that Mitochondrial Shape is dynamic change,
Reach dynamic equilibrium in continuous division and fusion.In human archeocyte, the albumen for adjusting mitochondrial fusion has Mfn1
(mitofusin 1) and Mfn2 (mitofusin 2) and Opa1 (optic atrophy protein 1) etc.；Adjust mitochondria
The albumen of division mainly has Drp1 (dynamin-related protein1), Fis1 (mitochondrial fission 1)
Deng.Mitochondria adjusts it in the subcellular positioning of intracellular by dividing and merging, and influences between mitochondria and mitochondria is inside and outside
The exchange of DNA and albumen, repair the mitochondria of damage and the mitochondrial DNA of mutation and change mitochondria shape and quantity etc.,
So that mitochondria, which is in, is adapted to state of the cell to its functional requirement, wherein, chondriokinesis protein D rp1 is regulation and control line grain
One of key protein of body division and fusion.
Drp1 albumen is a member in dynamins GTPase superfamilies, and as other family members, Drp1 also possesses
GTPase, middle and GTPase effector active regions.GTPase active regions mainly combine with GTP and hydrolyze GTP.
Middle and GTPase effector active regions project team mainly assembles with dynamin family proteins and regulates and controls GTPase
Related (Fig. 1).Drp1 albumen is mainly distributed on cell cytosol, and small part is distributed on mitochondria.When it is played function, Drp1
The division of mitochondria up regulation mitochondria is transferred to from endochylema.
Studies have shown that Drp1 can regulate and control the ability of chondriokinesis-fusion of its mediation by the modification after transcription.Most
Two articles of nearly Molecular cell show that the phosphorylation modification of Drp1 promotes the division of mitochondria and the growth of tumour cell,
Evidence is directly given and has shown that the chondriokinesis of Drp1 mediations is capable of the growth of modulate tumor.Meanwhile also there is Oncogene to grind
Study carefully and show, the transfer of breast cancer cell is subject to regulating and controlling for the mitochondria dynamic change of Drp1 mediations.The strong relatively low transfer of metastatic
Property breast cancer cell Mitochondria division protein D rp1 up-regulated expression and Drp1 substantially have stronger phosphorylation modification.Transfer
Property strong breast cancer cell mitochondria be presented significant disruptive features, and mitochondria be more easy to be distributed to shifted with breast cancer cell it is close
Relevant cell pseudopodium region is cut, the movement to pseudopodium provides required energy (ATP), thus promotes breast cancer cell
Transfer.The obvious transfer for inhibiting breast cancer cell of expression of interference Drp1.We display that Drp1 exists using same cell
Expression quantity is high in the strong breast cancer cell of metastatic, and the transfer of expression quantity and breast cancer cell is directly proportional.Disturb Drp1's
Obvious transfer and the invasive ability for inhibiting breast cancer cell of expression.But the upstream mechanism of Drp1 regulation and control Metastasis in Breast Cancer is still not
Understand, it is necessary to find new modulin.
In recent years, GBP families are because finding it in inducing apoptosis of tumour cell, suppression tumor cell proliferation, migration and invasion and attack
In play an important role and paid attention to be subject to researcher, but specific mechanism is not still fully aware of.GBP molecular weight of albumen is 65-
71kd, they belong to the dynamin superfamilies in big GTPase, find 7 and 11 hypotypes in people and mouse respectively at present.
Wherein, GBP1 and GBP2 is two important GBP albumen.GBP1 albumen possesses GTPase, middle and GTPase effector
Active region (Fig. 2), other GBP family proteins also possess same active region.GBP1 is found to be positioned at endochylema, and GBP2 is in born of the same parents
There is positioning on slurry and certain vesica film component, but this film component is not the cellular content of mitochondria, lysosome etc..
Have that researches show that GBP2 can be positioned on golgiosome.The structure and physiological property of GBP1, which is studied, must compare more, and the knot of GBP2
Structure research is also underway.But the sequence of GBP2 and GBP1 has 75% homology, and the activation domains of GBP2 are also shown
Show consistent with GBP1.In this way, the structure of GBP1 can be used for reference to study the biochemical characteristic of GBP2.GBP family proteins initially as
Interferon inducible protein, is found in autoimmune disease and plays an important role.But GBP1 albumen is found to suppress
The effect of Nasopharyngeal neoplasms.Research shows, its expression by suppressing MMP-1 in epithelial tumor cell is swollen to suppress epithelium
Oncocyte shifts, and suppresses the transfer of endothelial cell by inducing the expression of integrin alpha 4 in endothelial cell, also
Have been reported that display GBP1 can inhibit transfer of colon cancer cell etc..In the recent period, some researches show that GBP1 and incidence cancer, oophoroma are poor
Prognosis it is related, and the prognosis of breast cancer has part relations, and GBP1 can promote the migration of esophageal cancer cell.It is however, related
The isomers GBP2 albumen of GBP1 albumen is but studied less with Nasopharyngeal neoplasms relation.Breast cancer cell is furtherd investigate at us
In the research for shifting regulatory mechanism, the expression for finding to raise GBP2 in the high breast cancer cell of metastatic first can significantly reduce
The migration of cell and invasive ability.Therefore, it is necessary to explore the mechanisms of action of the GBP2 in Metastasis in Breast Cancer is suppressed.
The content of the invention
Prepare the medicine for the treatment of breast cancer in screening as drug targets the technical problem to be solved by the present invention is to provide Drp1
Application in thing, and provide new medicament selection for the treatment of breast cancer.
The technical scheme is that：
Applications of the Drp1 as drug targets in the medicine that screening prepares treatment breast cancer.
Further, the medicine refers to the reagent that can suppress Drp1 expression.
Further, the reagent of the suppression Drp1 expression includes suppressing the reagent of Drp1 protein stabilities, suppresses Drp1
The reagent of protein active, the reagent for suppressing Drp1 protein functions.
Further, the medicine includes the activator of GBP2 genes or GBP2 albumen.
Further, the activator can promote or strengthen GBP2 or be related to the material of GBP2 upstreams or downstream pathway
Expression or activity.
Further, the medicine includes GBP2 albumen and/or Mdivi-1.
Further, medicine of the invention further includes pharmaceutically acceptable carrier, can be binding agent, disintegrant, profit
It is lubrication prescription, excipient, diluent, thickener, filler, surfactant, gelling agent, adjuvant, preservative, antioxidant, steady
Determine agent one or more kinds of mixing therein.
The medicine of the present invention mode such as can take orally, inject and giving to breast cancer sufferer body.
Present invention also offers the method for breast cancer of the diagnosis characterized by Drp1 overexpressions and/or up-regulation, the side
Method comprises the following steps：(i) expression of Drp1 in Patient Sample A is measured；(ii) by Drp1 expressions and normal specimens into
Row compares；(iii) Patient Sample A is diagnosed into phase relative to the Drp1 expressions of normal specimens and the positive or negative of breast cancer
Association, the breast cancer are related to Drp1 overexpressions and/or up-regulation.
Present invention also offers a kind of screening technique of breast cancer medicines, by adding testing drug to breast cancer cell
Afterwards or breast is being measured using the expression of some period measurement Drp1 after testing drug to breast cancer tumour model animal
The effect of gland cancer Drug inhibition breast cancer cell transfer；More specifically, when the expression of Drp1 is adding or applying test
When being reduced after medicine or recovering normal level, the medicine may be selected as the medicine for suppressing breast cancer cell transfer.
The beneficial effects of the invention are as follows：The present invention provides Drp1 treatment breast cancer is prepared in screening as drug targets
Application in medicine, have devised new breast cancer target treatment medicine accordingly, and provide new medicine for the treatment of breast cancer
Thing selects.And then can to breast cancer carry out specific aim it is stronger, reach more effective treatment, further reduce breast cancer deaths
Rate, there is very high clinical medicine to be worth.
Brief description of the drawings
Fig. 1 is the active function domain structure of Drp1 and the modification figure after transcription.
Fig. 2 is the structure of GBP1.Wherein, N-terminal GTPase areas (the FEBS J.2012 Jul that LG-Domain is GBP1；279
(14):2544-54)。
Fig. 3 is the invasion and attack figure that chondriokinesis protein D rp1 promotes breast cancer cell.Wherein, A. is in the strong breast of transfer ability
Drp1 expression quantity is high in adenocarcinoma cell MDA-MB-231 and MDA-MB-436, and Drp1 expression quantity in the weak MCF-7 of transfer ability
It is low；When the expression of B, C, D, E, F.RNAi Drp1, the transfer of breast cancer cell MDA-MB-231 and MDA-MB-436 and invasion and attack energy
Power is suppressed.(n=3, mean ± S.D.*, P<0.05).
Fig. 4 is to show that GBP2 and Drp1 has the result figure of interaction.Wherein, A. is by the GBP2 albumen with GST labels
It is incubated with MDA-MB-231 cell pyrolysis liquids, the albumen that GST-pull down get off runs PAGE glue and then silver staining as a result, black arrow
Head is the difference band for sending to Mass Spectrometric Identification；B. Mass Spectrometric Identification pull-down silver stainings band Protein Information.C.GST and GST-GBP2
Transfect MDA-MB-231, GST pull-down detections；D.Flag and Flag-GBP2 transfection MDA-MB-231, IP detections.
Fig. 5 is that Drp1 influences result figures of the GBP2 to the transfer ability of breast cancer cell.Wherein, A. is stablized with GBP2 and expressed
MDA-MB-231 and MDA-MB-436 be model, after Drp1shRNA, GFP-Drp1 recovers the expression (reference of Drp1
Oncogene(2013)32,4814–4824)；B.Drp1shRNA and the influence after recovery to breast cancer cell invasion ability, with
MDA-MB-231 is model.(n=3, mean ± S.D.*, P<0.05).C, D stablize the MDA-MB-231 of expression as mould using GBP2
Influence after type, Drp1siRNA and recovery to Mitochondrial Shape.(n=3, mean ± S.D.*, P<0.05).
Embodiment
Applications of the Drp1 as drug targets in the medicine that screening prepares treatment breast cancer.
Wherein, the medicine refers to the reagent that can suppress Drp1 expression.
Wherein, the reagent of the suppression Drp1 expression includes suppressing the reagent of Drp1 protein stabilities, suppresses Drp1 albumen
The reagent of activity, the reagent for suppressing Drp1 protein functions.
Wherein, the medicine includes the activator of GBP2 genes or GBP2 albumen.The activator can promote or strengthen
GBP2 or be related to GBP2 upstreams or downstream pathway material expression or activity.
Wherein, the medicine includes GBP2 albumen and/or Mdivi-1.Pharmaceutically acceptable carrier is further included, can be viscous
Tie agent, disintegrant, lubricant, excipient, diluent, thickener, filler, surfactant, gelling agent, adjuvant, anti-corrosion
Agent, antioxidant, stabilizer one or more kinds of mixing therein.
Said medicine the mode such as can take orally, inject and giving to breast cancer sufferer body.
A kind of method for diagnosing the breast cancer characterized by Drp1 overexpressions and/or up-regulation, comprises the following steps：(i)
Measure the expression of Drp1 in Patient Sample A；(ii) by Drp1 expressions compared with normal specimens；(iii) by patient
Sample is associated relative to the Drp1 expressions of normal specimens and the positive or negative diagnosis of breast cancer, the breast cancer with
Drp1 overexpressions and/or up-regulation are related.
A kind of screening technique of breast cancer medicines, by after testing drug is added to breast cancer cell or to breast cancer
Tumor model animal suppresses breast using the expression of some period measurement Drp1 after testing drug to measure breast cancer medicines
The effect of adenocarcinoma cell transfer；More specifically, when Drp1 expression add or apply testing drug after reduce or
When recovering normal level, the medicine may be selected as the medicine for suppressing breast cancer cell transfer.
Related experiment and data
Experimental model：Breast cancer cell line (high migration breast cancer cell, low migration breast cancer cell, normal breast
Cell), experimental animal mouse and clinical sample.
Experimental method：The research methods such as cell biology, molecular biology and biophysics (such as immunofluorescence, RNAi
Technology, Laser Scanning Confocal).
First, the relation of the expression and breast cancer cell transfer of GBP2 and Drp1 is studied
Expression experiments of the 1.GBP2 and Drp1 in breast cancer clinical sample
(1) detection of clinical samples
Using the sample storehouse of hospital, the clinical sample of collection patient's different times breast cancer and normal breast, by immune
The methods of groupization, analyzes the expression of in situ tumor, Tumor-surrounding tissue, normal galactophore tissue and GBP2 and Drp1 in metastatic tumour,
And the relation of corresponding markers for breast cancer (such as ER, PR, Her2 etc.) and follow-up clinical prognosis, the expression for being GBP2 and Drp1
Evidence is provided with the relation of tumor of breast transfer.Breast cancer tissue and GBP2 of the research at the same time with different markers for breast cancer,
The relation of the expression of Drp1.All samples, which are obtained and handled, meets Ethics Committee's requirement.The tissue of company is also utilized at the same time
Chip is verified.
(2) primary tumor cell detects
Using the sample storehouse of hospital, Primary breast cancer cell line is separately cultured from different breast cancer tissues.Detect it
The expression of GBP2 and Drp1.Breast cancer tissue and primary cell and GBP2 of the research at the same time with different markers for breast cancer,
The relation of the expression of Drp1.
(3) primary cell Tumor formation and metastatic are compared in animal model
Primary breast cancer cell line is inoculated with by mouse model, compares Tumor formation and metastatic difference and GBP2, Drp1
Relation between expression quantity, it is further provided GBP2, Drp1 and the whether relevant evidence of Metastasis in Breast Cancer.
Expression experiments of the 2.GBP2 and Drp1 in high/low metastatic potential breast carcinoma cell strain
Using RT-PCR, Western blot equimoleculars biology and cytobiology technology, detection MCF-7, T47D,
ZR-75-1 (the low migration breast cancer cell of ER+/PR+), MDA-MB-231, MDA-MB-436, SUM149 (ER-/PR-/Her2-
The strong three cloudy breast cancer cells of migration) and MCF10A (normal breast cell of ER-/PR-/Her2-) in GBP2 and Drp1
Expression.
3. adjust influence experiment of the expression of GBP2 to breast carcinoma cell strain transfer ability
With molecular biology and cell biology means, the expression of research up-regulation GBP2 or the expression of RNAi GBP2,
Influence to abilities such as above-mentioned breast cancer cell cell cycle, apoptosis, invasion and attack and migrations with different transfer abilities.
2nd, GBP2 regulates and controls breast cancer cell mitochondria dynamic change and the expression of Drp1
Experiment finds that Drp1 may be the downstream molecules of GBP2, influences the molecule machine of breast cancer cell transfer to understand GBP2
System, intends further investigation and illustrates the molecular mechanism and its biological significance of the interaction regulation and control of GBP2 and Drp1.
Influence of the expression of 1.GBP2 to mitochondria dynamic change
The research methods such as combination cell biology, molecular biology and biophysics (such as immunofluorescence, RNAi technology,
Laser Scanning Confocal etc.), analyze GBP2 protein expressions under low metastatic breast cancer cell (such as MCF-7), metastatic mammary gland
The distribution of subcellular fraction (such as mitochondria, lysosome or kytoplasm) level of cancer cell (such as MDA-MB-231 and MDAMB-436) with
Positioning；Raise the expression of RNAi GBP2, fusion-division dynamic change characterization of comparative studies and analysis cell Mitochondria and
The quantity of difference, such as mitochondria, length, thickness, with a distance from nucleus and fusion speed.
The molecular mechanism of 2.GBP2 and Drp1 interactions
(1) interaction of clear and definite GBP2 and Drp1
By the technology such as fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (Co-IP) further clear and definite GBP2 and
The effect of Drp1；By GST pull-down, IP, protein groups joint mass-spectrometric technique, examines whether have other albumen to be recruited ginseng
With the interaction of GBP2 and Drp1.
(2) site of GBP2 and Drp1 interactions is analyzed
By biochemistry and molecular biology rite-directed mutagenesis or clip (truncation) domain and combine immune
Co-precipitation and Laser confocal scanning light microscopy resonance energy transfer research method, determine the pass with Drp1 interactions in GBP2 molecules
Key amino acid or domain；Vice versa, by the way that (truncation) is mutated or truncated to Drp1, specify Drp1 with
Key amino acid site or domain when GBP2 interacts；If other albumen participate in the combination of the two, with same
The binding function domain of the clear and definite albumen composition in side.
(3) influences of the GBP2 to Drp1 Subcellular Localizations
Research showed in the past, and Drp1 promotes the division of mitochondria must be from endochylema indexing to mitochondria, therefore, we are into one
Whether and how the interaction of step research GBP2 and Drp1 influences this indexable process, and the line grain of Drp1 mediations is regulated and controled for GBP2
Body divides provides further experimental evidence with merging dynamic change.
(4) influences of the GBP2 to Drp1GTPase activity
The GTPase activity of Drp1 is most important to its function, directly affects the dynamic change of mitochondria.Therefore, detect
Change of the interaction of GBP2 and Drp1 to the GTPase binding abilities and hydrolysis GTP activity of Drp1, to GBP2's and Drp1
Interaction provides the experimental evidence in terms of zymetology.
(5) whether GBP2 regulates and controls the posttranscriptional modification of Drp1
Preliminary experiment shows expression of the GBP2 expression without influence Drp1 in MDA-MB-231 and MDA-MB-436.In this way,
GBP2 may mediate the expression of Drp1 not over transcriptional level.But GBP2 is not excluded for by posttranscriptional modification to regulate and control
The activity of Drp1, after all Drp1 modified after being transcribed its activity (BBA-Biomembranes, (2013) 1833:1256–
1268).And GBP2 is also GTPase in itself.
Mass-spectrometric technique is combined by protein science, detects the posttranscriptional modification with Drp1, such as phosphorylation, sumoization etc.；Point
Analysing GBP2 or other which albumen influences the modification of Drp1.Pay close attention to some kinases, ERK, PKC etc..On this basis,
Disturbed with inhibitor or RNA, be overexpressed albumen, rite-directed mutagenesis, dominant negative mutations (dominant negative
The effect of these factor pairs of technical research Drp1 such as mutation).And then by these regulatory molecules of the technical research such as Co-IP and
The relation of GBP2, examines whether to participate in the effect of GBP2 and Drp1.
(6) influences of the GBP2 and Drp1 to mitochondria dynamic change and function
By cell biology means, study the heterogenous expression in breast cancer cell and lose and Drp1 interaction sites
Influence of the GBP2 albumen to mitochondria dynamic change, specifying GBP2 albumen, whether the logical interaction with Drp1 participates in adjusting line grain
The dynamic change of body.
(7) influence of the interaction of GBP2 and Drp1 to mitochondria dynamic change is checked
One section of synthesis imports breast cancer cell specifically for the small peptide for checking GBP2 and Drp1 interaction, research GBP2 with
The feature of mitochondria dynamic change in the case of Drp1 interactions are impacted.These experimental results will be to GBP2 and Drp1 phase interactions
Experiment on molecular level is provided with the accurate molecular mechanism for participating in breast cancer cell mitochondrial fusion-division dynamic change adjusting
Evidence.
3rd, effect of the GBP2 and Drp1 interactions in regulation and control breast cancer cell transfer
Previous experiments researches show that：In the strong breast cancer cell of metastatic, the up-regulated expression of chondriokinesis protein D rp1,
The expression for suppressing Drp1 then suppresses the transfer of breast cancer cell.Therefore, in above-mentioned breast cancer cell GBP2 expression and its and Drp1
On the basis of the results such as interaction, it is clear that how further research GBP2 and Drp1 interactions, which influence breast cancer cell, turns
What is moved is to illustrate it because of-the certainty of fruit relation.
The influence that 1.GBP2 and Drp1 interacts to breast cancer cell skeleton and pseudopodial movement
(1) influence that cytoskeletal filament microtubule arrays are changed
The microfilament microtubules of cytoskeleton and the movement of cell are closely related.Intend the method by cell biology, compare and grind
Study carefully GBP2 express or reduce in the case of, and by check GBP2-Drp1 interaction under the conditions of, shown with laser co-focusing
Micro mirror observation such as metastatic MDA-MB-231 and MDA-MB-436 breast cancer cell, low metastatic breast cancer cell MCF-7 microfilaments
Arrangement with micro-pipe whether or have which kind of change.
(2) distribution to mitochondria and the influence of function
With the observation of the means such as laser confocal microscope in the case where promoting breast cancer cell jump condition, GBP2 expression or reduction
In the case of, under the conditions of checking GBP2-Drp1 interactions, the pseudopodium region mitochondria of MDA-MB-231 and MDA-MB-436 cells
Form and changes in distribution, as the quantity of mitochondria, size, distribution taxis and in cell pseudopodium region film potential, ROS water
Change of gentle ATP synthesis etc..Influence mitochondria is interacted in cell by these researchs, the expression of GBP2 and its with Drp1
With the aggregation of pseudopodium, the generation of ROS, the reduction etc. of film potential and ATP synthesis provides experimental evidence, to disclosing how GBP2 participates in
The molecular mechanism that breast cancer carefully shifts regulation and control provides more full and accurate experimental evidence from subcellsular level and biochemical change.
2.Drp1 suppresses GBP2 the influence of breast cancer cell transfer
In view of the expression of up-regulation Drp1 can promote the transfer of breast cancer cell, intend attempting under GBP2 existence conditions, such as
Whether the overexpression Drp1 in MDA-MB-231 and MDA-MB-436, Metastasis in Breast Cancer ability caused by GBP2 can be reversed by seeing
Decline, this will from another side GBP2 and Drp1 interact participate in breast cancer cell transfer adjusting supply a clear proof.Meanwhile
By cell biology means, the dynamic for specifying the whether logical interaction participation adjusting mitochondria with Drp1 of GBP2 albumen becomes
Change.
3. the influence that the interaction for checking GBP2 and Drp1 is shifted to suppressing breast cancer cell
Intend the heterogenous expression in breast cancer cell and lose the GBP2 mutains with Drp1 interaction sites to breast cancer
Transcellular influence；Or one section of synthesis imports breast cancer cell specifically for the small peptide for checking GBP2 and Drp1 interactions,
Study the influence to breast cancer cell transfer ability.
4th, the effect of GBP2 regulation and control Metastasis in Breast Cancer is studied in animal model
1.GBP2 is on breast cancer Tumor formation and metastatic influence
By expressing or disturbing expression of the GBP2 in breast cancer cell, connect using these processed tumor cell lines
Kind mouse, the growth and transfer of tumour can be influenced by examining whether GBP2 expression changes in mouse model.Comparison sheet reaches external source
The neoplasm lung metastasis knot that the breast cancer cell (such as MDA-MB-231) with expressing empty carrier of GBP2 albumen is formed in Mice Body
Joint number, the transfer for regulating and controlling breast cancer cell in animal integral level for GBP2 albumen provide experimental evidence.
2. regulate and control Drp1 expression to breast cancer Tumor formation and metastatic influence
Compare the neoplasm lung metastasis tubercle number that the breast cancer cell of RNAi Drp1 expression is formed in Mice Body, in view of on
The transfer of breast cancer cell can be promoted by adjusting the expression of Drp1, intend attempting under GBP2 existence conditions, such as MDA-MB-231 with
Overexpression Drp1 in MDA-MB-436, sees the decline that whether can reverse Metastasis in Breast Cancer ability caused by GBP2, so that
Transfer in animal integral level to GBP2 and Drp1 regulation and control breast cancer cells provides strong experimental evidence.
5th, the upstream gene that regulation and control GBP2 is expressed in breast cancer cell is explored
It is negatively correlated that experiment shows that expression of the GBP2 and Drp1 in breast cancer is presented.In this way, it is presumed that whether shifting
Property strong mammary glandular cell in, exactly because GBP2 low expressions so that the chondriokinesis activity enhancing for causing Drp1 to mediate so that
Promote the transfer of breast cancer.How the expression that we intend studying GBP2 is adjusted in breast cancer.
With the promoter of bioinformatic analysis GBP2
The transcriptional control of GBP2 is analyzed, is detected with luciferase combination Real-Time Fluorescent Quantitative PCR Technique.
2. regulate and control other molecules of GBP2 expression
Using RNAi screenings come determine other molecules of regulation and control GBP2 expression (Neurosci Lett, (2013) 554:99–
104).We can use corresponding RNAi libraries, particularly some kinases libraries, after transfection library enters tumour cell, inspection
The differential expression of GBP2 is surveyed, then by Co-IP, with inhibitor, is overexpressed albumen, dominant negative mutations (dominant
Negative mutation) etc. biochemical and molecular biology method determine the expression of which molecule modulates GBP2.
3. the miRNA of screening regulation and control GBP2
Moreover, GBP2 have been observed that be miR-433 target for modulation (Leukemia, 27 (2013):344–352).Examining
This miRNA is tested whether while breast cancer plays regulation and control GBP2 expression.Other miRNA are also possible to the table of regulation and control GBP2
Reach, plan may act on the 3 '-UTR's of GBP2 by computerized algorithm screening (such as TargetScan, MicroInspector)
miRNA。
By the studies above, the medicine that we can design new effective treatment breast cancer for later use GBP2 provides in fact
Verify evidence.Follow-up research we can design the consistent peptide or small of GBP2 binding functions domain activity
This experiment mainly carrys out checking research in terms of In vitro cell experiment, interior animal experiment and tumor tissues detection three
Content.
Main experimental methods：
1. the research of breast cancer cell transfer
(1) cell scratch experiment
Cultivate to be overexpressed GBP2 or checked GBP2 and Drp1 in 6 well culture plates for spreading coverslip in advance and interact
Scratch experiment is carried out etc. the MDA-MB-231 cells before and after treatment conditions.It is small in 12 or 24 using cut healing as standard
When sampling take pictures, gained picture is analyzed and processed using Image-pro plus.Reflect that cell turns according to the change of scratch width
The change of shifting ability.
(2) breast cancer cell migration detection
To being overexpressed GBP2 or checking the MDA-MB-231 breast cancer before and after the treatment conditions such as GBP2 and Drp1 interaction
Cell, is detected with Transwell technologies, in the case where optics is just putting microscope (Leica, Germany) to quilt on Transwell
The cell of dyeing is taken pictures, and image carries out cell count by Image-pro Plus softwares, according to " culture under Transwell films
The change of the cell number of base " side, detects breast cancer cell transfer ability.
(3) breast cancer cell invasion detects
Transwell needs to carry out advance processing：The Matrigel of certain volume is taken to be diluted to final concentration with DMEM
The Matrigel solution of 0.3mg/ml, 100 μ l are added into each Transwell, 37 DEG C place 3 it is small when to ensure
Matrigel fully solidifies, and sucks the step of being detected by above-mentioned migration after liquid unnecessary in Transwell and carries out.
2. breast cancer cell mitochondrial fusion-division Dynamic change characteristics analysis and functional examination
(1) mitochondrial fusion-division Dynamic change characteristics analysis
1. Mitochondrial Shape observation of characteristics
The metamorphosis of mitochondria is also to reflect that it merges-divide one of index of behavioral characteristics change.Utilize tag line
The spy agent mitotracker of plastochondria, respectively to low metastatic breast cancer cell MCF-7 before and after the processing and metastatic breast cancer
The cell that cell MDA-MB-231 or MDA-MB-436 are dyed, each mitochondria shot at least 30 cells, uses Image
The quantitative analysis of J softwares simultaneously compares the mitochondrial morphology index between them (such as length, thickness and the distance of freestone) change
Or difference.
2. the measure of mitochondrial fusion speed
The above-mentioned several identical mammary gland for being quantitative determined and being compared before and after the processing using photo-activation technologies are thin
The fusion speed of born of the same parents' Mitochondria.We will contain the matter for being capable of amalgamation and expression PAGFP and mitochondrial matrix localization signal sequence
Grain Mito-PAGFP transfects the above-mentioned breast cancer cell with different transfer abilities, and mitochondria is excited with confocal laser microscope
On particular area (0.5 μm -0.75 μm of diameter), detect certain time in the diluted speed of fluorescence, melt so as to obtain mitochondria
The speed of conjunction.
(2) functional examination of mitochondria
1. mitochondrial respiratory rate determination
Using hippocampus mammalian cell mitochondria energy measuring device (Seahorse XF24-3), analyze and handling or hindering
Hold back low metastatic breast cancer cell MCF-7 and metastatic breast cancer cell MDA-MB- under the conditions of GBP2 and Drp1 interacts
The aerobic respiration speed of 231 or MDA-MB-436 Mitochondrias and the mitochondria limit respiratory rate under uncoupler effect
Change.
2. the measure of intracellular ATP
After above-mentioned several cells are cracked completely respectively, its a certain amount of albumen is taken to be added in 96 orifice plates,
Luminometer measures cell ATP concentration, is represented with nmol/mg albumen.
3. the measure of mitochondrial membrane potential in anoxic
With detection mitochondrial membrane potential molecular probe, such as DiOC6 (3) (3-Dihexy loxacarbocyanine
) or JC-1 (Tetrechlorotetraethylbenzimidazolcarbocyanine iodide) and other conditions iodide
After the cell incubation of processing, with flow cytometer (Becton Dickinson) detection film potential change, or with TMRM observation and
Compare cell fluorescence intensity change, and analysis comparison is carried out to result with IPP softwares.
4. the measure of cell mitochondrial ROS
Take CM-H2DCFDA (6-chloromethyl-2', 7'- of the above-mentioned each cell suspension respectively with detectable ROS changes
Dichlorodihydrofluorescein diacetate, acetylester) visit agent be incubated altogether, carried out with flow cytometer
Measure, its result is calculated with Cellquest softwares.
The experiment of 3.GBP2-Drp1 interactions
(1) measure of the domain of GBP2-Drp1 interactions or key amino acid
1. using the functional domain in the method analysis GBP2 or Drp1 albumen of bioinformatics, molecular biosciences is recycled
Learn to do section and obtain the different eukaryon expression plasmids for blocking (deletion) body gene of GBP2, turn with the Drp1 with full-length gene
Contaminate in cell, with the method for co-immunoprecipitation or laser energy resonance transfer, find in GBP2 albumen and interact with Drp1
Domain.Equally, with the above-mentioned domain blocked in body method measure Drp1 albumen with GBP2 interactions, this is checked for design
The small peptide or key amino acid of interaction between two albumen provide experiment basis.
2. it may determine phase in the domain to interact using the method analysis GBP2 albumen of bioinformatics with Drp1
The key amino acid of interaction, mutant is obtained using molecular biology method, is turned with co-immunoprecipitation or laser energy resonance
The method of shifting studies it.
(2) measure of the GBP2-Drp1 protein-interactings to the GTPase activity influences of Drp1
GBP2 albumen is expressed respectively with Drp1 albumen using Biochemistry and Molecular Biology technology, is purified, by Drp1
It is incubated with [α -32P] GTP, with GTPase before and after thin-layer chromatography and the above-mentioned sample addition GBP2 albumen of autoradiographic technique detection
Whether the difference of hydrolysing activity, determining the interaction of Drp1 and GBP2 influences its GTPase hydrolysing activity.
(3) posttranscriptional modification of Drp1
Combine mass-spectrometric technique using protein science, detect turn of the Drp1 after GBP2 is raised or lowered in tumour cell
Modified after record, such as phosphorylation, sumoization etc.；On this basis, disturbed with inhibitor or RNA, be overexpressed albumen, fixed point is prominent
Become, the effect of these factor pairs of technical research Drp1 such as dominant negative mutations.
The research experiment of 4.GBP2 expression
Using 3 '-UTR of the computer analysis and GBP2 miRNA combined and synthesize corresponding miRNA.Build GBP2's
The luciferase carrier and miRNA cotransfections of 3 '-UTR, detects the activity of luciferase, determines miRNA and the knot in 3 '-UTR areas
Close；And then by quantitative PCR, the methods of Western, detects the change of protein expression.
Screened for RNAi, using RNAi libraries, transfect siRNA therein and enter on 96 orifice plates the cell cultivated, detection
The wherein expression of the siRNA factors and the expression of GBP2, and then utilize and be overexpressed the skills such as albumen, rite-directed mutagenesis, dominant negative mutations
Art research.
5. Microfilaments In Cells reset the measure adjusted with the change of cell pseudopodium area mitochondrial membrane potential
(1) change of cytoskeletal filament microtubule arrays is detected
Locate in low metastatic breast cancer cell MCF-7 and metastatic breast cancer cell MDA-MB-231 or MDA-MB-436
Reason is front and rear or before and after checking the condition of GBP2 and Drp1 interactions, with the phalloidine flag F-actin with fluorescent marker or
Actin or tubulin carrier of person's cotransfection with GFP, observes and is promoting " conditioned medium " (NIH- of transfer
3T3conditinal medium) induction under, the difference of change or the rearrangement of actin and microtubule.
(2) the mitochondrial membrane potential measure of breast cancer cell pseudopodium part
In through being overexpressed GBP2 and checking the above-mentioned breast cancer cell of GBP2 and Drp1 interactions small peptide before and after the processing,
Dyed with TMRM, observed by living cells, measurement pseudopodium region mitochondria fluorescence intensity and the region endoplasm fluorescence intensity
Ratio, the mitochondrial membrane potential for calculating pseudopodium region change so as to reflect that pseudopodium region ATP is synthesized indirectly.
(3) use FCCP, GTP- γ S etc. to handle above-mentioned breast cancer cell, with immunofluorescence method detect its Mitochondrial Shape,
The change of Microfilaments In Cells, micro-pipe rearrangement etc..
6. the collection of breast cancer sample and the culture of primary tumor cell
(1) clinical samples are collected, pathological analysis, the trimming of sample, are preserved, fixed, embedding, immunohistochemical staining,
Correlation/conspicuousness statistical analysis：The collection of all clinical tumor samples, and pathological analysis are carried out in the court, all samples
Pass through stringent classification, sample is divided into two in principle, and a rapid to freeze and liquid nitrogen, the other half is fixed, embedding and
Immunohistochemical analysis.[we are generally according to 9 points of systems for immunohistochemical staining result：Staining power (is divided into 0-3 grade:Negative=
0, it is weak=1, in=2, strong=3) × cell dyeing very (is equally divided into 0-3 grades：0,1=1-25%, 2=26-50%, 3=
51-100%)] judged by two veteran Lab Technician, and carry out the statisticals such as final correlation and conspicuousness
Analysis.
(2) culture of Primary breast cancerous swelling oncocyte
Primary tumor cell presses the method (Breast Cancer Res, 16 (2014)) of document report, utilizes tumour cell
Separating kit carries out.The methods of obtained Primary breast cancer cell can be used immunohistochemistry, real-time quantitative PCR detects ER, PR
Deng the expression of the related tumor markers such as markers for breast cancer and Ki-67.
7. zoopery
(1) breast cancer orthotopic transplanting animal model
It is inoculated into SCID mice mammary fat pad and forms primary tumor, observe its one-tenth knurl ability, and at 30 days or so, dissection was small
Mouse, the protein staining (expression quantity) such as analysis in situ tumor growth curve, histopathological analysis, GBP2, Drp1, ER, PR, Her2,
The quantity of Nodules in lung and the difference of size etc..
(2) breast carcinoma cell strain animal model
The expression of expression or interference GBP2 in breast cancer cell, it is small using these processed tumor cell line inoculations
Mouse, the breast cancer cell (such as MDA-MB-231) with expressing empty carrier of comparison sheet up to exogenous GB P2 albumen are formed in Mice Body
Neoplasm lung metastasis tubercle number, while the tumour lung that the breast cancer cell for comparing RNAi Drp1 expression is formed in Mice Body turns
Move tubercle number, tumor growth curve, histopathological analysis, microvessel density analysis, apoptosis (cleaved caspase-3 dyeing
Deng), protein staining such as GBP2, Drp1 (expression quantity) etc..
Experimental studies results：
The research of Oncogene was displayed that in the strong breast cancer cell of metastatic in the past, chondriokinesis protein D rp1 expression
Up-regulation, Mitochondrial Shape tend to division (Oncogene, 32 (2013):4814–4824).And confirm that Drp1 mediations breast cancer is thin
The transfer of born of the same parents.Applicant also found that in the strong breast cancer cell of metastatic, Drp1 up-regulated expressions.And the result of applicant is also shown
Show that the expression for suppressing Drp1 can suppress the invasion and attack (Fig. 3) of breast cancer cell.
Researches show that the GBP2 albumen of GST labels gets off with the incubation of MDA-MB-231 cell pyrolysis liquids, GST-pull down
Albumen Mass Spectrometer Method, it is found that Drp1 albumen and GBP2 exist and interact.Moreover, the albumen that GST-pull down get off
With antibody test, display that Drp1 in the albumen of GST-GBP2pull down.Further, we transfect Flag-GBP2 entrance
Cell, Co-IP display that GBP2 and Drp1 interactions (Fig. 4).These experimental studies show that Drp1 and GBP2 being capable of phase interaction
With.
For GBP2 after MDA-MB-231 overexpressions, the mitochondria of cell tends to fusion (Fig. 5), and MDA-MB-231 itself and
The mitochondria of MDA-MB-436 is to tend to division (Oncogene, 32 (2013):4814–4824).We are in GBP2 expression
The expression of Drp1 is lowered in cell, finding the downward of Drp1 substantially makes the mitochondria of breast cancer cell more tend to merge.Drp1
The recovery of expression then makes breast cancer mitochondria recover normal and tends to splitting status.Moreover, the recovery of Drp1 expression is remarkably reinforced
The invasive ability of GBP2 expression breast cancer cells.
Test result indicates that GBP2 and Drp1 expression quantity in the strong breast cancer cell of metastatic is opposite.GBP2 is overexpressed
Inhibit the transfer ability of metastatic breast cancer cell MDA-MB-231 or MDA-MB-436.Drp1 is the new target of GBP2 effects
Point, GBP2 causes the division of mitochondria-merge dynamic change by the interaction with Drp1 albumen, so that it is thin to participate in breast cancer
Dysuria with lower abdominal colic transposition section.
Claims (8)
- Applications of the 1.Drp1 as drug targets in the medicine that screening prepares treatment breast cancer.
- 2. application according to claim 1, it is characterised in that the medicine refers to the reagent that can suppress Drp1 expression.
- 3. application according to claim 2, it is characterised in that the medicine includes the activation of GBP2 genes or GBP2 albumen Agent.
- 4. application according to claim 3, it is characterised in that the activator can promote or strengthen GBP2 or be related to The expression of the material of GBP2 upstreams or downstream pathway or activity.
- 5. application according to claim 2, it is characterised in that the medicine includes GBP2 albumen and/or Mdivi-1.
- 6. application according to claim 5, it is characterised in that the medicine includes GBP2 albumen and Mdivi-1.
- 7. diagnose the method for the breast cancer characterized by Drp1 overexpressions and/or up-regulation, it is characterised in that the described method includes Following steps：(i) expression of Drp1 in Patient Sample A is measured；(ii) Drp1 expressions and normal specimens are compared Compared with；It is (iii) Patient Sample A is associated relative to the Drp1 expressions of normal specimens and the positive or negative diagnosis of breast cancer, The breast cancer is related to Drp1 overexpressions and/or up-regulation.
- A kind of 8. screening technique of breast cancer medicines, it is characterised in that by breast cancer cell add testing drug after or Breast cancer is being measured using the expression of some period measurement Drp1 after testing drug to breast cancer tumour model animal The effect of Drug inhibition breast cancer cell transfer；More specifically, when the expression of Drp1 is in addition or using testing drug When reducing afterwards or recovering normal level, the medicine may be selected as the medicine for suppressing breast cancer cell transfer.
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Cited By (2)
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| CN113817776A (en) * | 2021-10-25 | 2021-12-21 | 中国人民解放军军事科学院军事医学研究院 | Application of GBP2 in regulating and controlling mesenchymal stem cell osteogenic differentiation |
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| CN109596585A (en) * | 2018-12-03 | 2019-04-09 | 贵州大学 | A kind of method of new high flux screening lysosome |
| CN113817776A (en) * | 2021-10-25 | 2021-12-21 | 中国人民解放军军事科学院军事医学研究院 | Application of GBP2 in regulating and controlling mesenchymal stem cell osteogenic differentiation |
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