Effect Of Fn14 On The Communication Of TRAF1 And TRAF2 Regulates Biological Behavior Of Breast Carcinoma Cells Via NF-κB Pathway

AbstractBreast cancer accounts for 25%of all female cancers,making it by far the most common malignant disease in world women.Metastasis of primary breast tumor cells through the blood or lymphatic system to distant organs is responsible for the majority of breast cancer deaths.Genomic characterization is beginning to define a molecular taxonomy for breast cancer;however,the molecular basis of invasion and metastasis remains poorly understood.Recent studies show that Fn14 is a tumor-specific cell surface marker,Fn14 gene was overexpressed in human esophageal cancer,liver cancer, breast cancer and brain cancer.It is presently unknown why the Fn14 gene is overexpressed in certain solid tumors relative to control non-tumorous tissue.A characteristic of Fn14 is that it contains a binding domain for TRAFs,adaptor cytoplasmatic molecules that activate downstream signalling.Fn14 can bind to four different TRAF proteins,TRAF 1,2,3 and 5.This association activates different intracellular pathways,including NF-kB,JNK,ERK and p38 activation.There is no study about the mechanism of fn14 and TRAFs.Plenty of studies show that TRAF1 and TRAF2 are the most important ones in all of the TRAFs.However,the exact action between TRAF1 and TRAF2 haven't known yet.As far there is no study about Fn14's effect on TRAF1 and TRAF2,then regulate the biological behavior in breast cancer cell lines. Materials and MethodsCells linesThe normal breast cell line MCF-10A,the low metastasis breast cancer cell line MDA-MB-231 and the high metastasis breast cancer cell line MDA-MB-435s.PlasmidsTargeting human Fn14 gene SiRNA sequences were designed using SiRNA Target Finder software program,pcDNA3.0-WT-Fn14 given by Shoji YAMAOKA(Tokey Medical and Dental University,JANPAN).The plasmid was verified by nucleotide sequence analysis.TransfectionCells were transfected with plasmid and siRNA,using Lipofectamine 2000 reagent according to the protocol of the manufacturer.Reverse transcriptase polymerase chain reaction(RT-PCR)To examine the mRNA of Fn14ImmunofluorescenceThe expressions of Fn14 in the above-mentioned cultured cell lines were examined through,immunofluorescence staining.Co-immunoprecipitationThe relationships between TRAF1 and TRAF2 with co-immuno precipitation, Fn14 and TRAF1,Fn14 and TRAF2Proliferation assays1×10³ cells were seeded in a 96-well plate,incubated for 24 to 120h,the MTT and dimethyl sulfoxide DMSO were added according to the protocol of the manufacturer. The OD value of each well was measured using a microplate reader with a test wavelength of 490 nm.Flow cytometryFlow cytometry were perfermed using Apoptosis Detection Kit according to the manufacturer's protocol on BD FACSCalibur^TM Flow Cytometer. Matrigel invasion assays100μl Cells were added to the upper chamber of an 8μm pore size coated with Matrigel.600μl RPMIDMEM/10%FBS was added to the lower chamber and cells were allowed to migrate for 24 hours.Cells were fixed in 4%Paraformaldehyde and stained with hematoxylin.The migrated cells were counted in 20 random high-power fields.Statistical analysisThe SPSS 14.0 software was employed to analyze the data.p＜0.05 was considered as statistical significance.Results1.Targeted up-regulation of communication of TRAF1 and TRAF2,the activation of NF-κB using pcDNA3.0-WT-Fn14 transfectionThe quantity of TRAF1 in combination with TRAF2 is significant higher in Fn14-overexpression cell line.2.Targeted down-regulation of communication of TRAF1 and TRAF2,the activation of NF-κB using Fn14SiRNA transfectionThe quantity of TRAF1 in combination with TRAF2 is significant lower in Fn14-inhibited by RNA interference cell line.3.Inhabition of Fn14 increases cell proliferation,invasion and reduces cell apoptosis in vitroMTT assay showed that the proliferation ability of cells transfected by Fn14SiRNA was significantly induced in comparison with those in the control cells. The apoptotic rate of the cells transfected with Fn14SiRNA was 13.3±1.05%, significantly lower than those of the control cells 41.5±2.15%(P＜0.01).The number of cells in control group that invade through Matrigel was 18.7±3.5.Overexpressionn of Fn14 increased significantly the number of the cells 67.0±2.6(P＜0.01).4.Overexpression of Fn14 decreases cell proliferation,invasion and induces cell apoptosis in vitroMTT assay showed that the proliferation ability of cells transfected by pcDNA3.0-WT-Fn14 was significantly reduced in comparison with those in the control cells.The apoptotic rate of the cells transfected with pcDNA3.0-WT-Fn14 was 42.6±4.56%,significantly higher than those of the control cells54.04±3.7%(P＜0.01). The number of cells in control group that invade through Matrigel was 29±1.6. Inhibiting Fn14 decreased significantly the number of the cells 7.6±3.5(P＜0.01).ConclusionspcDNA3.0-WT-Fn14 could effectively enhance the quantity of TRAF1 in combination with TRAF2 and induce breast carcinoma proliferation,invasive and metastasis,reduce cell apoptosis.Fn14SiRNA could effectively.inhibite the quantity of TRAF1 in combination with TRAF2 and reduce breast carcinoma proliferation, invasive and metastasis,induce cell apoptosis.