CN103285382B - PTPMeg2 albumen is preparing the application in Tumor suppression product - Google Patents

Abstract

The invention discloses a kind of PTPMeg2 albumen and prepare the application in Tumor suppression product.The invention provides PTPMeg2 albumen and have following 1 in preparation)-8) at least one function product in application: the 1) propagation of inhibition tumor cell; 2) Tumor suppression growth; 3) tumor is prevented and/or treated; 4) phosphorylation of STAT3 is suppressed; 5) stop pSTAT3 from Cytoplasm to nuclear position transfer; 6) dephosphorylation of pSTAT3 is promoted; 7) transcriptional activity of STAT3 is suppressed; Experiment of the present invention proves, PTPMeg2 specific effect is the substrate of PTPMeg2 in STAT3, STAT3, the propagation of process LAN T suppression cell of PTPMeg2 and the growth of tumor.

Description

PTPMeg2 albumen is preparing the application in Tumor suppression product

Technical field

The present invention relates to biomedicine field, particularly relate to a kind of PTPMeg2 albumen and preparing the application in Tumor suppression product.

Background technology

Signal transducer and activating transcription factor 3 (SignalTransducerandActivatorofTranscription3, STAT3) being a kind of bifunctional protein being present in cytoplasm and the coupling of tyrosine phosphorylation signalling channel, is one of STATs family seven members.STAT3 adjustment Growth of Cells, differentiation and to the reaction of many born of the same parents' external stimulus factors, somatomedin in most important.According to current existing report, a series of Hematopoietic Malignancies and comprise breast carcinoma entity tumor in observe the lasting phosphorylation phenomenon of STAT3.It is generally acknowledged, the Tyr705 residue of potential Cytoplasm STAT3 is activated by phosphorylation under the effect of cytokine receptor associated kinase or growth factor receptors related tyrosine kinases (Src).The STAT3 of phosphorylation is by Src homology tyrosine-2-phosphorylation interaction dimerization and assemble in nucleus, and therefore extensively activates as BCL-XL, transcribing of the protein gene such as cyclin D1 and c-Myc, SOCS3.STAT3 signalling channel may become an action target spot of therapy of tumor, and the research for the blocking treatment of STAT3 becomes the focus of oncotherapy research in recent years

Great majority research thinks that the lasting phosphorylation of STAT3 and jak kinase, the kinase whose activation of Src are relevant.But the phosphorylation of STAT3 closely depends on the dephosphorylation process that phosphatase (PTPs) mediates.A series of evidence support thinks that phosphatase plays a significant role in the signal path of multiple adjustment cell proliferation, apoptosis, adhesion and migration.PTPs is the extended familys enzyme of structural diversification, the dephosphorylation of catalytic phosphatase albumen.Previous research shows, Protein Tyrosine Phosphatases 1B (PTP1B) regulates cytokine signalling pathways by the dephosphorylation of JAK2, TYK2, STAT5a/b in nucleus.Other researchs show, SHP2 and TC45 in nucleus of STAT1, STAT3 and STAT5 and dephosphorylation.

Reported as can be seen from current research: stat protein can phosphatases different in Cytoplasm and nucleus and dephosphorylation, importantly, the unconventionality expression comprising PTPs in the development of the human diseases of cancer, diabetes, inflammation and infectious disease causes STATs to continue phosphorylation.

Research report is in the past thought, several PTPs is as PTPN1, PTPN3 and PTPN6 is proto-oncogene, but some PTPs comprising PTPN12 have tumor suppression function, wherein PTPL1/PTPN13 is by direct deactivation Src kinase regulatory breast carcinoma, PTPN12 is by multidigit point EGFR1, HER2 and Src kinase inhibition Metastasis in Breast Cancer.Previous research report is pointed out, PTPMeg2 and EGF-R ELISA and HER2 exist and interact.PTPMeg2 (PTPN9) is a kind of kytoplasm phosphatase, clones the homologous sequence from retinaldehyde binding protein and yeast Sec14p, it is reported the EGFR that can make phosphorylation, ErB2 and Fox-1 dephosphorylation.Functional study shows, in hematopoietic cell, PTPMeg2 promotes homotype Vesicle fusion in born of the same parents, the amplification of regulation of embryonic development and control erythroid cells.Other researchs show, PTPMeg2 regulates insulin generation, β Growth of Cells, and regulate insulin signaling by the dephosphorylation reducing Insulin receptor INSR in type 2 diabetes mellitus.

Summary of the invention

An object of the present invention is to provide a kind of application of PTPMeg2 albumen.

The PTPMeg2 of being applied as albumen provided by the invention has following 1 in preparation)-7) at least one function product in application:

1) propagation of inhibition tumor cell;

2) Tumor suppression growth;

3) tumor is prevented and/or treated;

4) phosphorylation of STAT3 is suppressed;

5) stop pSTAT3 from Cytoplasm to nuclear position transfer;

6) dephosphorylation of pSTAT3 is promoted;

7) transcriptional activity of STAT3 is suppressed;

The aminoacid sequence of described PTPMeg2 albumen is the sequence 1 in sequence table;

The aminoacid sequence of described STAT3 albumen is the sequence 3 in sequence table.

In above-mentioned application, above-mentioned tumor cell is the tumor cell containing endogenous PTPMeg2 albumen;

Above-mentioned tumor is breast carcinoma.In above-mentioned application, the above-mentioned tumor cell containing endogenous PTPMeg2 albumen is the breast cancer cell containing endogenous PTPMeg2 albumen;

Above-mentioned Tumor suppression growth is embodied in and reduces gross tumor volume and/or tumor weight;

Above-mentioned prevention pSTAT3 carries out IL-6 activates from Cytoplasm to nuclear position transfer;

The dephosphorylation of above-mentioned promotion pSTAT3 carries out under IL-6 activates;

The transcriptional activity of above-mentioned suppression STAT3 carries out under IL-6 activates.

In above-mentioned application, the dephosphorylation of described pSTAT3 is the 705th the Tyr dephosphorylation at described pSTAT3;

In addition, the phosphorylation of STAT3 is also the 705th the Tyr phosphorylation at STAT3.

The described breast cancer cell containing endogenous PTPMeg2 albumen is MDA-MB-231 cell;

Described each function all directly mediates pSTAT3 dephosphorylation by described PTPMeg2 and realizes.

Another object of the present invention is to provide the application of the material suppressing PTPMeg2 protein active.

Provided by the invention being applied as suppresses the material of PTPMeg2 protein active to have following 1 in preparation)-4) at least one function product in application:

1) propagation of tumor cell is promoted;

2) tumor growth is promoted;

3) phosphorylation level of STAT3 is improved;

4) transcriptional activity of STAT3 is increased;

The material of described suppression PTPMeg2 protein active is following A or B:

A, a kind of nucleotide, its nucleotides sequence is classified as the sequence 4 in sequence table;

B, carrier, transgenic cell line or recombinant adenovirus containing nucleotide described in A.

In above-mentioned application, described tumor cell is the tumor cell containing endogenous PTPMeg2 albumen;

Described tumor is breast carcinoma.

In above-mentioned application, the described tumor cell containing endogenous PTPMeg2 albumen is the breast cancer cell containing endogenous PTPMeg2 albumen;

Described promotion tumor growth is embodied in increases gross tumor volume and/or tumor weight.

In above-mentioned application, the described breast cancer cell containing endogenous PTPMeg2 albumen is MCF-7 cell.

Described PTPMeg2 albumen is also being the scope of protection of the invention as the application in antitumor medicine screening.

Experiment of the present invention proves, there is interaction in PTPMeg2 and STAT3, STAT3 is the substrate of PTPMeg2, the process LAN of PTPMeg2 plays direct negative regulation effect to pathology or physiological STAT3 phosphorylation, thus the growth of the propagation of T suppression cell and tumor, the hyperphosphorylation of the STAT3 that the loss of expression of PTPMeg2 then causes, and cause the propagation of cell and the acceleration of growth of tumor.

Accompanying drawing explanation

Fig. 1 is the interaction of PTPMeg2 and STAT3

Fig. 2 is that the STAT3 of PTPMeg2 and phosphorylation and unphosphorylated form exists interaction

Fig. 3 is the dephosphorylation that PTPMeg2 has to pSTAT3

Fig. 4 is the transcriptional activity that PTPMeg2 reduces STAT3

Fig. 5 is that PTPMeg2 suppresses the propagation of breast cancer cell and the growth of transplanted tumor in nude mice

Fig. 6 is the expression of PTPMeg2 and the dependency of pSTAT3 level

Detailed description of the invention

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Material used in following embodiment is exemplified below:

Anti-PTPMeg2 antibody (H300, catalog number (Cat.No.) sc-67049), anti-Myc antibody (9E10, catalog number (Cat.No.) sc-8334), anti-HA antibody (F-7, catalog number (Cat.No.) sc-7392), anti-green fluorescent protein antibody (FL, catalog number (Cat.No.) sc-8334), anti-pSTAT3 antibody (Tyr705, catalog number (Cat.No.) sc-8059), anti-pSTAT3 (Ser727) antibody, anti-STAT3 antibody (F-2, catalog number (Cat.No.) sc-8019), anti-STAT3 antibody (C-20, catalog number (Cat.No.) sc-482) antibody, rabbit source property IgG (SC-2027), all purchased from SantaCruz company.

Actin (AC-15) antibody available from Sigma (catalog number (Cat.No.) A5316).

Anti-rabbit FITC antibody (catalog number (Cat.No.) ZF-0311), against murine TRITC antibody (catalog number (Cat.No.) ZF-0313), all purchased from Beijing Technology Co., Ltd. in the morning of Zhong Shan Golden Bridge.

People recombinates IL-6 (CB407652) and IL-6 receptor (CB407653) purchased from CB company.

Protein I gG agarose particle (catalog number (Cat.No.) sc-2002) is purchased from SantaCruz company.

Glutathione-sepharose 4B granule (catalog number (Cat.No.) 17-0756-01), purchased from GE biotech firm.

Transfection reagent Lipofectamine ^tM2000 (purchased from Invitrogen, catalog number (Cat.No.) 18324012).

Cell pyrolysis liquid: 20mMTris-Cl, pH7.6,150mMNaCl, 5mMEDTA, 10mMMgCl2,0.5%NP40,10% glycerol, 1mMDTT, 0.1mMNa ₃vO ₄, 1mM phenylmethylsulfonyl fluoride, 1 μ g/mlaprotinin, 1 μ g/ml protease inhibitor and 1 μ g/ml pepsin inhibitor.

Method used in following embodiment is specific as follows:

1, Luciferase Assay

MCF7 cell is inoculated in 24 orifice plates, second day cell reporter plasmid pAPRE-luc or M67-luc and 5ng internal contrast plasmid pRL-TK cotransfection of 0.1 microgram.

Express STAT3 with the amount of every hole 0.4 microgram, the construct cotransfection MCF7 cell of PTPMeg2 and mutant thereof.

In order to remove endogenous PTPMeg2, also to MCF7 cell transfecting 0.8 microgram with the plasmid pSilencer-4.1 with shRNA structure for targeting sequence of PTPMeg2 (5 '-GATCCGGAAAGGCATTGTAAATTCAAGAGATTTACAATGCCTTCCTTCCTTA-3 ').

After transfection twenty four hours, adopt Dual-Luciferase report analysis system (VigofectInc. Beijing, China) carry out luciferase assay, with the activity value of renilla luciferase for internal reference, calculate the ratio of LUC Photinus pyralis LUC Photinus pyralis FL activity value and renilla luciferase activity value, what be reporter gene transcribes relative activity.

2, co-immunoprecipitation analysis and GST precipitation analysis method

Protein SDS-PAGE and Westernblot analyzes, operate according to (WangF, etal. (2008) VarpinteractswithRab38andfunctionsasitspotentialeffector. (Translatedfromeng) BiochemBiophysResCommun372 (1): 162-167 (ineng) .) record.

For immunoprecipitation experiment: grow the HEK293T cell expression plasmid transfection of specifying at 60 millimeters of culture dishs, and use cell pyrolysis liquid cracking on ice 30 minutes, then centrifugal 15 minutes of maximum speed.Cell lysate supernatant adds 2 micrograms and specifies antibody, 4 DEG C of overnight incubation.Then 30ul protein I gG agarose particle is added in conjunction with 4 hours, 4 DEG C.Rinse four granules with cell pyrolysis liquid, in conjunction with albumen adopt 2 × sample loading buffer eluting, carry out Westernblot analysis with appointment antibody.

GST precipitation analysis is identical with the program of immunoprecipitation experiment, and except the granule for precipitating is different, co-immunoprecipitation analysis protein I gG agarose particle, GST precipitation analysis glutathione-sepharose 4B granule precipitates.Second difference is exactly change cell pyrolysis liquid washing into PBST buffer solution (PBST buffer: 2mmol/L potassium dihydrogen phosphate (KH ₂pO ₄), 10mmol/L sodium hydrogen phosphate (Na ₂hPO ₄12H2O), 137mmol/L sodium chloride (NaCl), 2.7mmol/L potassium chloride (KCl), 0.05% tween 20, pH7.4).

3, immunocytochemistry

Test cell, through IL-6 process 30 minutes, is first inoculated in coverslip 24 hours and with serum starvation method process 18 hours before process.Cell, at 4 DEG C, fixes 20 minutes with 4% paraformaldehyde, the Tritonx-100 process with 0.3% 15 minutes.Under room temperature, close nonspecific binding site with 10% Normal Goat Serum, and in primary antibody 4 DEG C of night incubation.Primary antibody used is anti-PTPMeg2 or anti-Flag antibody.Secondary antibody used is the IgG antibody combining FITC, TRITC.With DAPI staining cell core.Flow cytometry is carried out with BDFACSC automatic analyzer.

4, cell growth assay

In order to process LAN PTPMeg2, by the MCF7 cell with shPTPMeg2 plasmid transfection, or the MDA-MB-231 cell of adenovirus infection is inoculated in 96 orifice plates with 1000/ porocyte density respectively, repeats 3 holes.Cell culture different time, the MTT that cell growth arrest adds with 5g/L for first 4 hours.

Before collection, with the DMSO Rong Xie Jia Za granule of 150 μ L.Relative optical density (OD the value)/hole under 570 nanometer detection wavelength is determined at the WELLSCANMK3ELIASA (Labsystems, Dragon, Finland) with 630 nanometers reference filters.Draw cell growth curve, the meansigma methods according to OD570-OD630 draws cell growth curve,

5, Transplanted tumor model

In order to PTPMeg2 overexpression, the MCF7 cell PTPMeg2shRNA carrier stabilizes transfection of exponential phase of growth, MDA-MB-231 infects adenovirus.

Cell suspension is used in 1 ml physiological saline in order to injection mice.By 1.0x10 ⁷(MCF-7) or5.0x10 ⁶(MDA-MB-231) cell is suspended from the normal saline of 0.1 ml volumes and carries out bilateral subcutaneous injection to BALB/c-nu/nu mice in 5 week age.

Gross tumor volume, is defined as (long x width ²)/2, a few days ago weighing with slide calliper rule to terminal.Draw tumor growth curve according to gross tumor volume meansigma methods (cubic millimeter), instruct according to zoopery and carry out all zooperies.

6, immunohistochemical assay

Formalin from human breast cancer patient is fixed tumor tissues paraffin embedding, section (3 microns).Human breast cancer patient Pathologic specimen is surgical resection specimen, derives from Tangshan, Hebei Province the People's Hospital.

Paraffin section dewaxes with dimethylbenzene respectively, ethanol serial dehydration, then 0.3% hydrogen peroxide/methanol inactivating endogenous or oxide enzyme is used, use 0.01M citrate buffer solution (pH6.0) Microwave method 10 minutes again, again by monoclonal antibody 4 degree of overnight incubation of instruction, with secondary antibody (biotin labeling; Santa Cruz) hatching, finally use three grades of antibody (peroxidase labeled antibodies; Santa Cruz) cultivate 60 minutes under room temperature (25 DEG C).

Sample with DAB (Santa Cruz) as substrate.Continue to use the tumor degree standards of grading that document (ZhouL, eta1. (2008) ExpressionofLAPTM4B-35:Anovelmarkerofprogression.invasiv enessandpoorprognosisofextrahepaticcholangiocarcinoma.Ca ncerLett264 (2): 209-217.) is recorded.Briefly, according to the ratio of cytoplasm positive stained cells, feminine gender is listed in 0 grade, 1 is weak (< 10%), 2 is moderate (10-25%), and 3 is that (26-75%) and 4 is very strong (> 75%) by force.Passing ratio is multiplied with density and obtains corresponding Di, the index obtained through scoring simplification carry out last assessment (namely 0 point, index 0-1; 1 point, index 2-4; 2 points, index 6-8; 3 points, index 9-12).The sample that dyeing scoring is at least 1 point is then listed in the positive.

The percent of the core positive cell of P-STAT3 is used to classify to Activity Rank, weak activity (+, < 25%), moderate activity (++, 26-50%), strong activity (+++, 51-75%) and very strong activity (++++, > 75%).

7, statistical analysis

All experiments at least in triplicate.Assess with t inspection and show difference between experimental group and matched group.With SPSS13.0, one way analysis of variance is carried out to data.Gene expression frequencies between PTPMeg2, STAT3 and pSTAT3 tumor sample is analyzed, adopts by the x of Fisher verification test correction ²explain frequency values < 5.Evaluated the dependency between protein level by Pearson's correlation coefficient and two-dimentional hierarchical clustering algorithm, the PS of all reports has dual character, with the level of P≤0.05 definition significance.

The interaction of embodiment 1, PTPMeg2 and STAT3

In order to study the negative regulatory factor of STAT3, carried out the examination of system to phosphatase, PTPMeg2 is considered to there is potential interaction with STAT3 albumen, studies the interaction of PTPMeg2 and STAT3 below.

One, the interaction of PTPMeg2 and STAT3

In order to clearly this interaction, co expression Myc-PTPMeg2 and Flag-STAT3 gene in HEK293T cell, and co-immunoprecipitation experiment and GST precipitation analysis have been carried out to the albumen of expressing.

Co-immunoprecipitation experiment in the body of 1, PTPMeg2 and STAT3

Prepare cell pyrolysis liquid from the HEK293T cell of exogenous expression Flag-STAT3 and Myc-PTPMeg2, with anti-Flag antibody sediment composite, detect the PTPMeg2 in sediment composite with anti-Myc antibody hybridization.

PcDNA3-Myc (purchased from Invitrogen company, catalog number V80020).

Myc-PTPMeg2 plasmid is that the encoding gene of PTPMeg2 (aminoacid sequence of PTPMeg2 is the sequence 1 in sequence table, and its encoding gene is the sequence 2 in sequence table) is inserted the plasmid that pcDNA3-Myc carrier obtains; Myc-PTPMeg2 plasmid (pcDNA3/Myc-PTPMeg2 plasmid) is documented in KrugerJM, etal.Protein-tyrosinephosphataseMEG2isexpressedbyhumanne utrophils.Localizationtothephagosomeandactivationbypolyp hosphoinositides.JBiolChem2002; 277:2620-8. the public can obtain from Tsing-Hua University.

Flag-STAT3 plasmid construction is on pXJ40 carrier, Flag-STAT3 plasmid (pXJ40/Flag-STAT3 plasmid) is documented in ZhangT, SeowKT, OngCT, CaoX.InterdomaininteractionofStat3regulatesitsSrchomolog y2domain-mediatedreceptorbindingactivity.JBiolChem2002; 277:17556-63.), the public can obtain from Tsing-Hua University, and the aminoacid sequence of STAT3 albumen is the sequence 3 in sequence table.

Specifically be divided into three groups:

Myc-PTPMeg2 group: by 1 microgram Myc-PTPMeg2 and 4 microgram pcDNA3-Myc empty carrier cotransfection HEK293T cells (purchased from basis institute of China Concord Medical Science University of Chinese Academy of Medical Sciences cell centre), cell culture in diameter be in the culture dish of 60mm.

Flag-STAT3 group: by 4 microgram Flag-STAT3 and 1 microgram pcDNA3-Myc empty carrier cotransfection HEK293T cell;

Myc-PTPMeg2 and Flag-STAT3 group: by 1 microgram Myc-PTPMeg2 and 4 microgram Flag-STAT3 transfection HEK293T cells;

After above-mentioned three groups of transfections after 24h, cell is according to co-immunoprecipitation experiment method above, carry out cracking, with anti-Flag antibody (purchased from SantaCruz company, catalog number: F-3165) sediment composite, detect the PTPMeg2 in sediment composite with anti-Myc antibody hybridization.

Result as shown in Figure 1A, illustrates that PTPMeg2 and STAT3 exists and interacts.

2, the external existence of PTPMeg2 and STAT3 interacts

GST plasmid (pXJ40/GST plasmid) is structured on pXJ40 carrier, is documented in RongYuetal.Wilms ' tumor1andsignaltransducersandactivatorsoftranscription3s ynergisticallypromotecellproliferation:apossiblemechanis minsporadicWilms ' tumor.CancerRes2006; 66:8049-57, the public can obtain from Tsing-Hua University.

GST-STAT3 plasmid (pXJ40/GST-STAT3 plasmid) is structured on pXJ40 carrier, is documented in RongYuetal.Wilms ' tumor1andsignaltransducersandactivatorsoftranscription3s ynergisticallypromotecellproliferation:apossiblemechanis minsporadicWilms ' tumor.CancerRes2006; 66:8049-57., the public can obtain from Tsing-Hua University.

By above-mentioned GST plasmid, GST-STAT3 plasmid and by the 1 Myc-PTPMeg2 plasmid obtained respectively cotransfection HEK293T cell carry out eukaryotic expression, transfection according to the method cell lysis of 1, obtained the cell pyrolysis liquid containing GST albumen respectively and contains the cell pyrolysis liquid of Myc-PTPMeg2 albumen, the cell pyrolysis liquid containing GST-STAT3 fusion rotein and the cell pyrolysis liquid containing Myc-PTPMeg2 albumen after 24 hours.

Above-mentioned two kinds of cell pyrolysis liquids are mixed rotation with 30ul glutathione agarose 4B granule 4 DEG C respectively spend the night, obtain GST complex and GST-STAT3 complex respectively, with SDS-PAGE glue protein isolate.

As shown in Figure 1B, the antibody hybridization (upper figure) indicated in figure, SDS-PAGE glue is with coomassie brilliant blue staining (figure below), and can find out, PTPMeg2 and STAT3 exists interaction in vitro for result.

3, there is endogenous interaction in PTPMeg2 and STAT3 albumen

Respectively that C57 mice is (magnificent purchased from dimension tonneau, lot number: SCXK (capital) 2011-0012) cerebral tissue and breast carcinoma MCF7 cell (purchased from basis institute of China Concord Medical Science University of Chinese Academy of Medical Sciences cell centre) carry out cracking according to the method for co-immunoprecipitation above, the antibody of hybridization is anti-STAT3 antibody (C20) and anti-PTPMeg2 antibody, and rabbit source property IgG is as negative control.

Result as shown in Figure 1 C, can be observed PTPMeg2 and STAT3 albumen and there is endogenous interaction.

4, there is location altogether in PTPMeg2 and STAT3 in cytoplasm

In order to observe the space orientation of two kinds of protein in intact cell more intuitively, be divided into 3 groups of experiments:

Flag-STAT3 group: 2 microgram Flag-STAT3 and 0.7 microgram pXJ40 empty carrier transfection MCF7 cell, cell culture is in six orifice plates;

Myc-PTPMeg2 group: by 0.7 microgram Myc-PTPMeg2 and 2 microgram pXJ40 empty carrier transfection MCF7 cells;

Flag-STAT3+Myc-PTPMeg2 group: by 2 microgram Flag-STAT3 and 0.7 microgram Myc-PTPMeg2 cotransfection MCF7 cell;

After transfection 24h, cell anti-Myc antibody (two resist for rabbit source FITC, display green fluorescence) and anti-Flag (two resist for Mus source TRITC, display red fluorescence) antibody hybridization, take pictures with laser confocal microscope.With the position of DAPI (display blue-fluorescence) showed cell core.The same visual field in intracellular position with the different albumen of three kinds of fluorescence records, is finally combined and is observed albumen whether there is common location in cell.Scale is 10 microns.

Result as shown in figure ip, can be found out, PTPMeg2 and STAT3 exists location altogether in Cytoplasm.

More than experiment proves that PTPMeg2 and STAT3 exists endogenous and ectogenic interaction.

Two, there is interaction in the STAT3 of PTPMeg2 and phosphorylation and unphosphorylated form

There are two kinds of forms in STAT3, the STAT3 of phosphorylation and unphosphorylated form in vivo, in order to determine the interaction type of PTPMeg2 and STAT3, is divided into 4 groups:

Myc-PTPMeg2: by 1 microgram Myc-PTPMeg2 plasmid and 4 microgram pcDNA3-Myc empty carrier transfection HEK-293T cells,

Flag-STAT3: by 4 microgram Flag-STAT3 plasmids and 1 microgram pcDNA3-Myc empty carrier transfection HEK-293T cell,

Flag-STAT3+Myc-PTPMeg2: by 4 microgram Flag-STAT3 and 1 microgram Myc-PTPMeg2 transfection HEK-293T cell,

Flag-STAT3+Myc-PTPMeg2+IL6: by 4 microgram Flag-STAT3 and 1 microgram Myc-PTPMeg2 transfection HEK-293T cell.

After transfection 24h, all cells change serum-free medium hungry 12 hours, and Flag-STAT3+Myc-PTPMeg2+IL6 group cell final concentration is 10ng/mlIL-6 process 30min, with the STAT3 of activating phosphatase.

The product of above-mentioned process is carried out cracking according to co-immunoprecipitation method above, cell pyrolysis liquid anti-Flag antibody co-precipitation, respectively with anti-Myc antibody hybridization sediment composite with anti-pSTAT3 (Tyr705) antibody hybridization sediment composite.

As shown in Figure 2 A, mutual immunity test shows result, and under IL-6 stimulates, the interaction of PTPMeg2 and STAT3 greatly increases.And an obvious pSTAT3 band is observed in the co-precipitation complex obtained with anti-Myc antibody (for catching Myc-PTPMEG2), this shows that the STAT3 of PTPMeg2 and phosphorylation and unphosphorylated form exists interaction, interacts stronger with the STAT3 of phosphorylation.

Three, PTPMeg2 reduces gathering of pSTAT3 in core

The same Myc-PTPMeg2 of plasmid PTPMeg2WT.

PTPMeg2CS plasmid (pcDNA3/PTPMeg2C515S plasmid) is documented in XuMJetal.PTP-MEG2isactivatedinpolycythemiaveraerythroidp rogenitorcellsandisrequiredforgrowthandexpansionoferythr oidcells.Blood2003; 102:4354-60. the public can obtain from Tsing-Hua University.

Be divided into 3 groups:

STAT3: by 1 microgram Flag-STAT3 and 4 microgram pcDNA3-Myc empty carrier transfection MCF7 cells;

STAT3+PTPMeg2CS: by 1.5 microgram Flag-STAT3 and 4 microgram PTPMeg2CS transfection MCF7 cells;

STAT3+PTPMeg2WT: by 1.5 microgram Flag-STAT3 and 0.5 microgram PTPMeg2WT transfection MCF7 cell;

Transfection, after 24 hours, is 10mg/mlIL-6 process 30min with final concentration, is contrast with what do not add IL-6.

Then by the cell after above-mentioned process, with anti-PTPMeg2 antibody, (two resist for anti-rabbit FITC, display green fluorescence) and anti-Flag (forFlag-STAT3, two resist for against murine TRITC, display red fluorescence) antibody hybridization, take pictures with laser confocal microscope.With DAPI (display blue-fluorescence) showed cell nuclear location.With the celluar localization of the indicated albumen of laser confocal microscope display.Merge the common location that image shows two kinds of albumen.Scale is 10 microns.

As shown in Figure 2 B, STAT3 is positioned at Cytoplasm to result under normal circumstances, but enters nucleus (Fig. 2 B, b3) after IL-6 stimulates.When STAT3 and PTPMeg2 coexpression, in kytoplasm, obviously can observe two kinds of albumen (Fig. 2 B, c3).What is interesting is, observe when with during PTPMeg2 coexpression by IL-6 stimulate under STAT3 still in Cytoplasm (Fig. 2 B, d1-3).This result shows, under IL-6 stimulates, PTPMeg2 hinders STAT3 from Cytoplasm to nuclear position transfer.As evidence, a kind of mutant PTPMeg2CS losing the ability of dephosphorylation STAT3, under the stimulation of IL-6, fails to stop STAT3 from Cytoplasm to nuclear translocation.

Above-mentioned all experimental datas show, PTPMeg2 causes low-level pSTAT3.

Four, PTPMeg2 has the dephosphorylation to pSTAT3

1, PTPMeg2 reduces the level of pSTAT3

Check PTPMeg2 whether dephosphorylation STAT3, HEK293T cell transfecting Flag-STAT3 and Myc-PTPMeg2 stimulate 30 minutes with 10ng/mlIL-6.

Specifically be divided into 3 groups:

Flag-STAT3: by 4 microgram Flag-STAT3 and 2 microgram pcDNA3-Myc empty carrier transfection HEK293T cells;

Flag-STAT3+PTPMeg2CS: by 4 microgram Flag-STAT3 and 2 microgram PTPMeg2CS transfection HEK293T cells;

Flag-STAT3+PTPMeg2WT: by 4 microgram Flag-STAT3,0.5 microgram PTPMeg2WT and 1.5 microgram pcDNA3-Myc empty carrier transfection HEK293T cells;

Transfection, after 24 hours, is (10ng/ml) IL-6 process 30min with final concentration, is contrast with what do not add IL-6.

Carry out cell lysis according to method above after process, antibody hybridization indicated in cell pyrolysis liquid figure, detect the expression of pSTAT3 (Tyr705).

Indicated antibody is as follows:

The antibody of pSTAT3 (Tyr705) is anti-pSTAT3 (Tyr705) antibody;

The antibody of Flag-STAT3 is anti-Flag antibody;

The antibody of PTPMeg2 is anti-PTPMeg2 antibody;

The antibody of B-Actin is anti-B tubulin (E-19);

The antibody of GFP is anti-green fluorescent protein (FL);

As shown in Figure 3A, the PTPMeg2 of wild type can make the reduced levels of pSTAT3 to result, and the PTPMeg2CS of saltant type does not then affect pSTAT3 level.Beta-Actin is as loading internal reference, and GFP is as the contrast of transfection efficiency.Result shows, during PTPMeg2 and STAT3 coexpression, pSTAT3 level declines (Fig. 3 A).In contrast, the transfection of mutant PTPMeg2CS fails to reduce pSTAT3 level (Fig. 3 A).

2, PTPMeg2 has the dephosphorylation ability of pSTAT3

Be divided into 2 groups:

Empty carrier group: 0.5 microgram pcDNA3-myc empty carrier and 2 microgram Flag-STAT3 are transfected in HEK293T cell;

PTPMeg2 group: 0.5 microgram Myc-PTPMeg2 and 2 microgram Flag-STAT3 is proceeded in HEK293T cell;

Above-mentioned two groups of respectively transfections after 24 hours hungry 12 hours, use IL-6 (10ng/ml) to stimulate again respectively 30 minutes and do not add IL-6 and cultivate 30 minutes, then the different time removes the factor, and add proteasome inhibitor MG132 (purchased from CB company, catalog number (Cat.No.): CB474791) (25 μMs) process 1 hour or 2 hours.

Cell lysis is carried out according to method above, antibody hybridization indicated in cell pyrolysis liquid figure after process.Indicated antibody is as follows:

The antibody of pSTAT3 (Tyr705) is anti-pSTAT3 (Tyr705) antibody;

The antibody of Flag-STAT3 is anti-Flag antibody;

The antibody of Myc-PTPMeg2 is anti-Myc antibody;

The antibody of B-Actin is anti-B tubulin (E-19);

Result as shown in Figure 3 B, pSTAT3 (Tyr705) lowers to some extent in different time points, this attenuating is not because add with not changing because of adding with proteasome inhibitor MG132 (25 μMs), prove that this attenuating is because dephosphorylation, instead of cause because of protein degradation.

Whether induce by the degradation process of dephosphorylation or protein in order to clear and definite pSTAT3 level declines, remove IL-6 and detect pSTAT3 level after adding protein degradation inhibitor MG132.Result shows: decline faster (Fig. 3 B) than pSTAT3 level when not having a PTPMeg2 during PTPMeg2 process LAN.Meanwhile, whether proteasome inhibitor MG132 exists all does not affect pSTAT3 level.These data representations, the dephosphorylation of the induced phosphorylated STAT3 of PTPMeg2 instead of degraded.

3, PTPMeg2 makes the dephosphorylized dynamic process of pSTAT3

Empty carrier group: 1 microgram pcDNA3-myc empty carrier and 2 microgram Flag-STAT are transfected in HEK293T cell;

PTPMeg2 group: 1 microgram Myc-PTPMeg2 and 2 microgram Flag-STAT is transfected in HEK293T cell;

Above-mentioned two groups of respectively transfections after 24 hours hungry 12 hours, use IL-6 (10ng/ml) to stimulate again respectively 30 minutes and do not add IL-6 and cultivate 30 minutes, different time (10,30,60min) removes the factor to observe the process of inhibition of phosphorylation.

Cell lysis is carried out according to method above, antibody hybridization indicated in cell pyrolysis liquid figure after process.

Indicated antibody is as follows:

The antibody of pSTAT3 (Tyr705) is anti-pSTAT3 (Tyr705) antibody;

The antibody of pSTAT3 (Ser727) is anti-pSTAT3 (Ser727) antibody;

The antibody of Flag-STAT3 is anti-Flag antibody;

The antibody of Myc-PTPMeg2 is anti-Myc antibody;

The antibody of B-Actin is anti-B tubulin (E-19);

The antibody of GFP is anti-green fluorescent protein (FL);

As shown in Figure 3 C, the dynamic change of pSTAT3 (Tyr705) level shows the dephosphorylation speed of pSTAT3 to result; Experimental data shows, and but the process LAN of PTPMeg2 promotes the dephosphorylation of pSTAT3 in Tyr705 site the not impact (Fig. 3 C) on Ser727 site, improves the dephosphorylation of pSTAT3 in Tyr705 site.

4, PTPMeg2 mediates the dephosphorylation of pSTAT3 in dose-dependent mode

(62.5,15.5,250, the 500 and 1000 μ g/ hole Myc-PTPMeg2 of HEK293T cell cotransfection Flag-STAT3 (2 μ g/ hole) and various dose, then IL-6 (10ng/ml) stimulates 30 minutes, not add IL-6 for contrast;

Cell lysis is carried out according to method above, antibody hybridization indicated in cell pyrolysis liquid figure after process.

Indicated antibody is as follows:

The antibody of pSTAT3 (Tyr705) is anti-pSTAT3 (Tyr705) antibody;

The antibody of Flag-STAT3 is anti-Flag antibody;

The antibody of Myc-PTPMeg2 is anti-Myc antibody;

The antibody of B-Actin is anti-B tubulin (E-19);

As shown in Figure 3 D, when showing PTPMeg2 and STAT3 coexpression, pSTAT3 level declines result.PTPMeg2 is confirmed in dose-response experiments further to pSTAT3 (Tyr705) dephosphorylation.These results show, PTPMeg2 is the phosphatase of a special pSTAT3 tyrosine residue.

Five, PTPMeg2 reduces the transcriptional activity of STAT3

1, PTPMeg2 suppresses IL-6 to stimulate the STAT3 transcriptional activity caused

Based on the interaction of PTPMeg2 and STAT3, query the transcriptional activity whether PTPMeg2 regulates and controls STAT3.For this reason, the APRE luciferase reporter gene of reflection STAT3 transcriptional activation is adopted, to detect the impact of PTPMeg2 on the transcriptional activation of STAT3.

MCF7 cell transfecting Myc-PTPMeg2 and Flag-STAT3, transfection APRE-Luc reporter plasmid and pRL-TK plasmid (as internal reference) Dual-Luciferase reporter plasmid method detect relative luciferase activity (ratio of detected value and internal reference value) simultaneously.Result, with mean ± standard deviation record, repeats independently to test for 3 times.

Totally 4 groups:

Matched group (control): with the reporter plasmid pGL3-(APRE) of 100ng ₄-luc plasmid (RongYuetal.Wilms ' tumor1andsignaltransducersandactivatorsoftranscription3s ynergisticallypromotecellproliferation:apossiblemechanis minsporadicWilms ' tumor.CancerRes2006; 66:8049-57., the public can obtain from Tsing-Hua University.) and 5ng internal reference plasmid pRL-TK (purchased from Promega, catalog number: E6241) cotransfection MCF7 cell;

PTPMeg2 group: with every hole 100ngMyc-PTPMeg2 transfection MCF7 cell;

STAT3 group: with every hole 400ngFlag-STAT3 transfection MCF7 cell;

PTPMeg2+STAT3 group: with every hole 100ngMyc-PTPMeg2 and 400ngFlag-STAT3 cotransfection MCF7 cell;

In order to each group of silencing endogenous PTPMeg2, RNAi is to MCF7 cell transfecting 800 μ gshPTPMeg2/pSilencer-4.1 plasmids (targeting sequence is 5 '-GATCCGGAAAGGCATTGTAAATTCAAGAGATTTACAATGCCTTCCTTCCTTA-3 ' (sequence 4)) (purchased from Shanghai Ji Ma companies).

Transfection is carried out adding/do not add IL-6 respectively and is stimulated 12 hours after 24 hours.

Adopt Dual-Luciferase report analysis system (VigofectInc. Beijing, China) carry out luciferase assay, with the activity value of renilla luciferase for internal reference, calculate the ratio of LUC Photinus pyralis LUC Photinus pyralis FL activity value and renilla luciferase activity value, what be reporter gene transcribes relative activity.Result is recorded by mean (average) ± standard deviation (S.D).

As shown in Figure 4 A, the APRE luciferase reporter gene relative activity of matched group (control) is 6.02 ± 0.32 to result; The post-stimulatory relative activity of IL-6 is 16.00 ± 0.16;

PTPMeg2 group APRE luciferase reporter gene relative activity is 2.38 ± 0.01; The post-stimulatory relative activity of IL-6 is 4.12 ± 0.21;

STAT3 group APRE luciferase reporter gene relative activity is 9.61 ± 0.96; The post-stimulatory relative activity of IL-6 is 54.12 ± 3.10;

PTPMeg2+STAT3 group APRE luciferase reporter gene relative activity is 2.76 ± 0.14; The post-stimulatory relative activity of IL-6 is 5.53 ± 0.48;

As seen from the figure, in MCF7 cell, the PTPMeg2 of process LAN causes the transcriptional activity of STAT3 to decline (Fig. 4 A).

2, PTPMeg2 suppresses the transcriptional activity of STAT3 in dose-dependent mode

PTPMeg2 and the STAT3 coexpression of various dose is in MCF7 cell, and adding/do not add IL-6 stimulates 12 hours.The PTPMeg2CS of various dose also with STAT3 cotransfection, specific as follows:

Identical with the method for above-mentioned 1, unlike being divided into 8 groups:

Matched group (control): with reporter plasmid pAPRE-luc and the 5ng internal contrast plasmid pRL-TK cotransfection MCF7 cell of 100ng;

STAT3: with every hole 400ngFlag-STAT3 transfection MCF7 cell;

PTPMeg2WT (50): with every hole 50ngMyc-PTPMeg2 transfection MCF7 cell;

PTPMeg2WT (100): with every hole 100ngMyc-PTPMdg2 transfection MCF7 cell;

PTPMeg2WT (200): with every hole 200ngMyc-PTPMdg2 transfection MCF7 cell;

PTPMdg2CS (100): with every hole 100ngMyc-PTPMdg2 transfection MCF7 cell;

PTPMeg2CS (200): with every hole 200ngMyc-PTPMdg2CS transfection MCF7 cell;

PTPMdg2CS (400): with every hole 400ngMyc-PTPMeg2CS transfection MCF7 cell;

Transfection total amount empty carrier pXJ40 furnishing balances.

Testing result as shown in Figure 4 B,

Matched group (control): APRE luciferase reporter gene relative activity is 6.02 ± 0.32; The post-stimulatory relative activity of IL-6 is 16.00 ± 0.16;

STAT3:APRE luciferase reporter gene relative activity is 9.61 ± 0.96; The post-stimulatory relative activity of IL-6 is 54.12 ± 3.10;

PTPMeg2WT (50): APRE luciferase reporter gene relative activity is 3.51 ± 0.62; The post-stimulatory relative activity of IL-6 is 21.85 ± 0.85;

PTPMeg2WT (100): APRE luciferase reporter gene relative activity is 3.55 ± 0.07; The post-stimulatory relative activity of IL-6 is 13.80 ± 0.64;

PTPMeg2WT (200): APRE luciferase reporter gene relative activity is 2.76 ± 0.14; The post-stimulatory relative activity of IL-6 is 7.49 ± 0.89;

PTPMeg2CS (100): APRE luciferase reporter gene relative activity is 6.82 ± 0.21; The post-stimulatory relative activity of IL-6 is 34.55 ± 3.15;

PTPMeg2CS (200): APRE luciferase reporter gene relative activity is 6.85 ± 0.41; The post-stimulatory relative activity of IL-6 is 62.72 ± 1.37;

PTPMeg2CS (400): APRE luciferase reporter gene relative activity is 10.32 ± 0.38; The post-stimulatory relative activity of IL-6 is 115.28 ± 0.48;

Can find out, result display is along with the increase of PTPMeg2 dosage, and PTPMeg2 can reduce the transcriptional activity of STAT3, and PTPMeg2CS then loses the inhibitory action of PTPMeg2 to STAT3 transcriptional activity.Therefore, the inhibitory action of this PTPMeg2 to the activity of the luciferase that STAT3 mediates has dose dependent (Fig. 4 B, left column).What is interesting is, when mutant PTPMeg2CS increases expression gradually, the activity of the luciferase of STAT3 mediation also increases (Fig. 4 B, right row).Result shows, the inhibitory action of PTPMeg2 to STAT3 transcriptional activity has dose dependent.

4, reticent PTPMeg2 can increase the transcriptional activity of STAT3

Mock plasmid: be pSilencer4.1 empty carrier, purchased from Shanghai Ji Ma company;

ShPTPMeg2 plasmid: shPTPMeg2/pSilencer-4.1.

Identical with the method for above-mentioned 1, unlike being divided into 6 groups:

Matched group (control): with reporter plasmid pAPRE-luc and the 5ng internal contrast plasmid pRL-TK cotransfection MCF7 cell of 100ng;

PTPMdg2+STAT3: with every hole 400ngMyc-PTPMdg2+400ngFlag-STAT3 transfection MCF7 cell;

Mock: with every hole 1200ngMock transfection MCF7 cell;

ShPTPMdg2+STAT3: with every hole 800ngshPTPMdg2+400nggFlag-STAT3 transfection MCF7 cell;

Testing result as shown in Figure 4 C,

The APRE luciferase reporter gene relative activity of matched group (control) is 6.02 ± 0.32; The post-stimulatory relative activity of IL-6 is 16.00 ± 0.16;

STAT3 group APRE luciferase reporter gene relative activity is 9.61 ± 0.96; The post-stimulatory relative activity of IL-6 is 54.12 ± 3.10;

PTPMdg2+STAT3 group APRE luciferase reporter gene relative activity is 2.76 ± 0.14; The post-stimulatory relative activity of IL-6 is 5.53 ± 0.48;

Mock:APRE luciferase reporter gene relative activity is 4.53 ± 0.93; The post-stimulatory relative activity of IL-6 is 10.65 ± 0.82;

ShPTPMeg2:APRE luciferase reporter gene relative activity is 5.25 ± 0.97; The post-stimulatory relative activity of IL-6 is 20.65 ± 1.22;

ShPTPMeg2+STAT3:APRE luciferase reporter gene relative activity is 6.24 ± 0.95; The post-stimulatory relative activity of IL-6 is 69.24 ± 1.06;

Can find out, shPTPMeg2 and STAT3 coexpression is in MCF7 cell, and transfection APRE-Luc reporter plasmid and pRL-TK internal reference simultaneously, application IL-6 stimulates 12 hours, and the disappearance of the PTPMeg2 caused by shRNA increases the transcriptional activity of STAT3.

Five, the level of PTPMeg2 and pSTAT3 is negative correlation in breast cancer cell

Choose the expression that breast cancer cell MCF7 and MDA-MB-231 (purchased from basis institute of China Concord Medical Science University of Chinese Academy of Medical Sciences cell centre) detect endogenous pSTAT3 (Tyr705) and PTPMeg2.

Respectively breast cancer cell MCF7 and breast cancer cell line MDA-MB-231 are carried out the albumen in antibody hybridization SDS-PAGE indicated in cell lysis, cell pyrolysis liquid figure according to method above.

Indicated antibody is as follows:

The antibody of pSTAT3 (Tyr705) is anti-pSTAT3 (Tyr705) antibody;

The antibody of STAT3 is anti-STAT3 antibody (F-2);

The antibody of PTPMeg2 is anti-PTPMeg2 antibody;

The antibody of B-Actin is anti-B tubulin (E-19);

As shown in Figure 5A, display endogenous pSTAT3 (Tyr705) is lower at MCF7 cells, and endogenous PTPMeg2 is relatively high for result.In MDA-MB-231 cell, endogenous pSTAT3 (Tyr705) is expressed higher, and endogenous PTPMeg2 is relatively low.Result shows, the level of PTPMeg2 albumen in MDA-MB-231 is lower than MCF7 cell, and the level of pSTAT3 then contrary (Fig. 5 A) simultaneously.This result prompting PTPMeg2 expresses and the expression of pSTAT3 exists certain relation.

Relation research between embodiment 2, PTPMeg2 and tumor growth or growth of tumour cell

Due to the phosphorylation of STAT3 and tumor is closely related, explores the impact of PTPMeg2 in the generation evolution of tumor.For this reason, research is derived from MCF7 (the low pernicious cell strain) cell of human breast cancer the changes of function obtained with the PTPMeg2 albumen of MDA-MB-231 (a kind of cell strain of Highly invasive) and after disappearance.

One, reticent PTPMeg2 is promoting the application in growth of tumour cell

1, reticent PTPMeg2 causes pSTAT3 level to increase increases with the expression of its downstream gene

Scramble plasmid is insert on pSilencer4.1 carrier, purchased from Shanghai Ji Ma company by the sequence (5 '-gATCCgAgTgCggATAgAgTAAATTCAAgAgATTTcTcAcGccTATcTcATTA-3 ') mixing shRNA;

ShPTPMeg2/pSilencer4.1 plasmid is the same;

Respectively by Scramble plasmid, shPTPMeg2/pSilencer4.1 plasmid and pSilencer4.1 (purchased from Shanghai Ji Ma company) empty carrier transfection MCF7 cell, after transfection after 24 hours, be digested to and be unicellularly laid on 96 orifice plates, screen with 800mg/LG418.Select positive colony, obtain MCF7/Scramble cell, MCF7/shPTPMeg2 cell and MCF7/Mock stable cell lines respectively.

Above-mentioned MCF7/Scramble, MCF7/shPTPMeg2 and MCF7/Mock stable cell lines is carried out the albumen in antibody hybridization SDS-PAGE indicated in cell lysis, cell pyrolysis liquid figure according to method above.

Indicated antibody is as follows:

The antibody of pSTAT3 (Tyr705) is anti-pSTAT3 (Tyr705) antibody;

The antibody of STAT3 is anti-STAT3 antibody (F-2);

The antibody of PTPMeg2 is anti-PTPMeg2 antibody; Result as shown in Figure 5 B, in order to verify that the pSTAT3 of rise is the reason that PTPMeg2 lowers, passes through the expression using the reticent PTPMeg2 of shRNA in MCF7 cell.During immunoblot experiment display PTPMeg2 disappearance, the expression of pSTAT3 sharply increases (Fig. 5 B, the right swimming lane).

2, reticent PTPMeg2 can cause the propagation of MCF7 cell to be accelerated

MCF7/Scramble, MCF7/shPTPMeg2 and MCF7/Mock stable cell lines obtained above-mentioned 1 plants in 96 orifice plates (1000 cells/well), and each treatment conditions establish 10 multiple holes, cultivate 5 days.After inoculating cell, get at interval of 24h the vegetative state that 96 orifice plates measure cell.Discard culture medium, every hole adds the MTT100ul (0.05mg/ml) of serum-free medium preparation, continues to cultivate 4h; And then discard whole culture medium, in each culture hole, add 150 μ lDMSO Rong Xie Jia Za granules.Drafting board within 10min, measure the proliferation activity of 570/630nm absorbance reflection cell, result adopts mean ± standard deviation record.

Result as shown in Figure 5 C, can be found out, MCF7/Scramble cell is respectively 0.270 ± 0.028,0.692 ± 0.056,1.116 ± 0.058,1.821 ± 0.057,1.376 ± 0.060 at the cultivation OD570/630nm of 1,2,3,4,5 days;

MCF7/Mock cell is respectively 0.271 ± 0.024,0.771 ± 0.110,1.009 ± 0.101,1.697 ± 0.056,1.318 ± 0.060 at the cultivation OD570/630nm of 1,2,3,4,5 days;

MCF7/shPTPMeg2 cell is respectively 0.258 ± 0.026,1.140 ± 0.087,1.916 ± 0.161,2.568 ± 0.172,2.188 ± 0.170 at the cultivation OD570/630nm of 1,2,3,4,5 days;

Cell proliferation experiment result shows, during PTPMeg2 disappearance, MCF7 Growth of Cells increases (Fig. 5 C).

3, reticent PTPMeg2 increases the growth of tumor

By above-mentioned 2 obtain 1 × 10 ⁷mCF7/shPTPMeg2 cell injects the oxter (n=6) of Female nude mice in 6 week age (purchased from dimension tonneau China, lot number: SCXK (capital) 2010-0009).Above-mentioned 2 of equal number obtains the opposite side oxter of MCF7/Mock cell subcutaneous injection to nude mice, every 2 days diameters by vernier caliper measurement tumor, within the 34th day, terminates experiment and weighs tumor weight.

The nude mice model tumor weight of nude mice bilateral oxter as shown in Figure 5 D, can be found out, MCF7/shPTPMeg2 cell infusion posterior tuberosity is heavily 1.28 ± 0.47 grams; MCF7/Mock cell infusion posterior tuberosity is heavily 0.22 ± 0.13 gram;

The transplanted tumor in nude mice size of nude mice bilateral oxter is as shown in Fig. 5 E and 5F, can find out, MCF7/shPTPMeg2 cell infusion posterior tuberosity is respectively 0 ± 0 at the volume of the 0th, 7,21,25,28,31,34 day, 2.9 ± 1.2,112.2 ± 57.1,337.6 ± 208.0,1199.6 ± 607.7,2518.0 ± 902.0mm ³; MCF7/Mock cell infusion posterior tuberosity is respectively 0 ± 0,0 ± 0,0.56 ± 0.19,0.70 ± 0.40,4.83 ± 2.25,21.5 ± 10.4,91.1 ± 81.6mm at the volume of the 0th, 7,21,25,28,31,34 day ³;

The Vitro Tumor Growth test of mice-transplanted tumor model shows, the MCF7 cell of PTPMeg2 disappearance forms larger tumor cell (Fig. 5 D-E) than compared with control cells, and growth more rapidly (Fig. 5 F).

Two, the application of process LAN PTPMeg2 in Tumor suppression and growth of cancer cells

1, process LAN PTPMeg2 suppresses the expression of pSTAT3 and downstream gene thereof

Build adenovirus:

Ad/PTPMeg2 plasmid records QiY, etal.Purificationandcharacterizationofproteintyrosinepho sphatasePTP-MEG2.JCellBiochem2002; 86:79-89., the public can obtain from Tsing-Hua University, compared with Ad/GFP, many PTPMeg2 albumen.

Ad/GFP is purchased from Invitrogen company, catalog number 954505.

MDA-MB-231/WT cell: the MDA-MB-231 cell not infecting adenovirus;

MDA-MB-231/Ad/PTPMeg2 cell (Ad/PTPMeg2): for by 10 ¹⁰the Ad/PTPMeg2 adenovirus infection MDA-MB-231 cell of VP obtains;

MDA-MB-231/Ad/GFP cell (Ad/GFP): for by 10 ¹⁰the Ad/GFP adenovirus infection MDA-MB-231 cell of VP obtains;

By each group of cell seeding of rear for above-mentioned infection 24 hours (1000 cells/well) in 96 orifice plates, cultivate 5 days.

Then each group of cell after above-mentioned process is carried out the albumen in antibody hybridization SDS-PAGE indicated in cell lysis, cell pyrolysis liquid figure according to method above.

Indicated antibody is as follows:

The antibody of pSTAT3 (Tyr705) is anti-pSTAT3 (Tyr705) antibody;

The antibody of STAT3 is anti-STAT3 antibody (F-2);

The antibody of PTPMeg2 is anti-PTPMeg2 antibody; Result as depicted in fig. 5g, uses adenoviral expression systems at MDA-MB-231 cell process LAN PTPMeg2.Result shows, the MDA-MB-231 cellular expression PTPMeg2 infecting adenovirus has comparatively low phosphorylation STAT3 level (Fig. 5 G, right lanes) than the cell infecting contrast adenovirus (GFP).

2, process LAN PTPMeg2 suppresses MDA-MB-231 cell proliferation

Mtt assay measures 570/630nm absorbance, and for detecting the quantity of above-mentioned MDA-MB-231/WT cell in cultivation 5 days, MDA-MB-231/Ad/PTPMeg2 cell, MDA-MB-231/Ad/GFP cell, result adopts mean ± standard deviation record.

Result as illustrated in fig. 5h, can be found out, MDA-MB-231 cell (WT) is respectively 0.267 ± 0.014,0.581 ± 0.087,1.205 ± 0.210,2.231 ± 0.224,1.377 ± 0.216 at the OD570/630nm of the 1st, 2,3,4,5 day;

MDA-MB-231/Ad/PTPMeg2 cell (Ad/PTPMeg2) is respectively 0.253 ± 0.027,0.540 ± 0.067,1.078 ± 0.045,1.748 ± 0.046,1.284 ± 0.049 at the OD570/630nm of the 1st, 2,3,4,5 day;

MDA-MB-231/Ad/GFP cell (Ad/GFP) is respectively 0.274 ± 0.023,0.339 ± 0.058,0.683,0.253 ± 0.126,0.267 ± 0.127,0.173 ± 0.137 at the OD570/630nm of the 1st, 2,3,4,5 day.

3, process LAN PTPMeg2MCF7 reduces the growth of tumor

Adopt the 2x10 that the method for above-mentioned one 3 obtains above-mentioned 1 ⁶mDA-MB-231/Ad/PTPMeg2 cell (Ad/PTPMeg2) and MDA-MB-231/Ad/GFP cell (Ad/GFP) inject oxter, female Balb/c-nu nude mice both sides in 6 week age respectively, within 34th day, terminate experiment and weigh tumor weight, and detect tumor volume betwixt.

Tumor size as shown in fig. 5i;

Tumor is heavy as indicated at figure 5j: be heavily 0.27 ± 0.11 gram the tumor of 34 days after MDA-MB-231/Ad/PTPMeg2 cell (Ad/PTPMeg2) cell infusion;

Tumor at 34 days after MDA-MB-231/Ad/GFP cell (Ad/GFP) injection is heavily 1.05 ± 0.12 grams;

Tumor volume is as it can be seen from figure 5k: at the tumor volume of 5,12,15,18,21 days respectively 0 ± 0 after MDA-MB-231/Ad/PTPMeg2 cell (Ad/PTPMeg2) cell infusion; 12.26 ± 5.15,36.80 ± 9.96,89.28 ± 34.89,168.07 ± 60.60mm ³;

Tumor volume at 5,12,15,18,21 days after MDA-MB-231/Ad/GFP cell (Ad/GFP) injection is respectively 3.45 ± 1.41,65.19 ± 26.76,163.59 ± 48.86,374.85 ± 101.75,677.96 ± 211.65mm ³;

As can be seen from the above, consistent with the low-level of pSTAT3 is, the MDA-MB-231 growth and proliferation of cell speed of overexpression PTPMeg2 slows down (Fig. 5 H), the transplanted tumor in nude mice volume formed reduces (Fig. 5, I-J), the speed of growth comparatively slow (Fig. 5 K) and tumor weight less (Fig. 5 J).These results show, the expression changing PTPMeg2 can affect growth and the propagation of tumor, and namely PTPMeg2 is a kind of antioncogene.

Three, the expression of PTPMeg2 becomes negative correlation with pSTAT3 level in adenocarcinoma of breast

Whether regulating the phosphorylation of STAT3 to understand PTPMeg2 in human tumor, exploring the relation that in human breast cancer, pSTAT3 and PTPMeg2 express.

The formalin detected from human breast cancer patient according to immunohistochemical method above fixes pSTAT3 in tumor tissues (being provided by Tangshan City People's Hospital of Hebei province), the expression of STAT3 and PTPMeg2 in people's adenocarcinoma of breast.

Representational immunohistochemical staining (Fig. 6) shows PTPMeg2, the staining conditions of pSTAT3 and STAT3.PTPMeg2 and STAT3 positive cell is positioned in endochylema, and pSTAT3 is positioned in nucleus.All tumor tissues are all operating Operated Specimens.Cancer other (P) or tumor tissues (T) separate by dotted lines.Scale, 50 microns.

Result is as shown in Table 1 and Table 2:

Table 1 is the expression of PTPMeg2 in breast carcinoma

X ²＝53.262，p＝0.000

Table 2 is pSTAT3 and the dependency of PTPMeg2 in breast carcinoma

r ^*＜0.05，r ^**＜0.01.

Result shows, in cancer beside organism, PTPMeg2 expresses strong (90%=37/41), and (83%=59/71) is expressed in the passiveness in tumor tissues.In contrast, in cancer beside organism, pSTAT3 maintains the level of lower (or negative), but maintains higher level at relative tumor tissues.By the coherent detection of Spearman, observe PTPMeg2 express and pSTAT3 level be that (Spearman's correlation coefficient is-3.33 to negative correlation, p value=0.004), this analysis also shows, in breast carcinoma, the level increase of STAT3 reduces be proportionate (correlation coefficient is-2.65, p value=0.023) with PTPMeg2.All results show, in breast carcinoma, the expression of PTPMeg2 and the expression of pSTAT3 have stronger dependency.

Above-mentionedly can to find out because the tyrosine residue of STAT3 to continue phosphorylation relevant to dissimilar human cancer (comprising breast carcinoma), targeting pSTAT3 has become the Critical policies for the treatment of of cancer.No matter the lasting phosphorylation of STAT3 albumen is by tyrosine kinase excessive activation or tyrosine phosphatase function is impaired to be caused.Although reported that many kinases activate STAT3 in tumor, it has still been necessary to identify special STAT3 phosphoprotein phosphatase.Up to the present, according to known, do not report the phosphatase of Direct Phosphorylation STAT3, although propose the dephosphorylation that PTP-TCs reconciles STAT3 and STAT1.In the present invention, find PTPMeg2 be one powerful directly and the phosphatase of STAT3 effect and mediate the dephosphorylation of pSTAT3.

The present invention has provided proves that STAT3 is several evidences of the substrate of PTPMeg2:

First, PTPMeg2 and STAT3 has the interaction on physiology and pathology.It should be noted that PTPMeg2 has powerful affinity to STAT3 and pSTAT3.

Secondly, PTPMeg2 dephosphorylation pSTAT3 has time-dependent and dose dependent.

3rd, the catalytic force effect to pSTAT3 negative regulation that has been the PTPMeg2 mutant loss of inertia.

The lipid binding territory of the four, PTPMeg2 lacks dephosphorylation function without impact.On the contrary, the PTPMeg2 of disappearance can strengthen pSTAT3 level, and causes making the propagation of nude mouse inner cell and the growth of tumor.With the data consistent of biochemistry and cytobiology, observe PTPMeg2 and pSTAT3 in mankind mastopathy cell's sample and express in negative correlation between the two.

The all result of the present invention has concordance and comparability.

Research is in the past thought, several PTPs is as PTPN1, PTPN3 and PTPN6 has carcinogenecity, but comprise PTPN12 (SunT, etal. (2011) ActivationofMultipleProto-oncogenicTyrosineKinasesinBrea stCancerviaLossofthePTPN12Phosphatase. (Translatedfromeng) Cell144 (5): 703-718 (ineng). have tumor suppression function at other interior PTPs.In the present invention, find, PTTMeg2 is the tumor-inhibiting factor of a kind of dephosphorylation STAT3.Have employed several cell model in the present invention to go to prove that PTPMeg2 forces expression inhibiting growth of tumour cell and PTPMeg2 disappearance to lead oncogenic expansion.What is interesting is, find the negative expression of PTPMeg2 in human breast cancer, and keep high expressed in Tumor-surrounding tissue.This expression pattern and PTPN7 (FridbergM, etal. (2008) ImmunohistochemicalanalysesofphosphatasesinchildhoodB-ce lllymphoma:lowerexpressionofPTENandHePTPandhighernumbero fpositivecellsfornuclearSHP2inB-celllymphomacasescompare dtocontrols. (Translatedfromeng) PediatrHematolOncol25 (6): 528-540 (ineng). with PTPN13 (Glondu-LassisM, etal. (2010) PTPL1/PTPN13regulatesbreastcancercellaggressivenessthrou ghdirectinactivationofSrckinase. (Translatedfromeng) CancerRes70 (12): 5116-5126 (ineng) is similar, PTPN7 and PTPN13 is reported in breast carcinoma, renal carcinoma, thoroughly lack in esophagus cancer.Recently, it is reported, PTPN13 in some tumors by somatic mutation, allelic loss, or promoter methylation and losing activity.But whether PTPMeg2 has this kind of mutation type still unclear in tumor.

It is reported, PTPL1/PTPN13 is by the aggressiveness (Glondu-LassisM of direct deactivation Src kinase regulatory breast carcinoma, etal. (2010) PTPL1/PTPN13regulatesbreastcancercellaggressivenessthrou ghdirectinactivationofSrckinase. (Translatedfromeng) CancerRes70 (12): 5116-5126 (ineng) .), PTPN12 comprises EGFR1 by multiple target spot, HER2 and Src kinases suppresses Metastasis in Breast Cancer at interior multiple target spots.A previous research report is artificial, and the action target spot of PTPMeg2 is EGFR and HER2.Under study for action, find PTPMeg2 directly and STAT3 interact.What is interesting is, when the phosphorylation using V-Src to activate STAT3, observe the dephosphorylation that PTPMeg2 mediates STAT3 strongly.But, do not observe the interaction that V-Src and PTPMeg2 is any.This result shows, PTPMeg2 directly locates STAT3.Because STAT3 and EGF-R ELISA or HER2 accompany, likely (YuanT as observed in forefathers, WangY, ZhaoZJ, & GuH (2010) Protein-tyrosinephosphatasePTPN9negativelyregulatesErbB2 andepidermalgrowthfactorreceptorsignalinginbreastcancerc ells. (Translatedfromeng) JBiolChem285 (20): 14861-14870 (ineng) .), PTPMeg2 is and the mutual effect of STAT3/EGFR composite bulk phase.

Another interesting observed result is, at PTPMeg2 special mediation STAT3 at the dephosphorylation of Try705 residue on the phosphorylation of STAT3Ser727 residue without impact.This is seemingly rational, because PTPMeg2 belongs to Protein Tyrosine Phosphatases family.STAT3 is very important to many cellular physiological processes processes, believes that the dephosphorylation of other tyrosine residue of STAT3 is mediated by other phosphatases not yet proved.

In a word, present invention demonstrates that the pSTAT3 dephosphorylation that Cytoplasm phosphatase PTPMeg2 directly mediates, the activity of negative regulation STAT3.PTPMeg2 passes through the growth of dephosphorylation STAT3 Tumor suppression consumingly.

Claims (3)

1.PTPMeg2 albumen has following 1 in preparation) or 2) application in the product of function:

1) stop pSTAT3 from Cytoplasm to nuclear position transfer;

2) transcriptional activity of STAT3 is suppressed;

The aminoacid sequence of described PTPMeg2 albumen is the sequence 1 in sequence table;

The aminoacid sequence of described STAT3 albumen is the sequence 3 in sequence table;

Described each function all directly mediates pSTAT3 dephosphorylation by described PTPMeg2 and realizes.

2. application according to claim 1, is characterized in that:

Described prevention pSTAT3 carries out IL-6 activates from Cytoplasm to nuclear position transfer;

The transcriptional activity of described suppression STAT3 carries out under IL-6 activates.

3. suppress the material of PTPMeg2 protein active to have following 1 in preparation)-4) at least one function product in application:

1) propagation of tumor cell is promoted;

2) tumor growth is promoted;

3) phosphorylation level of STAT3 is improved;

4) transcriptional activity of STAT3 is increased;

The material of described suppression PTPMeg2 protein active is following A or B:

A, a kind of nucleotide, its nucleotides sequence is classified as the sequence 4 in sequence table;

B, carrier, transgenic cell line or recombinant adenovirus containing nucleotide described in A;

Described tumor cell is MCF-7 cell;

Described tumor is breast carcinoma.