TCEA1 Regulates Tbe Proliferation And Differentiation Potentials Of Mouse Granulocytes

BackgroundAcute myeloid leukemia(AML)is a hematological malignancy resulting from uncontrolled proliferation and impaired differentiation of hematopoietic stem cells or progenitor cells.Generally,translocation,aberrant gene expression,envirement and age of body as usually are overwhelming factors that are responsible for the ocurrence and prognosis of AML.Although many therapies such as induced chemotherapy,chemotherapy after remission therapy,targeted therapy and hematopoietic stem cell transplantation have been applied in the treatment of AML.However,AML is still a disease with high mortality rate and variable prognosis.Thus,the discovery and validation of new regulators that regulate proliferative potentials of myeloid cells could be of value to improve the understanding of leukemogenesis,to predict outcome,and to provide novel targets for therapy and diagnosisDuring the past decades,most of researchers were devoted into exploring the transcriptional regulation of the initiation stage,and elongation was thought to be a step without regulation in which bases were mechanically added to mRNA strand.But this idea was changed in recent years.Researchers have focused on the investigation of elongation,and it has been clear that elongation is a key regulatory step to control gene expression.Moreover,researchers have provided ample evidences and indicated that RNAP ? is often paused at promoter-proximal by DRB sensitivity-inducing factor(DSIF)and negative elongation factor(NELF).It is therefore that a subset of pause-release factors recruit positive elongation factors to stimulate the release of paused RNAP ? to promote elongation.Transcription elongation factor S-?,also named as TFIIS,is relatively conservation in eukaryotes.S-? is capable of restoring the paused RNAP ? to productive elongation and strongly enhancing transcription effects.It has well proposed that S-? has three family members,TCEA1(Transcription elongation factor A1,also named as TFIIS.o)encoded by Tceal,is the most common form of S-? and distributes widely in all tissues in eukaryotic.TCEA1 perhaps is the first general transcription elongation factor reported to associate with the pause-releasing of RNAP ?.Studies have showed that S-? promotes elongation of gene transcription by stimulating arrested polymerase RNAP ? to cleave nascent transcript.It is a well-known fact that a lot of transcription factors are required and expressed at specific phase in the development of myeloid cells.For differentiation,the expression of genes maintaining proliferation should be switched off,whereas,the expression of genes controlling differentiation should be switched on.The dysregulation of related factors in cells might disrupt the balance between the production and destruction of specific genes,thereby it is likely shifting towards cancer.Currently,studies on TCEA1 mainly focus on the structure,mechanism of releasing the paused RNAP ? and roles in solid tumors and erythropoiesis.Howeverthe role of TCEA1 in the development of granulocytes is still unclear.In the present study,we began with the screening of functional genes that regulate the proliferation potentials of granulocytes by a shRNA library(cancer 1000 library).We then explored the roles of TCEA1(an important candidate gene)in proliferation,differatiation and apoptosis of mouse model cells.In our study,we identified 18 candidate genes and showed that functional genes are capable of regulating granulocyte proliferation,Moreover our results demonstrated the important role of TCEA1 in regulation of myeloid cell fates.These findings extended our knowledge of TCEA1 function.Moreover,the discovery of the proliferative potentials of TCEA1 during the development of granulocytes is expected to provide new diagnostic indicator in the clinical therapy of myeloid cell derived leukemia,and provide a new insight into the targeted therapyObjectives1.To discover potential functional genes that regulate proliferative potentials of granulocytes by the combination of shRNA library screening with the methylcellulose colony formation2.To validate the regulating function of important candidate genes identified in the large-scale primary screening by combining cell biology experiments with biochemistry strategiesMaterials and Methods1.Cell culture1.1.BOSC23 cells were maintained in DMEM medium containing 10%FBS(Fetal Bovine Serum),100 u/ml penicillin and 100 ?g/ml streptomycin.1.2.32Dcl3 cells were maintained in RPMI 1640 containing 10%FBS(Fetal Bovine Serum),100 u/ml penicillin,100 dg/ml streptomycin and 1 ng/ml IL-3.2.shRNA screen2.1.Adult FVB/n mice were injected with 150 mg/kg 5-FU 5 days before harvest their BM cells.At day 5,mice BM cells were harvested under the aseptic condition2.2.The Establishment of screening system:As negative control group,5-FU treated FVB/n mouse BM cells(2×106)were cultured in methylcellulose containing 100 ng/ml G-CSF and 100 ng/ml SCF.As positive control group BM cells(1×106)transfected with MycT58A were cultured as mentioned above.5 days after plating colonies were collected and counted,and their ability of proliferation and differentiation was assessed by cell counting and morphological analysis.Then cells in the primary plating were re-plated for secondary culture.7 days after re-plating colonies were collected and counted,and their ability of proliferation and differentiation were assessed as mentoned above.2.3.shRNA library retrovirus packaging:BOSC23 cells were plated in the 10cm culture dish one day before transfection.At the transfection day,shRNAs were transfected into BOSC23 cells by lipo2000 regent.The virus supernatant was collected after 48-72 hours and stored at-80?.2.4.The transduction of BM cells:Virus was thawed.2 million BM cells were transduced by incubation with 1 million shRNA viral particles and centrifugation at 2400 rpm for 2 hours,infected cells were resuspended with pre-stimulation mediun and incubated at 37? 5%CO2.At the next day,the transduced procedure was repeated again.2.5.Methylcellulose colony-forming assay:After 1 day of culture,for staining 40 ug/ml 7-AAD was used.The transducted BM cells(GFP+7AAD-)were sorted into methylcellulose medium supplemented with recombinant murine G-CSF at 100 ng/ml and SCF at 100 ng/ml and cultured at 37? 5%CO2 for 5 days.After 5 days,cells were re-plated and grown in secondary culture for 7 days.At day 5 and day 7,colonies were collected and counted.Wright-Giemsa staining was used for morphology analysis.2.6.Identifying shRNAs in the screening:Colonies were removed into RNase/DNase-free centrifuge tube from the methylcellulose medium by 20-200 ul pipet tip and centrifuged at 1000rmp for 5 minutes.DNA extraction was performed according to a conventional protocol.The amplified polymerase chain reaction(PCR)was used to collect shRNAs fragment.For DNA sequencing,the shRNA-containing fragments were re-sub-cloned into the TA TOPO vector.Sequence alignment method was used to obtain genes information.3.Expression plasmids construction3.1.shRNA fragments obtained from shRNA library screening were cloned into the MSCV-LTR-miR30-SV40-GFP(LMS)expression vector.These shRNAs stable expression cell lines(32Dcl3-shTCEA1,32Dcl3-shEPS8,32Dcl3-shZNF212)were also constructed.3.2.We generated additional shRNAs targeting Tceal gene.These shRNA fragments and control shRNA were cloned into the MSCV-LTR-miR30-SV40-GFP(LMS)expression vector.We also established stable knockdown TCEA1 or control cell lines in 32Dcl3 by retroviral transfection.4.Resultant cell lines construction4.1.Retrovirus was obtained as mentioned above.4.2.Virus was thawed and added with 2ug/ml polybrene.32Dcl3 cells were transduced by incubation with virus and centrifugation at 2400 rpm for 2 hours,the transduced procedure was repeated the next day.Two days later,colonies were selected with 2 ug/ml puromycin and incubated 37? 5%CO2.5.Protein extraction5.1.Cell precipitation was collected and washed three times by PBS buffer for protein extraction.Next,cell precipitation was lysed on ice for 20 minutes by RIPA lysis buffer supplemented with 1 x protease inhibitor cocktail.Protein supernatant was obtained by centrifuging at 4? 12000rpm.Protein concentrations was determined by using the Bio-Rad BCA protein assay kit.6.Western blot6.1.Protein was denatured at 97?.30 ug of protein extracts per lane were electrophoresed with denaturing SDS-polyacrylamide gels(10%),and transferred to PVDF membranes.Membranes were blocked in TBST containing of 5%BSA for 2 hours at room temperature,and incubated with TCEA1 antibody at 1:1000 dilution,then washed three times with TBST followed by incubation with HRP-conjugated secondary antibody at 1:10000.TCEA1 expression level of resultant cell lines was detected by using ECL detection reagent and FluorChem E system.7.RNA extraction7.1.32Dcl3-shTCEA1.1669,32Dcl3-shTCEA 1.538 and control cells were cultured in differentiation medium for 24 hours.Cell precipitation was collected and incubated with 1ml TRIzol regent for 5 minutes at RT.Next,200ul chloroform was added,and incubation the homogenized sample for 5 minutes at RT for phase separation.Sample was centrifuged at 12000 rpm for 15 minutes at RT.Supernatant was transferred into a new tube,and RNA was precipitated by 500ul isopropyl alcohol.RNA pellet was collected and washed by 75%ethanol.Air-dry RNA pellet for 10 minutes and RNase-free water was used to dissolve RNA pellet,RNA concentration and quality were determined by Nanodrop machine.8.Quantitative real-time PCR(qPCR)8.1.The extracted RNA was used as temple,cDNA was prepared from RNA using a First Strand Synthesis kit.S YBR Green supermix and 50 ng of each cDNA was used to determined mRNA level of MPO,PRTN3,ELANE,LTF,MMP9 and CRISP3 in 32Dcl3-shTCEA1.1669,32Dcl3-shTCEA 1.538 and control cells lines9.Cell proliferation assay9.1.Trypan Blue staining and cell counting:32Dcl3-shTCEA1.1669,32Dcl3-shTCEA1.538 and control cells were cultured in growth medium supplemented with 1 ng/ml for 1,3,5 and 7 days or in differentiation medium containing 50 ng/ml G-SCF for 5 days.At the indicated day 10 ul cell sample was stained with 10 ul 0.4%Trypan Blue solution for 3 minutes.To determine proliferation/survival of cells,cell number was counted under microscope.Each sample were performed in triplicate.9.2.Cell cycle analysis:1×106 cells cultured in medium with 1 ng/ml IL-3 or 50 ng/ml G-CSF were collected and washed two times with PBS,and then fixed with 70%ice-cold ethanol for overnight.Cell sample was incubated with 200 ug/ml RNase A for 30 minutes at 37? and stained with 50 ug/mL propidiumiodide for 5 minures on ice.Cell cycle was analyzed by flow cytometry.10.Cell apoptosis assay10.1.1×106 cells cultured in medium with 1 ng/ml IL-3 or 50 ng/ml G-CSF were collected and washed two times with PBS.Cell apoptosis was quantified by flow cytometry with Annexin V-PE and 7-AAD staining.11.Morphological characterization11.1.Cells in differentiation medium were collected and resuspended in 10 ul PBS.Cytospin slides were made and stained with Wight-Giemsa regents.Specially,cytospin slides were fixed with 600 ul regent 1 for 1 minute,and stained with 1200 ul regent 2 for 7 minutes,washed with double distilled water for 30-40 seconds,air-dry and sealed with neutral resin.Microscopic examination was used to evaluate cell differentiation12.GEO database analysis12.1.GSE47044 database was reanalyzed to gain insight into the expression level of TCEA1 in normal and cancer cells.12.2.To analyze the influence of TCEA1 silencing on the change of important transcription factors.13.Statistical analysis13.1.The mean plus and minus the standard deviation(SD)were used in data.Data were analyzed by Graphpad Prism 5 software.Differences between groups were assessed by t-text.Comparisons between different groups were calculated with the Student t-test.P values less than 0.05 means significant.Results1.Identification of candidate genes that may increase the proliferative potentials of granulocytes by shRNA library screening:Retroviral particles of the shRNA library were transducted into 5-FU treated FVB/n mouse BM cells,and positive transducted BM cells(GFP+7-AAD-)were sorted by flow cytometry.Through methylcellulose colony-forming assay,we obtained 28 positive clonies phenotyped with increasing proliferative potentials,and information of shRNAs fragments obtained by sequencing and sequence alignment fall into 18 genes Thses genes involve in various cellular functions such as gene expression regulation,signal transduction,cell cycle control and transcription regulation.We then validated the ability of promoting proliferation of genes indentified by shRNA library by in vitro model cell line and showed that silencing Tceal,Eps8 and Znf212 are all capable of enhancing proliferative potentials.Percentages of cells in S phase in shTCEA1.shEPS8 and shZNF212 transfected cells were increased by 13.89%,9.39%,5.3%respectively than control cells.Cell survival of these cells was also increased by 271.95%,254.88%,60.98%respectively than control cells.These results indicated that we successfully screened functional genes which are capble of enhancing granulocyte proliferative potentials by shRNA library screening.Moreover,we showed that Tcea1 is the functional gene that performed the greatest influence on the proliferative potentials of granulocytes.Nextly,we used TCEA1 as a paradigm to validate shRNA library screening results in vitro.2.Expression of TCEA1 was downregulated in lymphoma(a kind of blood cancer)by using GEO databse(GSE47044):This result indicated a potential link between TCEA1 inactivation with proliferation of blood cells3.Silencing TCEA1 promotes granulocyte proliferation/survival:Cells were maintained in medium containing 1 ng/ml IL-3 or 50 ng/ml G-CSF.Compared with control cells,viability of resultant cells(32Dcl3-shTCEA1.1669 and 32Dcl3-shTCEA1.538)was increased significantly.Consistently,cell cycle analysis showed that percentage of cells in S phase of resultant cells(32Dcl3-shTCEA1.1669 and 32Dcl3-shTCEA 1.538)was respectively elevated by 8%,12%in medium with IL-3 and 5%.8%in medium with G-CSF.4.Silencing TCEA1 expression impairs granulocyte differentiation:TCEA1 depletion cells had charaters of immature myeloid cells.The expression of markers which is maninly expressed in myeloblast and promyelocyte stages such as myeloperoxidase(MPO),proteinase 3(PRTN3)and neutrophil elastase(ELANE)was elevated significantly in Tceal knockdown cell lines(32Dcl3-shTCEAl.1669,32Dcl3-shTCEA1.538).Consistently,Tceal knockdown cells expressed lower level of secondary granule protein lactoferrin(LTF),as well as the tertiary granule protein gelatinase(MMP9)and the cysteine-rich secretory protein 3(CRISP3)compared with control cells.Moreover,32Dcl3-shTCEA1.1669,32Dcl3-shTCEA1.538 cells exhibited immature cell morphology.They characterized with high nuclear:cytoplasmic ratio,round or ovoid nucleus,clear boundaries of nucleus,very small central hole and basophilic cytoplasm(According to qPCR results The cytoplasm is full with primary azurophilic granules).On the contrary,the expression of lactoferrin(LTF),gelatinase(MMP9)and the cysteine-rich secretory protein 3(CRISP3)was increased in control cells Of note,control cells exhibited mature phenotype at least more mature than TCEA1 knockdown cells.Specially,control cells have nucleus shaped with odd or segmented,and segmented nucleus have one to five segments.Combining with qPCR results,the cytoplasm of control cells is less basophilic and prominently express secondary and tertiary granule proteins.5.Silencing TCEA1 inhibits granulocyte apoptosis:Cells were maintained with medium containing IL-3 or G-CSF.In medium supplemented with 1 ng/ml IL-3,apoptosis of TCEA1 silent cells 32Dcl3-shTCEA 1.1669 and 32Dcl3-shTCEA1.538 was decreased by 8.17%and 7.77%respectively compared with control cells.Similar results were observed when cells were maintained in medium supplemented with 50 ng/ml G-CSF.Notably,apoptosis of TCEA1 silent cells 32Dcl3-shTCEA1.1669 and 32Dcl3-shTCEA1.538 was decreased by 37.5%and 3 1.37%respectively compared with control cells.6.TCEA1 knockdown influences the expression of some transcription factors analyzed by GEO databse analysis(GSE31912):Inactivation of TCEA1 led to concomitant elevating of C/EBP? and GFI-1,whereas reducing of C/EBP? and IRF8.Conclusions1.18 candidates(typically as TCEA1,EPS8,ZNF21 2)identified by shRNA library have potential capacity to regulate proliferation of granulocytes.2.TCEA1 plays a critical role in the development of granulocytes:TCEA1 silence promotes proliferation of granulocytes,while,losing of TCEA1 impairs differentiation and inhibits apoptosis of granulocytes.This disorder of development leads to accumulation of immature cells and absence of mature immune cells.3.TCEA1 silencing is related with tumorgenesis in blood cancer.4.TCEA1 regulates granulopoiesis probably by controlling related gene expression.Background Breast cancer is one of the prevalent malignances and characterized with complex pathogenesis and various subtype.The incidence rate of breast cancer continues to rise as a rate of 3%.More than 1 million women were diagnosed with breast cancer per year,and more than 450 thousand patients of them died of breast cancer in the worldwide.Currently,the therapeutic strategies for breast cancer are maily categorized into three groups: convertional surgical resection combined with chemotherapy,endocrine therapy for estrogen positive patients and target therapy for HER2 positive or HGFR mutant patients.However,it is difficult for distant-metastasis breast cancer such as Triple Negative Breast Cancer(TNBC)to benefit from such treatments.Therefore,reveal new mechanisms for breasr cancer pathogenesis is valuable to reveal the new molecular therapeutic target and its application for the clinical treatment of breast cancer,especially for triple negative breast cancer.C2ORF40(Chromosome 2 open reading frame 40)also named as ECRG4(Esophageal cancer-related gene 4)is widely expressed in human tissues.As a tumor suppressor,C2ORF40 regualtes cell proliferation,differentiation,senescence,invasion,migration,innate immunity and inflammation.Previous study revealed that the expression of C2ORF40 is down-regulated in esophageal carcinoma,breast cancer,colorectal cancer,nasopharyngeal carcinoma and glioma.Studies also showed that the level of C2ORF40 positively related with the disease free survival and metastasis free survival.Thus,it is reseanable that low level of C2ORF40 was used as an important biomarker for poor prognosis in breast cancer and nasopharyngeal carcinoma.C2ORF40 suppresses tumorgenesis by inhibiting cell proliferation,migration,invasion and promoting apoptosis.Currently,C2ORF40 was thought to suppress tumor by paracrine mechanism.However,it is unclear that how C2ORF40 transduces its tumor suppression signaling into intracellular and then regulates biological response.Receptor tyrosine kinases(RTKs)are generally comprised of a ligand binding region in the extracellular domain,a signling transmembrane helix and a cytoplasmic domain containing a tyrosine kinase domain which is responsible for the transduction of extracellular signals into cells.RTKs and their downstream signaling pathway were considered to be important in regulating cell proliferation,differentiation,invasion,migration and apoptosis.RTKs aberrations including activation mutation,over?expression and amplification were reported in malignances such as breast cancer.Epidermal Growth Factor Receptor 1(EGFR,also named as ErbBl),a member of type I receptor tyrosine kinase,EGFR over-expresion or signaling aberration is a mechanism to promote cell proliferation in various breast cancer.And positve of 'EGFR was considered as an independent poor prognostic factor in Triple Negative Breast Cancer(TNBC).Fibroblast Growth Factor Receptor 1(FGFR1),another member of RTKs family,conserved in envolution and regulated development and angiogenesis.Abnormal regulation of FGFR1 is significantly associated with breast tumorgenesis.Researches indicates that there is an association between the abnormal activation of FGFR1 signaling with tumorgenesis and drug resistance.Studies have showed that upregulation of FGF2-FGFR1 and its downstream signaling is responsible for drug resistance in cancer such as non-small cell lung cancer.Vascular Endothelial Growth Factor Receptor 1(VEGFR1,also named as Fltl),an important RTK belong to the vascular endothelial growth factor receptor family,was thought to participate in the regulation of angiogenesis,revascularization,tumorgenesis and metastasis.In recent years,studies have showed that VEGFR1 is not only a decoy receptor but also a positive regulator in angiogenesis.Meawhile,over-expression of VEGFR1 and abnormal activation of its signaling promoted cell proliferation and were associated with high risk of metastasis and recurrence in breast cancer.Of note,specific antibody of VEGFR1 or F56(an anti-angiogenic small peptide against VEGFR1)treatment inhibited tiie growth and metastasis of tumor cells by inhibiting signaling transduction.This study mainly identified the interaction partner of C2ORF40 by tandam affinity purification-mass spectrometry in breast cancer cells.Moreover,we primarily explored the signaling transduction mechanism underlying the tumor suppression action of C2ORF40.The establishment of the transmembrane signal transduction mechanism of C2ORF40 further reveals the mechanism of tumor suppression of C2ORF40.Objectives1.Tandam affinity purification-mass spectrometry(TAP-MS)was used to co-purify proteins associated with C2ORF40.Our findings provides a foundation to further explore transmembrane signaling transduction mediated by C2ORF40.2.To establish the signaling transduction mechanism of C2ORF40 tumor surpression.Materials and Methods1.Cell culture1.1.HEK293 T and human breast cancer cells MDAMB468 were maintained with DMEM medium containing 10% FBS(Fetal Bovine Serum),100 u/ml penicillin and 100)ig/ml streptomycin.1.2.Human breast cancer cells BT549 and MDAMB231 were maintained with RMPI1640 medium containing 10% FBS(Fetal Bovine Serum),100 u/ml penicillin and 100 pg/ml streptomycin.2.Expression plasmids construction2.1.C2ORF40 sequence was amplified from pBabe-puro-C2ORF40 plasmid.Retrovirus package plasmids pBabe-puro-3xFlag-HA-C2ORF40,pBabe-3xFlag-C2ORF40-HA and the relative empty vector were constructed by molecular colone technology.All plasmids were verified by sequencing technology.2.2.Transient expression plasmids pCMV 10-3xFlag-C20RF40-HA,pCMV10-3xFlag-HA-C2ORF40 and there respective control vectors were also constructed and verified by sequencing technology.3.The construction of cell lines stably expressing C2ORF403.1.Phi-NX cells were used to package retrovirus of pBabe-puro-3xFlag-HAC2ORF40,pBabe-3xFlag-C2ORF40-HA or the respective control plasmids.BT549 and MDAMB231 cells were transducted with the mentioned retrovirus to construct BT549-C2ORF40.MDAMB231-C2ORF40 and corresponding contro] ce?I lines.C2ORF40 silencing cells were constructed in our previous research.4.Tandem affinity purifying interaction partners of C2ORF404.1.Plasmids were amplified and extracted by Plasmid Maxprep Kit.Plasmids were transfected into HEK293 T cells by lip2000 reagent,and protein were collected after 48 h.Affinity purification was according to the FLAG? HA Tandem Affinity Purification Kit.Specifically,the anti-3xFlag M2 resin was used to coimmunoprecipitate 3xFlag-HA-C2ORF40 co-fusion protein and its copurification proteins.For the first elution,3xFlag peptide was used to elute the binding protein complex.The elution protein complex was purified further by second purification by binding to anti-HA resin.Final purified protein complex was subjected to separate by 10% SDS-PAGE and stained with ProteoSilver?Plus Silver Stain Kit.Special bands from sample in which over-express C2ORF40 were collected and stored at 4°C.5.Identifing the co-purification proteins of C2ORF40 by Mass Spectrometry5.1.Protein bands were destained and digested in-gel according to typic digestion protocol.The digested peptides were desalted by Zip Tip and analyzed by LCMS/MS.5.2.LC-MS/MS analysis: High pressure liquid chromatography associated with hybrid line ion trap mass spectrometer was used to separate and analyse the enzymolysis sample.MS data were searched in human protein database Mascot and Sequest,then analyzed by Proteome Discovererl.4.6.Verifying the results of TAP-MS by co-immunopreciptation(Co-IP)6.1.HEK293 T cells were transfected with pCMV10-3xFlag-C20RF40-HA or empty vector.After 48 hours,co-immunoprecipitation(Co-IP)of HEK293 T cells lysates with antibody to Flag was used to precipitate C2ORF40 and its partner protein GBR2.GRB2 protein was detected by Western blots technology.7.Co-immunopreciptation was used to detect the interaction between C2ORF40 withRTKs7.1.HEK293 T cells were transfected with pCMV10-3xFlag-C20RF40-HA or empty vector.After 48 hours,co-immunoprecipitation(Co-IP)of HEK293 T cells lysates with antibody to Flag was used to precipitate C2ORF40 and co-purified RKTs protein.The conrresponding RTKs antibodys were used to detect RTKs that specifically interacted with C2ORF40.8.Co-immunoprecipitation assay was used to verify the specific interaction between C2ORF40 with GRB2,VEGFR1,FGFR1 and EGFR in breast cancer cells8.1.Co-immunoprecipitation of MDAMB231-C2ORF40 cell lysates with Flag antibody was used to precipitate C2ORF40 and co-purified proteins.VEGFR1 antibody,FGFR1 antibody and EGFR antibody were used respectively to detect expression of VEGFR1,FGFR1 and EGFR.In addition,reverse coimmunoprecipitation of MDAMB231-C2ORF40 cell lysates with antibodies to FGFR1 ? VEGFR1 and EGFR to confirm the interaction between C2ORF40 with these RTKs.9.The shRNAs targeted on VEGFR1,FGFR1 and EGFR were used to explore the interaction between C2ORF40 with VEGFR1,FGFR1 and EGFR9.1.MDAMB231-C2ORF40 cells were transfected with shRNAs targeted on VEGFR1,FGFR1,EGFR or their corresponding control.Coimmunoprecipitation of cell lysates with Flag antibody was used to detect interaction between C2ORF40 with GRB2,VEGFR1,FGFR1 and EGFR.10.We detected the influencing of C2ORF40 over-expression on activation of RTKs and their downstream signaling10.1.C2ORF40 over-expression or corresponding control protein sample was prepared.We first analyzed phosphorylation of FGFR1,VEGFR1 and EGFR with or without C2ORF40 expression in breast cancer cells by immunoprecipitation with antibodies to FGFR1,VEGFR1 or EGFR and western blots with p-Tyr-100 antibody.10.2.We evaluated the phosphorylation of ERK under condition with C2ORF40 expression or C2ORF40 silence in breast cancer cells.11.We detected the influencing of C2ORF40 over-expression on RTKs phosphorylation induced by growth factors11.1.MDAMB231-C2ORF40 and corresponding control cells were treated with VEGF,FGF-2 and EGF respectively for 20 minutes.Co-immunoprecipitation of cell lysates with antibody to VEGFR1,FGFR1 and EGFR was used for purification.Western blots with p-Tyr-100 antibody was used to detect phosphorylation of VEGFR1,FGFR1 and EGFR induced by VEGF,FGF-2 and EGF respectively.12.The influencing of C2ORF40 on activation of RTKs downsteam protein underthc condition treated with RTKs inhibitors alone or combination12.1.C2ORF40 expression cells and the corresponding cells were treated with VEGFR1 inhibitor(ZM 306416),FGFR1 inhibitor(PD173074)and EGFR inhibitor(Gefitinib)alone or combination(the combination of ZM306416 with PD173074 used as paradigm)for 48 hours.Phosphorylation of ERK was detected by western blots.13.The influencing of C2ORF40 over-expression on breast cancer cell proliferation,migration and invasion with the treatment of RTKs inhibitor13.1.Cells expressing C2ORF40 or the corresponding empty vector were treated with VEGFR1 inhibitor(ZM 306416),FGFR1 inhibitor(PD173074)and EGFR inhibitor(Gefitinib)respectively.Cells proliferation was evaluated by MTT assay and clone formation assay.13.2.Cells expressing C2ORF40 or the corresponding empty vector were treated with VEGFR1 inhibitor(ZM 306416),FGFR1 inhibitor(PD173074)and EGFR inhibitor(Gefitinib)respectively.Migration of cells was evaluated by wound?healing assay and transwell assay.13.3.Cells expressing C2ORF40 or the corresponding empty vector were treated with VEGFR1 inhibitor(ZM 306416),FGFR1 inhibitor(PD173074)and EGFR inhibitor(Gefitinib)respectively.Invasion of cells was evaluated by matrigel assay.14.Statistical analysis14.1.Results of western blots were analyzed by ImageJ software of.Data shown in figures represents the mean plus and minus the standard deviation(SD)and analyzed by GraphPad Prism 5.Comparisons between different groups were calculated with the Student t-test The criteria of statistical significances was P values less than 0.05.Results1.Establishment of both transient over-expression and the retrovirus expression plasmids of C2ORF40 as well as their corresponding resultant cell lines: We constructed pCMV10-3xFlag-C20RF40-HA? pCMV10-3xFlagHA-C2ORF40,pBabe-puro-3xFlag-C2ORP40-HA? pBabe-puro-3xFlag-HAC2ORF40 and their corresponding control plasmids.For stably expressing,resultant breast cancer cells were also constructed and named as MDAMB231-C2ORF40,MDAMB231(expressing corresponding control plasmid).BT549-C2ORF40,BT549(expressing corresponding control plasmid).2.Identification of the combination of C2ORF40 with GRB2 by tandem affinity purification coupled with LC-MS/MS: pCMV10-3xFlag-C20RF40-HA plasmid was introduced into HEK293 T cells by Iip2000 reagent,and protein were collected after 48 h.Co-immunoprecipitation coupling anti-Flag M2 resin-with anti-HA resin was used to co-purify C2ORF40 protein and its binding proteins.Final purified protein complex was subjected to separate by 10% SDSPAGE.Protein bands were visualized by silver staining.These protein bands were destained and digested into peptides with trypsin in gel slice.Finally,we identified GRB2 as the interaction partner of C2ORF40.3.C2ORF40 interacted with VEGFR1,FGFR1 and EGFR: We discovered VEGFR1,FGFR1 and EGFR co-precipitated with C2ORF40 by coimmunoprecipitation both in HEK293 T and MDAMB231 breast cancer cells.But the other familiar RTKs such as VEGFR2,FGFR2,HER2,HER4 and ICiFlR didn't co-precipitate with CO2RF40.In addition,reverse co-immunoprecipitation of MDAMB231-C2ORF40 cell lysates with antibodies to FGFR1,VEGFR1 and EGFR confirmed that C2ORP40 interacts with VEGFR1,FGFR1 and EGFR.When specific shRNAs targeting FGFR1,VEGFR1 and EGFR respectively were transfected into MDAMB231-C2ORF40 resultant cells,there is reducing or even no detectable of FGFR1,VEGFR1 and EGFR protein in C2ORF40 co?precipitated complex.Taken together,those findings demonstrated that FGFR1.VEGFR1 and EGFR specially interacted with C2ORF40.4.C2ORF40 over-expression impaired activation of RTKs as well as their downstream signaling protein: MDAMB231-C2ORF40 cells exhibited a reduction of phosphorylated-FGFRl(p-FGFRl),phosphorylated-VEGFRl(pVEGFR1)and phosphorylated-EGFR(p-EGFR).Besides,the level of phosphorylated-ERK(p-ERK)was decreased significantly in C2ORF40 expression breast cancer cells compared with cells expressing empty vector.Whereas,the level of p-ERK was obviously increased in C2ORF40 silent cells.5.Abrogating activation of RTKs(VEGR1,FGFR1,EGFR)could not be reversed by corresponding growth factors: Cells were incubated with 50ng/ml VEGF for 20 minutes,co-immunoprecipitation coupled with western blots showed that the level of p-VEGFRl was significantly increased in MDAMB231 cells with the stimulation of VEGF.Whereas,level of p-VEGFRl is not changed obviously in MDAMB231-C2ORF40 cells with the stimulation of VEGF;Cells were incubated with lOng/ml FGF-2 for 20 minutes,co-immunoprecipitation coupled with western blots showed that the level of p-FGFRl was significantly increased in MDAMB231 cells with the stimulation of FGF-2.Whereas,level of p-VEGFRl is not changed obviously in MDAMB231-C2ORF40 cells with the stimulation of FGF-2;Cells were incubated with lOOng/ml EGF for 20 minutes,co-imxmmoprecipitation coupled with western blots showed that the level of pEGFR was significantly increased in MDAMB231 cells under the stimulation with EGF.Whereas,level of p-EGFR is not changed obviously in MDAMB231-C20RF40 cells with the stimulation of EGF.6.C2ORF40 inhibits ERK activation by multi-targeting RTKs: Breast cancer cells expression C2ORF40 or empty vector were treated with FGFR1 inhibitor PD173074(35 nM)? VEGFR1 inhibitor ZM306416(0.3...