CN106492191A - Application of the WWC3 protein moleculars in antitumor drug is prepared - Google Patents
Application of the WWC3 protein moleculars in antitumor drug is prepared Download PDFInfo
- Publication number
- CN106492191A CN106492191A CN201611124240.8A CN201611124240A CN106492191A CN 106492191 A CN106492191 A CN 106492191A CN 201611124240 A CN201611124240 A CN 201611124240A CN 106492191 A CN106492191 A CN 106492191A
- Authority
- CN
- China
- Prior art keywords
- wwc3
- dvl2
- domains
- lats1
- protein
- 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.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
Abstract
The invention belongs to biological technical field, the molecular mechanism that WWC3 protein moleculars suppress Wnt and activation Hippo signal paths is related generally to, and sought protection in the application for prepare antitumor drug using the principle of this mechanism.WWC3 protein moleculars are the type albumen containing WW domains and C2 domains；WWC3 is interacted by the PY motifs of WW domains and Dvl2, the PDZ domain interactions of ADDV domains and Dvl2 can also be passed through, hinder CK1 δ/phosphorylation of the ε kinases to DVLs, promote the degraded of Wnt path effect protein molecule β catenin, reduce β catenin and enter core, play the effect for suppressing Wnt pathway activities.WWC3 is interacted with Hippo paths central authorities kinases LATS1 by the WW domains of its own, is promoted LATS1 phosphorylations, and then is made Hippo effect protein YAP be stranded in endochylema, plays the effect for promoting Hippo pathway activities.The combination of LATS1 and WWC3 has competitiveness with Dvl2.
Description
Technical field
The invention belongs to biological technical field, is related generally to the mechanism of action of WWC3 protein moleculars and is existed using this mechanism
Prepare the application in terms of antitumor drug.
Background technology
Wnt signal paths both participate in biological fetal development with Hippo signal paths, and histoorgan is formed, the propagation of cell,
Differentiation and the inactivations of important physiological process, the abnormal activation of Wnt paths and Hippo paths such as apoptosis and the occurrence and development of tumor
There is close contact, disclose their internal relation and important theoretical and experiment basis will be provided for preparing antitumor drug.
The Wnt pathway activities of exception are usually present in tumor cell.The core factor of Wnt paths is beta-catenin
（β-catenin）, when classical Wnt path signal is activated, CK1 δ/ε activation, CK1 δ/ε phosphorylations Dishevelled of activation
（Dvls）, ultimately result in β-catenin in endochylema and do not accumulated by ubiquitination, enter the β-catenin after core and combined simultaneously with Tcf-4
The transcriptional activities such as the target gene c-myc of activation Wnt paths, CyclinD1, MMP7, promote propagation and the invasion and attack of tumor cell.When
When β-catenin are degraded by proteasome or can not enter core, then Wnt pathway activities are interrupted.
The propagation of Hippo paths and tumor cell, invasion and attack and the relation attention for shifting.The molecular composition bag of the path
Include：Molecules upstream（Fat, NF2, WWC1, AMOT albumen etc.）, central kinase complex（MST1/2-WW45-LATS1/2-MOB）
With downstream effect molecule（YAP albumen）Deng.Then suppress propagation and the invasion and attack of tumor cell when Hippo paths are activated, and work as
When Hippo pathway activities are suppressed, dephosphorylized YAP is then combined with TEAD (coactivator altogether) after entering core, starts which
Target gene（CTGF and CyclinE etc.）Transcriptional activity, promote tumor cell propagation and invasion and attack.
Thus, it is found that Wnt signal paths activity can be suppressed and can activate the factor of Hippo pathway activities, and disclose which
The molecular mechanism of effect, it will provide preferable action target spot and important theory for preparing anti-tumor drug or targeted drug
Basis.
WWC3 is one of mankind's WWCs protein families (WWC1, WWC2, WWC3), and WWCs contains：Nearly N-terminal pair " WW " structure
Domain（Can interact with PPxY motifs, participate in the activation of Hippo paths）, middle body is the C2 domains related to film, nearly C
Hold as aPKC binding sites（Participate in the regulation of cell polarity）, C-terminal is ADDV domains（PDZ binding motif）.Mesh
Before, the effect about WWCs families in human tumor is only limitted to WWC1 and participates in the research of Hippo paths.And WWC2 and WWC3
Whether have an impact with the relation of human tumor and to Wnt signal paths activity etc. and to have no report.
Content of the invention
Goal of the invention：Object of the present invention is to provide WWC3 protein moleculars are swashed while Wnt signal paths are suppressed
The effect of Hippo signal paths living and molecular mechanism, and the method for proving to there are these mechanism, are to be prepared using these mechanism
Antitumor（Targeting）Medicine provides foundation.
Technical scheme：
Application of the WWC3 protein moleculars in antitumor drug is prepared, it is characterised in that：The WWC3 protein moleculars are containing WW
Domain and C2 domainsType albumen；
The WWC3 suppresses Wnt activity by DVLs and activates Hippo pathway activities by LATS1.
Application of the described WWC3 protein moleculars in antitumor drug is prepared, it is characterised in that：The WWC3 albumen point
Son is interacted with important upstream protein molecule DVLs in Wnt paths by its own WW domains and ADDV domains,
The interaction hinders CK1 δ/phosphorylation of the ε kinases to DVLs, promotes the drop of Wnt path effect protein molecule β-catenin
Solution, reduces β-catenin and enters core, play the effect for suppressing Wnt pathway activities.
Application of the described WWC3 protein moleculars in antitumor drug is prepared, it is characterised in that：Mutation using WWC3
Body and Dvl2 spliceosomes, demonstrate WWC3 by WW domains and the PY motif phase interactions of Dvl2 using the method for co-immunoprecipitation
With, can also pass through the PDZ domain interactions of ADDV domains and Dvl2, performance suppresses the effect of Wnt pathway activities.
Application of the described WWC3 protein moleculars in antitumor drug is prepared, it is characterised in that：
The mutant of the WWC3 includes WWC3- Δ WW, WWC3- Δ ADDV, WWC3- Δ WW＆ADDV；
The Dvl2 spliceosomes include Dvl2- Δ PDZ, Dvl2- Δ PY, Dvl2- Δ PDZ＆PY.
Application of the described WWC3 protein moleculars in antitumor drug is prepared, it is characterised in that：The WWC3 passes through which
The WW domains of itself are interacted with Hippo paths central authorities kinases LATS1, are promoted LATS1 phosphorylations, and then are imitated Hippo
Answer protein Y AP to be stranded in endochylema, play the effect for promoting Hippo pathway activities.
Application of the described WWC3 protein moleculars in antitumor drug is prepared, it is characterised in that：WWC3 and LATS1 swashs
Enzyme interacting has simultaneously played the function of activating Hippo pathway activities, and lacks the WWC3 of WW domains then without this function；
Increase the expression of LATS1, reduce can the binding capacity of WWC3 and Dvl2；Reduce LATS1 expression, then WWC3 with
The binding capacity of Dvl2 increases, i.e. the combination of LATS1 and WWC3 has competitiveness with Dvl2.
Advantages of the present invention：
The invention provides a kind of application of WWC3 protein moleculars.The present invention is tried based on molecular biology and cell function
Test, design is rigorous, verity is strong, and with certain novelty, WWC3 is suppressed by affecting Wnt and Hippo pathway activities
The propagation of lung carcinoma cell, invasive ability, and verified there is the application prospect of higher translational medicine, is in nude mice model
Clinical development target therapeutic agent provides foundation.
Description of the drawings
Comparative result figure (* * * Ps of the Fig. 1 for impacts of the overexpression WWC3 to lung carcinoma cell Colony forming ability<0.001)；
Fig. 2 is comparative result figure (the * * * P for knocking out impacts of the WWC3 to lung carcinoma cell Colony forming ability<0.001)；
Comparative result figure (* * Ps of the Fig. 3 for impacts of the overexpression WWC3 to lung carcinoma cell invasion ability<0.01)；
Fig. 4 is comparative result figure (the * * P for knocking out impacts of the WWC3 to lung carcinoma cell invasion ability<0.01)；
Fig. 5 is comparative result figure (the * * P that mtt assay investigates impact of the expression of double regulation control WWC3 to proliferation of lung cancer cells ability<
0.01)；
Fig. 6 be overexpression WWC3 to transplanted tumor in nude mice Forming ability（Transplanted tumor volume and weight）Impact comparative result figure
(***P<0.001, * * P<0.01, n=5)；
Fig. 7 is to knock out WWC3 to transplanted tumor in nude mice Forming ability（Transplanted tumor volume and weight）Impact comparative result figure (* * P
<0.01, n=5)；
Fig. 8 is that protein immunization imprinting investigation WWC3 is expressed in six kinds of lung cancer cell lines and normal bronchial epithelial cell HBE
Comparative result figure；
Fig. 9 is comparative result figure (the * * P that luciferase reporter gene investigates that double regulation control WWC3 is affected on Wnt pathway activities<
0.01,*P<0.05)；
Figure 10 is comparative result figure (the * * P that luciferase reporter gene investigates that double regulation control WWC3 is affected on Hippo pathway activities
<0.01,***P<0.001)；
Figure 11 is that protein immunization imprinting investigates double regulation control WWC3 to Wnt paths and the impact of Hippo path target gene proteins level
Comparative result figure;
Figure 12 is the result that real-time quantitative PCR investigates that double regulation control WWC3 is affected with Hippo path target genes mRNA level in-site on Wnt
Comparison diagram (* P<0.05);
Figure 13 is the protein blot figure that co-immunoprecipitation investigates that WWC3 and DVL2 has interaction;
Figure 14 is the structural modelss figure of WWC3 wild plasmids and mutant plasmids;
Figure 15 is the structural modelss figure of DVL2 wild plasmids and mutant plasmids;
Figure 16 is the Diagnosis of Sghistosomiasis that co-immunoprecipitation investigates that WWC3 is interacted with DVL2 by its WW domain and ADDV domains
Note figure;
Figure 17 is the immunoblotting analysis that co-immunoprecipitation investigates that DVL2 is interacted with WWC3 by its PDZ domain and PY motifs
Figure;
Figure 18 is that the WW domains and ADDV domains of co-immunoprecipitation investigation WWC3 are tied with the PY motifs and ADDV of DVL2 respectively
The immunoblotting analysis figure that structure domain interacts;
Figure 19 is the comparative result figure that co-immunoprecipitation investigates that double regulation control WWC3 is affected on DVL2 phosphorylation levels;
Figure 20 is that luciferase reporter gene investigates common knockout WWC3 and impact (* * * Ps of the CK1 δ/ε to Wnt pathway activities<
0.001);
Figure 21,22 is that protein immunization imprinting is investigated on the basis of WWC3 is knocked out, and suppresses CK1 δ/ε activity or knocks out CK1 δ/ε's
Expression, the change comparison diagram of DVL2 phosphorylation levels.
Figure 23 is the change that co-immunoprecipitation investigates CK1 δ/ε and DVL2 binding capacities after gradient transfection WWC3；Gradient is transfected
The change comparison diagram of WWC3 and DVL2 binding capacities after CK1 δ/ε.
Figure 24 is the change that luciferase reporter gene investigates Wnt pathway activities after H1299 transfects WWC3 and mutant
Comparison diagram (* * P<0.01,*P<0.05);
Figure 25 is that protein immunization imprinting investigates DVL phosphorylation levels and Wnt path target bases after H1299 transfects WWC3 and mutant
Because of the change comparison diagram that expresses
Figure 26 is that protein immunization imprinting investigates the expression of double regulation control WWC3 to Hippo paths major protein LATS and YAP phosphoric acid
The comparative result figure of the impact of change level
Figure 27 is that the change of luciferase reporter gene investigation Hippo pathway activities after H1299 transfects WWC3 and mutant is right
Than scheming (* P<0.05);
Figure 28 is the immunoblotting analysis figure that co-immunoprecipitation investigates that WWC3 and LATS1 has interaction
Figure 29 is the comparative result figure that WWC3 is changed with LATS1 binding capacities after co-immunoprecipitation investigates overexpression DVL2
Figure 30 is the comparative result figure that WWC3 is changed with LATS1 binding capacities after co-immunoprecipitation investigates knockout DVL2
Figure 31 is the comparative result figure that WWC3 is changed with DVL2 binding capacities after co-immunoprecipitation investigates overexpression LATS1
Figure 32 is the comparative result figure that WWC3 is changed with DVL2 binding capacities after co-immunoprecipitation investigates knockout LATS1
Figure 33 is that protein immunization imprinting is investigated on the basis of overexpression WWC3, transfects DVL2, LATS1 and YAP phosphorylation levels
The comparative result figure of change
Figure 34 is that protein immunization imprinting is investigated on the basis of overexpression WWC3, knocks out DVL2, LATS1 and YAP phosphorylation levels
The comparative result figure of change
Figure 35 is investigated on the basis of overexpression WWC3 for protein immunization imprinting, transfects DVL2 and mutant, LATS1 and YAP
The comparative result figure that phosphorylation level changes
Specific embodiment：
1. WWC3 has the propagation for suppressing tumor cell and invasion and attack effect low expression in lung carcinoma cell
（1）In vitro with experiment in vivo in, WWC3 can suppress propagation and the invasion and attack of tumor cell
In order to verify the impact of WWC3 biological functions in vivo and in vitro, the H1299 lung cancer cell lines of WWC3 low expressions are selected, turned
Dye WWC3 overexpression plasmids (G418 screenings)；In the A549 cells of relatively high expression WWC3, (G418 is sieved transfection shRNA- WWC3
Choosing), cell function experiment is carried out after screening.
(1-1）Colony forming is tested：Matched group and transfection (knockout) group cell are inoculated into 6cm2(1000 in culture dish
Individual/ware), after 1640 culture medium culturings 12 days, cleaned with phosphate buffer twice, ice methanol fixes 20min, then Gimsa
Dyeing 10min, last count number.Experiment in triplicate, is averaged.We have found that overexpression WWC3 can significantly inhibit lung
The clonality (Fig. 1) of cancerous cell, knocks out WWC3 rear clones Forming ability and strengthens（Fig. 2）.
（1-2）Matrigel invasion is tested：With 1640 culture medium of double nothings according to 1 in 24 orifice plates：3 dilution proportion substrate
Glue (BD Biosciences), spreads the upper room (Costar) of 8 micron pore sizes.Then by transfection after A549, H1299 micro- with 100
Rise and contain 5 × 105Individual cell density is seeded in room and is incubated 16 hours.Put the culture medium containing 10% calf serum in lower room.
After cultivating 48 hours after plantation, methanol fixes 15 minutes, haematoxylin dyeing.10 visuals field are randomly selected, invasion and attack is counted and is arrived cell
Under cell number.Experiment in triplicate, is averaged.We have found that overexpression WWC3 can significantly inhibit the invasion and attack of lung carcinoma cell
Ability（Fig. 3）, after knocking out WWC3, lung carcinoma cell invasion ability strengthens（Fig. 4）.
（1-3）MTT experiment：After transfection in 96 orifice plates with the culture medium culturing about 3000 or so containing 10% serum
A549, H1299 cell, each hole add 20 microlitres of 5 mg/ml MTT (Thiazolyl Blue) solution, are incubated at 37 DEG C
After 4 hours, solution is removed, the MTT crystallizations generated with 150 microlitres of DMSO dissolvings detect 490nm wavelength with spectrophotometer
Absorption peak is carried out quantitatively.Proliferation of lung cancer cells reduced capability after overexpression WWC3 is we have found that, lung carcinoma cell after WWC3 is knocked out
Multiplication capacity strengthens（Fig. 5）.
（1-4）Nude mice by subcutaneous is tested into tumor：H1299 the and A549 cell suspension of above-mentioned transfection carries out nude mice (BALB/c)
Dorsal sc is inoculated with (matched group and each five nude mices of experimental group), every nude inoculation 0.2ml cell suspension（Containing about cell 1 ×
107Individual）Kept under sterile conditions afterwards, so as to observation impacts of the WWC3 to tumor cell proliferation in vivo situation.Observe and measure and be swollen
Tumor size and Mouse Weight（Measured once per 3 days）, draw tumor growth curve and mouse survival curve.Unified execution in 8 weeks, takes
Tumor body weighs knurl weight, sees whether there is metastasis.Tumor makes paraffin section.We have found that the tumor body constitution of overexpression WWC3 groups
Amount is significantly less than blank control group with volume（Fig. 6）, the tumor weight and volume that knock out WWC3 groups are significantly greater than blank control group
（Fig. 7）.
（1-5）Through testing into tumor in nude mice tail vein body：H1299 the and A549 cell suspension of above-mentioned transfection carries out nude mice tail
Intravenous inoculation, every nude inoculation 0.1ml cell suspension（Containing about cell 1 × 106Individual）Kept under sterile conditions afterwards, to detect
Impacts of the WWC3 to tumor cell whole body transfer case.Observe and measure Mouse Weight（Measured once per 3 days）, draw mice life
Deposit curve.Unified execution in 8 weeks, takes mouse systemic main organs (heart, liver, spleen, lung, kidney), weighs, and perusal organ surface is
No have metastasis, and fixed internal organs make paraffin section.Whether there is tumor cell in application HE staining Microscopic observation internal organs
Transfer.Such as there is transfer, the neoplasm metastasis area sum purpose for comparing transfection group and blank control group is how many.We have found that：
Compared with blank control group, (lung) neoplasm metastasis number and area are substantially reduced overexpression WWC3 groups；Knock out WWC3 groups, Lung metastases
Number and size are dramatically increased.
（2）WWC3 is in cancerous lung tissue（Cancerous cell line）In be low expression
RIPA lysates with pre-cooling（RIPA ：PMSF=100：1）To 6 kinds of lung cancer cell lines（A549、H1299、H460、H292、
LK2、Calu1）And the carrying out of normal bronchial epithelial cell HBE cracks, ultrasound 3 times on ice, placement 10 minutes, 4 DEG C,
12000rpm is centrifuged 15 minutes, takes supernatant, with BCA kit measurement total protein contents, with BSA as standard substance.By 8% concentration
SDS-PAGE carry out Protein Separation, applied sample amount is 60 micrograms, and electrophoresis 1 hour, by the protein electrotransfer in gel to PVDF
On film, with phosphate Tween buffer after taking-up（0.1% (v/v) polysorbas20 is added in PBS）After film washing liquid washes film 5 minutes, will
Film is put in the phosphate Tween buffer containing 5% defatted milk powder, and on shaking table, room temperature is closed 120 minutes；Phosphate tween is buffered
After liquid washes film 5 minutes, film is put into an anti-WWC3 1：500, Sigma, GAPDH 1：10000, Sigma, 4 °C overnight；Phosphoric acid
Salt Tween buffer washes film 10 minutes × 3 times；Radix Cochleariae officinalises enzyme labelling goat anti-rabbit igg (1:3000, Zhong Shan Golden Bridge), mountain sheep anti mouse
IgG(1:3000, Zhong Shan Golden Bridge) incubation vibration 120 minutes on room temperature shaker, phosphate Tween buffer washes film 10 minutes ×
3 times；Luminous using luminescent solution (Pierce).After ECL is luminous, band gray value measure is carried out using ImageJ, with WWC3/
The ratio of GAPDH takes average in triplicate as relative expression quantity, experiment（Fig. 8）.
There is the effect for suppressing Wnt paths, promoting Hippo pathway activities
The H1299 cell lines of selection WWC3 low expressions carry out the overexpression experiment of WWC3.In order to more intuitively observe result, I
Employ Wnt3A（50ng/mL）Having carried out pre-stimulation to the Wnt activity of cell amplifies its signal.Utilize in 24 orifice plates
The Super 8x Top Flash of the WWC3 of 0.5 microgram of liposome Lipo3000 (Invitrogen) cotransfection and 0.5 microgram
(Wnt path reporter plasmids) and 50 nanogram control plasmid pRL-TK (sea pansy element), pEGFP-C2 empty carriers are used as table excessively
Negative control up to plasmid.After 37 °C of incubations 30 hours, using double fluorescein prunus mume (sieb.) sieb.et zucc. reporter gene detecting systems（Promega,
Madison, WI）The expression of examining report gene is (using Super 8x TOP Flash reporter gene activity and pRL-TK Stichopus japonicuss
The value of the Wnt pathway activities that the ratio of plain reporter gene activity is mediated as TCF-4), as a result show that WWC3 can be significantly inhibited
The Wnt pathway activities of Wnt3A inductions（Fig. 9）.Equally, in H1299 cells, cotransfection WWC3 and pGL3b_8xGTIIC
(Hippo path reporter plasmids) and pRL-TK (method, dosage are the same), luciferase reporter gene show that WWC3 can
Significantly inhibit YAP activity (promoting Hippo pathway activities)（Figure 10）.
The A549 cell lines of the relatively high expression WWC3 of selection carry out the interference experiment of WWC3.Cotransfection shRNA- in A549
(method, dosage are same for WWC3 and Super 8x TOP Flash plasmids (Wnt path reporter plasmids) and pRL-TK plasmids
Before), as a result show the Wnt pathway activities for knocking out that WWC can dramatically increase Wnt3A inductions（Fig. 9）.In the same manner, corotation in A549
(method, dosage are same with pRL-TK plasmids for dye shRNA-WWC3 and pGL3b_8xGTIIC (Hippo path reporter plasmids)
Before), as a result show that knockout WWC3 can promote YAP activity (suppressing Hippo pathway activities)（Figure 10）.
WWC3 is transfected in H1299 cells, albumen is extracted after 48h and RNA, Western Blot and qPCR technology for detection is sent out
The overexpression of existing WWC3 can significantly lower Wnt path major protein non-phosphorylated β-catenin, and target
Gene c-myc, the albumen of MMP7, cyclinD1 and mRNA expressions, while lower the albumen of Hippo path target gene CTGF
And mRNA expressions（Figure 13,14）.ShRNA-WWC3 is transfected in A549, and Western Blot and qPCR technology for detection finds
Knock out WWC3 and can significantly raise Wnt path major protein non-phosphorylated β-catenin, and target gene c-
The albumen of myc, MMP7, cyclinD1 and mRNA expressions, while raise the albumen and mRNA of Hippo path target gene CTGF
Expression（Figure 11,12）.
Therefore, WWC3 is the protein molecular that a kind of suppression Wnt paths simultaneously facilitate Hippo pathway activities.
By WW domains and ADDV domains PY motifs and PDZ domain interactions respectively with Dvl2
Twice of 5mL PBSs of A549 cell lines, and with 1mL NP-40 lysates ice bath 15 minutes after, cell lysis is produced
Thing is transferred in new 1.5mL EP pipes from culture plate, after 4 DEG C of 16000g of pyrolysis product are centrifuged 20 minutes is shifted supernatant
Into new pipe, protein (A+G) beads (Santa Cruz Technology) is added, after 4 DEG C of rotary shaker 2h, supernatant is taken
To new centrifuge tube, Dvl2 antibody (2 μ L/mg) is added, is settled as bait protein, 4 DEG C of rotations are overnight.Next day adds
Protein (A+G) besds, after 4 DEG C of rotation 6h, 4 DEG C of 1000rpm 5min abandon supernatant, retain precipitation, add 1mLRIPA to split
Solution liquid (RIPA:PMSF=100:1) magnetic bead is washed, repeatedly after 3 times, 2 × Loading Buffer is added, is boiled sample 10min, carry out
Western Blot are detected.As a result show that endogenouss WWC3 and Dvl2 is present to interact.Equally, cotransfection GFP- in A549
WWC3 and FLAG-Dvl2 plasmids, the ectogenic WWC3 and Dvl2 of co-immunoprecipitation discovery can equally interact, and (method is same
Before)（Figure 13）.
Build WWC3 spliceosomes (WWC3- Δ WW, WWC3- Δ ADDV, WWC3- Δ WW＆ADDV) and Dvl2 spliceosomes
(Dvl2-ΔPDZ、Dvl2-ΔPY、Dvl2-ΔPDZ&PY)（Figure 14,15）, to determine the combination of WWC3 and Dvl2 interactions
Site.
Cotransfection WWC3 wild plasmids and Dvl2 wild types and mutant plasmid in H1299 cell lines, after transfection
48h, collection cell, co-immunoprecipitation shows Dvl2, and by its PDZ domain and PY motifs, (method is same with WWC3 interactions
Before)（Figure 17）；Equally, cotransfection Dvl2 wild plasmids and WWC wild types and mutant plasmid in H1299, immunity are coprecipitated
Form sediment and show that WWC3 interacts (method is the same) by its WW domain and ADDV domains with Dvl2（Figure 16）.
Cotransfection WWC3- Δs WW and Dvl2- Δ PY plasmids in H1299, co-immunoprecipitation show reservation ADDV domains
WWC3 and retain PDZ domains Dvl2 can interact（Figure 18）；In H1299 cotransfection WWC3- Δs ADDV with
Dvl2- Δ PDZ plasmids, co-immunoprecipitation show that the WWC3 and the Dvl2 for retaining PY domains that retain WW domains still are able to mutually
Effect（Figure 19）.
Therefore：WWC3 is that WW domains and ADDV domains by its own are tied with the PY motifs and PDZ of Dvl2 respectively
Structure domain interacts.
The interaction of 4.WWC3 and Dvl2 hinders CK1 δ/ε, and to the phosphorylation of Dvl2, negativity regulates and controls Wnt pathway activities
WWC3 plasmids are transfected in H1299 cells, albumen after 48h, is extracted, Western Blot detections find：The overexpression of WWC3
The phosphorylation of Dvl2 can significantly be lowered；ShRNA- WWC3 are transfected in A549, and Western Blot detections find striking for WWC3
Except the phosphorylation that can significantly raise Dvl2（Figure 20）.
ShRNA-WWC3 is transfected in A549 cells, CK1 δ/epsilon inhibitor (IC261, Santa Cruz is simultaneously introduced
Technology), or transfection siRNA-CK1 δ/ε, 37 DEG C after 30 hours, luciferase reporter gene and Western Blot
(method is the same) shows：Knock out CK1 δ/ε or suppress its activity significantly reverse the Wnt paths caused due to WWC3 silences to live
The rise of property（Figure 20）And the rise of Dvl2 phosphorylations（Figure 21,22）.
Gradient transfection WWC3 in H1299 cells, co-immunoprecipitation show：With the gradually increase that WWC3 is expressed, CK1 ε
Gradually decrease with the binding capacity of Dvl2（Method is the same）（Figure 23）；On the other hand, gradient transfection CK1 ε in H1299, immunity is altogether
Precipitation finds：With the gradually increase that CK1 ε are expressed, the binding capacity of WWC3 and Dvl2 is gradually decreased（Method is the same）（Figure 23）
Last transfection WWC3 and WWC3 mutants in H1299 cells, luciferase reporter gene show that WWC3 wild types can
Significantly inhibit Wnt pathway activities, and WWC3- Δ WW＆ADDV（Can not be combined with Dvl2）Then act on without this（Method is the same）（Figure
24）；Protein immunization imprinting finds：Wild type WWC3 can lower p-Dvl2 in Wnt paths, the β-catenin of activation and target base
Because of protein expression level, and WWC3- Δ WW＆ADDV（Can not be combined with Dvl2）Then act on without this（Method is the same）（Figure 25）
5.LATS1 and Dvl2 competitive bindings WWC3 and promote Hippo pathway activities
WWC3 plasmids are transfected in H1299 cells, albumen after 48h, is extracted, Western Blot detections find：YAP and LATS swashs
The phosphorylation level of enzyme is raised（Figure 24）；Conversely, after knocking out WWC3 in A549, Western Blot detections find：YAP with
The phosphorylation level of LATS kinases is lowered（Figure 26）；The above results positive regulatory factors of this proof WWC3 for Hippo paths.
Transfect three kinds of mutant plasmids of WWC3 and WWC3 in H1299 respectively, tested using luciferase reporter gene,
It was found that：Compared with matched group, WWC3 can significantly lower YAP activity (activation Hippo paths), and lack WW domains
Mutant1 and Mutant3 then can not（Figure 27）；Simultaneously we apply co-immunoprecipitation experiment to find that wild type WWC3 can be with
LATS1 kinase interactions, and the WWC3 for lacking WW domains then can not（Figure 28）.The WW domains of this explanation WWC3 promote
Play an important role in Hippo Pathway Activations.
This experimental result also can be combined with the WW domains of WWC3 according to Dvl2, illustrated that LATS1 and Dvl2 can be with
The same site of WWC3 combines.Therefore, we predict：The LATS1 and very possible competitive binding WWC3 of Dvl2, and then affect
Hippo pathway activities.In order to prove this supposition, on the one hand overexpression Dvl2 in H1299, co-immunoprecipitation find WWC3 with
The combination of LATS1 is reduced（Figure 29）；Dvl2 is knocked out in H1299, and co-immunoprecipitation experiment finds the binding capacity of WWC3 and LATS1
Increase（Figure 30）.On the other hand our overexpression LATS1 in H1299, co-immunoprecipitation have found the binding capacity of WWC3 and Dvl2
Reduce（Figure 31）；In H1299, disturb the expression of LATS1, co-immunoprecipitation to find that the binding capacity of WWC3 and Dvl2 increases（Figure
32）.These demonstrate that LATS1 and Dvl2 being capable of competitive binding WWC3.
In order to what kind of has affect after probing into LATS and Dvl2 competitive binding WWC3 on Hippo pathway activities, in H1299 altogether
Transfection WWC3 and two kinds of plasmids of Dvl2, after 48h, Western Blot detections find that Dvl2 can reverse the p- that WWC3 causes
LATS1, p-YAP level is raised（Figure 33）；Cotransfection WWC3 plasmids and siRNA-Dvl2, Western Blot inspections in H1299
Survey the phosphorylation for finding to knock out that Dvl2 can be dialled further up the LATS1 and YAP that WWC3 causes（Figure 34）.
Last cotransfection WWC3 plasmids and Dvl2 wild types and mutant plasmid in H1299, Western Blot are detected
It was found that the wild type Dvl2 plasmids that can be combined with WWC3 can reverse the rise of the p-LATS1 that WWC3 causes and p-YAP levels,
And the Dvl2 Mutant3 plasmids that can not be combined with WWC3 can not then be reversed（Figure 35）.
Claims (6)
- Application of the 1.WWC3 protein moleculars in antitumor drug is prepared, it is characterised in that：The WWC3 protein moleculars be containing WW domains and C2 domainsType albumen；The WWC3 suppresses Wnt activity by DVLs and activates Hippo pathway activities by LATS1.
- 2. application of the WWC3 protein moleculars according to claim 1 in antitumor drug is prepared, it is characterised in that：Described WWC3 protein moleculars are by its own WW domains and ADDV domains and important upstream protein molecule DVLs in Wnt paths Interact, the interaction hinders CK1 δ/phosphorylation of the ε kinases to DVLs, promote Wnt path effect protein molecule β- The degraded of catenin, reduces β-catenin and enters core, play the effect for suppressing Wnt pathway activities.
- 3. application of the WWC3 protein moleculars according to claim 1 in antitumor drug is prepared, it is characterised in that：Utilize The mutant of WWC3 and Dvl2 spliceosomes, demonstrate WWC3 by WW domains and the PY of Dvl2 using the method for co-immunoprecipitation Motif interacts, and can also pass through the PDZ domain interactions of ADDV domains and Dvl2, play and suppress Wnt pathway activities Effect.
- 4. application of the WWC3 protein moleculars according to claim 1 in antitumor drug is prepared, it is characterised in that：The mutant of the WWC3 includes WWC3- Δ WW, WWC3- Δ ADDV, WWC3- Δ WW＆ADDV；The Dvl2 spliceosomes include Dvl2- Δ PDZ, Dvl2- Δ PY, Dvl2- Δ PDZ＆PY.
- 5. application of the WWC3 protein moleculars according to claim 1 in antitumor drug is prepared, it is characterised in that：Described WWC3 is interacted with Hippo paths central authorities kinases LATS1 by the WW domains of its own, promotes LATS1 phosphorylations, and then Make Hippo effect protein YAP be stranded in endochylema, play the effect for promoting Hippo pathway activities.
- 6. application of the WWC3 protein moleculars according to claim 1 in antitumor drug is prepared, it is characterised in that： WWC3 with LATS1 kinase interactions and has played the function of activating Hippo pathway activities, and lacks the WWC3 of WW domains then Without this function；Increase the expression of LATS1, reduce can the binding capacity of WWC3 and Dvl2；Reduce LATS1 expression, then WWC3 with The binding capacity of Dvl2 increases, i.e. the combination of LATS1 and WWC3 has competitiveness with Dvl2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611124240.8A CN106492191A (en) | 2016-12-08 | 2016-12-08 | Application of the WWC3 protein moleculars in antitumor drug is prepared |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611124240.8A CN106492191A (en) | 2016-12-08 | 2016-12-08 | Application of the WWC3 protein moleculars in antitumor drug is prepared |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106492191A true CN106492191A (en) | 2017-03-15 |
Family
ID=58330289
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611124240.8A Pending CN106492191A (en) | 2016-12-08 | 2016-12-08 | Application of the WWC3 protein moleculars in antitumor drug is prepared |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106492191A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115951051A (en) * | 2022-10-18 | 2023-04-11 | 北京卓诚惠生生物科技股份有限公司 | High-sensitivity novel coronavirus antigen colloidal gold detection kit and preparation method thereof |
| WO2023193739A1 (en) * | 2022-04-07 | 2023-10-12 | 复旦大学 | Polypeptide for activating hippo signaling pathway and uses thereof |
-
2016
- 2016-12-08 CN CN201611124240.8A patent/CN106492191A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| DIRK OLIVER WENNMANN ET AL.: "Evolutionary andMolecular Facts Link the WWCProtein Family to Hippo Signaling", 《MOL. BIOL. EVOL.》 * |
| 宋娟: "Hippo-YAP信号通路为靶点的肿瘤治疗研究进展", 《中国肿瘤临床》 * |
| 许传铭等: "哺乳动物Hippo 信号通路：肿瘤治疗的新标靶", 《遗传》 * |
| 郭海强等: "癌蛋白YAP1 的研究进展", 《生物化学与生物物理进展》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023193739A1 (en) * | 2022-04-07 | 2023-10-12 | 复旦大学 | Polypeptide for activating hippo signaling pathway and uses thereof |
| CN115951051A (en) * | 2022-10-18 | 2023-04-11 | 北京卓诚惠生生物科技股份有限公司 | High-sensitivity novel coronavirus antigen colloidal gold detection kit and preparation method thereof |
| CN115951051B (en) * | 2022-10-18 | 2024-01-12 | 北京卓诚惠生生物科技股份有限公司 | Novel high-sensitivity coronavirus antigen colloidal gold detection kit and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Huang et al. | The miR-26a/AP-2α/Nanog signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma | |
| Xiong et al. | Effects of microRNA‐29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma | |
| Yang et al. | TGF-β-miR-34a-CCL22 signaling-induced Treg cell recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma | |
| Moyano et al. | αB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer | |
| Fan et al. | Late SV40 factor: a key mediator of Notch signaling in human hepatocarcinogenesis | |
| Yang et al. | MiR-133b regulates the expression of CTGF in epithelial-mesenchymal transition of ovarian cancer. | |
| Tang et al. | LncRNA DNAJC3-AS1 regulates fatty acid synthase via the EGFR pathway to promote the progression of colorectal cancer | |
| Lu et al. | Inhibition of STAT6/Anoctamin-1 activation suppresses proliferation and invasion of gastric cancer cells | |
| Liu et al. | FBXO17 promotes malignant progression of hepatocellular carcinoma by activating wnt/β-catenin pathway. | |
| Zhang et al. | Exosomal miR-211 contributes to pulmonary hypertension via attenuating CaMK1/PPAR-γaxis | |
| Wang et al. | DCLK1 promotes malignant progression of breast cancer by regulating Wnt/β-Catenin signaling pathway. | |
| CN106492191A (en) | Application of the WWC3 protein moleculars in antitumor drug is prepared | |
| Huang et al. | The study on expression of CIAPIN1 interfering hepatocellular carcinoma cell proliferation and its mechanisms. | |
| Passi et al. | Mechano-signaling aspects of hepatocellular carcinoma | |
| Wang et al. | MiR-375 impairs the invasive capabilities of hepatoma cells by targeting HIF1α under hypoxia | |
| Du et al. | High STRN expression promotes HCC invasion and migration but not cell proliferation or apoptosis through facilitating epithelial-mesenchymal transition | |
| Xu et al. | LncRNA XIST promotes proliferation and epithelial-mesenchymal transition of retinoblastoma cells through sponge action of miR-142-5p. | |
| CN103920164B (en) | MiR-424-5p is suppressing the application in secondary liver cancer | |
| CN104726584A (en) | Application of miR-425 in tumor diagnosis, treatment and prognosis | |
| Xiang et al. | Tanreqing injection regulates cell function of hypoxia-induced human pulmonary artery smooth muscle cells (HPASMCs) through TRPC1/CX3CL1 signaling pathway | |
| Zhang et al. | Effect of Codonopsis pilosula polysaccharides on the growth and motility of hepatocellular carcinoma HepG2 cells by regulating β-catenin/TCF4 pathway | |
| He et al. | Desmocollin-2 inhibits cell proliferation and promotes apoptosis in hepatocellular carcinoma via the ERK/c-MYC signaling pathway | |
| CN108642179A (en) | The MiR-210 experimental methods that related target is verified in glioma | |
| CN112410429B (en) | Application of FXYD3 as gastric cancer diagnosis marker and treatment target | |
| CN112083171B (en) | Ku70 protein T455 locus phosphorylation inhibitor and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170315 |
|
| RJ01 | Rejection of invention patent application after publication |