| Objective:According to statistics from the International Agency for Research on Cancer(IARC)of the World Health Organization in 2020,breast cancer has surpassed lung cancer to become the world's most common malignant tumor,with 2.26 million new cases in 2020,accounting for 11.7%of the total number of new cancers.Exploring the related mechanisms of the occurrence and development of breast cancer has always been a popular research in tumor-related science.Eg5 is a mitosis-related protein that plays an important role in the separation of the centrosome and the formation of the bipolar spindle in the early stage of mitosis.Studies have shown that Eg5 is highly expressed in breast cancer and other tumor tissues,and plays an important role in promoting the development of tumors.Down-regulating the expression of Eg5 protein will lead to the formation of a monopolar spindle during mitosis,induce cell apoptosis,and inhibit cell proliferation.At present,inhibitors targeted Eg5(such as monastrol)have been used as potential anti-tumor drugs in clinical trials,but studies have shown that Eg5 inhibitors are not very effective in treating tumors,and the combination of drugs has not yet found a synergistic effect.Therefore,it is of great significance to explore new Eg5 interacting proteins to provide new ideas for guiding clinical combination drugs.Tumor necrosis factor receptor associated factor 4(TRAF4)is one of the members of the TRAFs family of cytoplasmic adaptor proteins,and was first discovered in the metastatic lymph nodes of breast cancer.Studies have found that it is highly expressed in a variety of tumor tissues,and plays an important role in promoting tumorigenesis and regulating tumor cell proliferation,apoptosis,migration,invasion and other biological behaviors.It has been reported in the literature that TRAF4 can enhance the nuclear expression of PRMT5 in breast cancer cells,thereby promoting the proliferation of breast cancer cells.It also participates in the regulation of PI3K/AKT and TGF-? signaling pathways to promote tumor invasion and metastasis.However,the relevant mechanism of TRAF4 regulating tumor cell apoptosis has not yet been fully clarified.Rozan et al.detected Eg5 as a candidate protein that interacts with TRAF4 through yeast two-hybrid experiment and mass spectrometry experiment.In summary,the main purpose of this study is to detect the expression of TRAF4 and Eg5 in breast cancer tissues and cells and whether there is any interaction between the two protein;explore the role of TRAF4 in mitotic spindles assembly;explore The binding domain of TRAF4 and Eg5,and the related mechanism of TRAF4 regulating the expression of Eg5;finally,we will explore how TRAF4 and Eg5 regulate the biological behavior of breast cancer cells.Methods:1.Immunohistochemistry Surgically removed tumor specimens were fixed in 10%neutral formalin and embedded in paraffin.The specimens were then cut into 4-?m thick sections and baked in an oven at 70? for 2 h.The tissue sections were dewaxed in xylene,absolute ethanol,gradient alcohol,and distilled water,and then boiled in 0.01M citrate buffer(pH 6.0)at high temperature and pressure for 2min.Endogenous peroxidase activity was blocked with 0.3%hydrogen peroxide,and non-specific binding was blocked with 5%normal goat serum for 30min at 20?.Tissue sections were then incubated with TRAF4 rabbit polyclonal antibody(H2818,1:100,Santa Cruz Biotechnology)and Eg5 rabbit polyclonal antibody(ab37814,1:100,Abcam)at 4?overnight.Immunochemical reactions were developed using an Elivision super HRP(mouse/rabbit)immunohistochemistry kit(Maixin-Bio,Shenzhen,China)and 3,3-diaminobenzidine(DAB).The nuclei were stained with hematoxylin,and then the sections were dehydrated in ethanol before mounting.TRAF4 and Eg5 expression levels were evaluated based on the percentage of positive cells(PP)and staining intensity(SI)within the whole tissue section.Staining intensity was evaluated semi-quantitatively using the immune response score(IRS)and calculated using the equation:IRS=PP × SI.PP:0=no staining;1=1-25%;2=26-50%;3=51-75%;and 4=76-100%.SI:0=no staining;1=weak;2=moderate;3=strong staining.Each case was then defined as negative(IRS<3),low expression(3=<IRS<6),or high expression(6=<IRS?12).2.Cell lines and cell culture The non-tumorigenic mammary epithelial cell line MCF 10A,and the breast cancer cell lines MCF-7,MDA-MB-231,SK-BR-3,and MDA-MB-453 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences(Shanghai,China)and identified by short tandem repeat(STR)DNA analysis.MCF-10A cells were cultured in 1:1 Dulbecco's modified Eagle's medium(DMEM)/F12(Gibco,Waltham,MA,USA)supplemented with 5%serum,10?g/mL insulin(Sigma-Aldrich Co,St.Louis,MO,USA),and 20 ng/mL epidermal growth factor(EGF).MCF-7,SK-BR-3 and MDA-MB-453 cells were cultured in DMEM(Gibco,Waltham,MA,USA)supplemented with fetal bovine serum(FBS).MDA-MB-231 cells were cultured in L15(Gibco,Waltham,MA,USA)supplemented with 10%FBS.All cells were cultured at 37? in a humidified incubator with 5%CO2.3.Plasmid,siRNA,and transfection Plasmids for full-length TRAF4(wild type,WT),TRAF4 Zinc fingers domain deletion mutant(TRAF4 ? Zn)provided by Dr.Bert W.O'Malley.Plasmids for TRAF4 RING domain deletion mutant(TRAF4 ? R),TRAF4 TRAF domain deletion mutant(TRAF4 ? T)and HA-ubiquitin were purchased from Addgene(Cambridge,USA).Plasmids for Eg5 were purchased from OriGene(Rockville,MD,USA).TRAF4 siRNA,Smurf2 siRNA,and Eg5 siRNA were purchased from RiboBio(Guangzhou,China).Cells were transfected with plasmids using the Attractene Transfection Reagent or with siRNA using HiPerFect Transfection Reagent(Qiagen,Hilden,Germany)according to the manufacturer's protocols.The empty plasmid and scrambled sequences were used as controls.4.Screening of stable expression cell lines The Eg5-shRNA plasmid was purchased from Suzhou Gema Gene(puromycin resistant).Cells were seeded in a 24-well plate,and different concentration gradients of G418(1?1100?g/ml)or puromycin(1-11 ?g/ml)were added to determine the optimal screening concentration of the cell line.After 48 hours of cell transfection,add G418 or puromycin-containing culture medium(in this experiment,G418 screening concentration is 500?g/ml,puromycin screening concentration is ?g/ml),and the culture medium is replaced every 3 days.After about 4 weeks of screening,a cell line stably expressing the target gene was obtained.5.Western blot analysis Total protein was extracted from cell lines using lysis buffer(Thermo Fisher Scientific),containing a protease and phosphatase inhibitor cocktail(Beyotime,Shanghai,China),and quantified using the Bradford method.Next,40?g of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis(8%),and the separated proteins were transferred to polyvinylidene fluoride membranes(EMD Millipore,Billerica,MA,USA).After blocking with 5%non-fat milk in PBS,the membranes were incubated overnight at 4? with primary antibodies against:TRAF4(1:100),Eg5(1:1000),Smurf2(F0641,1:100,Santa Cruz Biotechnology),HA(1:1000),?-tubulin(1:1000),and GAPDH(1:2000).Next,the membranes were incubated with secondary HRP-conjugated antibody,anti-mouse immunoglobulin G(IgG),or anti-rabbit IgG(Santa Cruz Biotechnology Inc.)at 37? for 2h.Finally,antibody binding was visualized using electro-chemiluminescence(Thermo Fisher Scientific),and quantified using ImageJ(National Institute of Health,Bethesda,MD,USA).6.Co-Immunoprecipitation(Co-IP)Cell lysates were obtained as described above and precleared by rocking for 2h at 4? with 20?l(50%slurry)agarose A/G beads.After the beads were removed,the lysates were incubated with the appropriate antibodies(1-2?g antibodies per 200?g protein)at 4? overnight.Next,20?l(50%slurry)agarose A/G beads was added and the samples were rocked for 6 h at 4?.Finally,the immune complexes were washed with cell lysis buffer and protein bands were detected using immunoblotting assays.7.Immunofluorescence staining Breast cancer cells were cultured in 24-well plates for 24 h.The cells were fixed in 4%paraformaldehyde for 15min,and then treated with 0.2%Triton X-100 for 15min to ensure permeabilization.After blocking in 3%BSA for 1h at room temperature,the cells were incubated overnight at 4? with primary antibodies against:TRAF4(5100900,1:100,Biosciences),Eg5(1:100),and ?-tubulin(ab52866,1:100,Abcam).Next,the cells were incubated with a tetramethylrhodamine-labeled secondary antibody for 2 h at room temperature in the dark.The nuclei were then stained with DAPI.Finally,representative images were captured using an Olympus LH100-3 microscope(Olympus,Tokyo,Japan)..8.Flow cytometry measurement of cell apoptosis Cells were seeded in a sterile six-well plate and transfected.After 48 hours of culture,the cells were collected by centrifugation at 2000rpm for 5 minutes.Wash twice with PBS,add 500?l buffer to resuspend,add 5?l FITC-AnnexinV dye and 5 ?l PI dye to each tube,keep in the dark for 15 minutes,and measure by flow cytometer.9.Cell proliferation and colony formation assays Cell viability was measured by the mitochondrial reduction of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide(MTT)assay.Cells(3,000 cells/well)were seeded in 96-well plates in medium containing 10%FBS.Samples were treated with MTT solution(10?l/well)for 4h.The medium was aspirated from each well and 150?l DMSO was added.The absorbance was then measured at 490nm using a microplate reader.The OD value of a blank was subtracted from the absorbance obtained at each given time.We then calculated the relative ratio,and used these values to plot the cell proliferation curve.For colony formation experiments,cells(1,000/dish)were seeded in 40-mm dishes and incubated for 10-15 days.The cells were then fixed with methanol and stained with crystal violet.The number of colonies with>50 cells were counted.10.Tumor formation experiment in nude mice 15 BALB/c Nude nude mice aged 4 to 5 weeks were randomly divided into three groups.The tumor cells in the logarithmic growth phase were collected,prepared into a cell suspension with a cell number of 5 ×107/ml,and 200 ?l was subcutaneously injected into the armpit of a nude mouse.The tumor volume was measured and recorded every 3 days.11.Statistical analyses The Pearson chi-square test and Fisher's exact test were used to analyze the relationship between TRAF4 and Eg5 expression.Differences between the groups were analyzed using a paired t-test.Differences with P-values<0.05 were considered statistically significant.All experiments were performed at least three times.Results:1.TRAF4 and Eg5 are highly expressed in breast cancer,and the expression levels of these proteins are positively correlated.In order to detect the expression of TRAF4 and Eg5 in breast cancer tissues we used immunohistochemistry to detect the expression of TRAF4 and Eg5 in 16 normal breast tissues and 78 breast cancer tissues,and through western blot experiments we detect the expressions of TRAF4 and Eg5 in 16 fresh breast cancer tissues and its paired adjacent tissues,as well as normal breast epithelial cells and 4 breast cancer cell lines.Experimental results show that TRAF4 and Eg5 are highly expressed in breast cancer tissues and breast cancer cell lines.In addition,we statistically analyzed the correlation between the expression of the two proteins in 78 cases of breast cancer tissues.The results showed that there was a positive correlation between the expression of TRAF4 and Eg5 in breast cancer tissues.2.TRAF4 interacts with Eg5 and up-regulates the expression of Eg5 in breast cancer cells.We used immunofluorescence experiments to detect the subcellular localization of TRAF4 and Eg5 in four breast cancer cell lines,and found that both of them are co-localized in the cytoplasm in the four breast cancer cell lines,suggesting that TRAF4 and Eg5 may interact with each other.We selected Luminal A breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231 for follow-up experiments.We detected by co-immunoprecipitation experiment that both TRAF4 and Eg5 interact with each other in the two cell lines.Subsequently,in order to study the significance of the interaction between TRAF4 and Eg5,we transfected TRAF4 to detect the expression of Eg5 and found that the expression of Eg5 protein increased.Similarly,when we interfere with the expression of TRAF4,the expression of Eg5 protein decreases.The above results indicate that TRAF4 up-regulates the expression of Eg5.3.TRAF4 participates in spindle assembly by up-regulating the expression of Eg5 protein during mitosis.Because Eg5 is a mitosis-related protein,and TRAF4 interacts with Eg5 and up-regulates its expression,so we detected whether TRAF4 participates in the assembly of the mitotic spindle by regulating Eg5.We synchronize the cells and detect whether TRAF4 is differentially expressed between the interphase and the mitotic phases by western blot experiments.The results showed that TRAF4 was highly expressed during mitosis,suggesting that TRAF4 may be a mitosis-related protein.Next,we used immunofluorescence experiments to detect the subcellular localization of TRAF4 and Eg5 during mitosis.The results showed that TRAF4 and Eg5 co-localized in the throughout every stage of mitosis.Combined with the results of co-immunoprecipitation above,these results suggest that TRAF4 and Eg5 interact with each other during mitosis.We then detectected the expression of Eg5 in thymidine synchronized MCF-7 cells which were transfected with or without TRAF4 si-RNA.The results showed that the expression of Eg5 during 8-10h after DTB release was significantly reduced in TRAF4-depleted cells comparing with in control cells(Fig.3C).These data indicate that TRAF4 mainly positively regulates the expression of Eg5 protein during mitotic phase,and may be involved in the regulation of cell mitosis by up-regulating the expression of Eg5.Studies have confirmed that Eg5 plays an important role in the process of spindle assembly,and interference with the expression of Eg5 leads to the formation of monopolar spindles during mitosis.We have previously confirmed that TRAF4 regulates the expression of Eg5 protein during mitosis,so we speculate whether TRAF4 is involved in spindle assembly by regulating Eg5?We interfered with the expression of TRAF4 and detected the spindle morphology through immunofluorescence experiments.The results show that interference with TRAF4 can also lead to the formation of monopolar spindles,and this abnormality can be corrected by overexpression of Eg5 at the same time,indicating that TRAF4 participates in spindle assembly by up-regulating the expression of Eg5 during mitosis.4.The up-regulation of Eg5 by TRAF4 depends on its zinc finger and ring finger domains.In order to further study the binding domain of TRAF4 and Eg5,and the related mechanism of TRAF4 regulating Eg5 protein expression,we constructed plasmids with TRAF4 RING domain deletion,Zinc finger domain deletion and TRAF domain deletion.We carried out co-immunoprecipitation and found TRAF4 with Zinc fingers domain deletion mutant could not be dragged down by Eg5 antibody.Subsequently,we transfected wild type TRAF4,RING domain deleted(TRAF4 ? R);Zinc fingers domain deleted(TRAF4 ? Zn)or TRAF domain deleted(TRAF4 ? T)mutants into MCF7 and detected the expression of Eg5 protein by wsetern blot and found that the Zinc finger domain deleted and the RING domain deleted TRAF4 could not regulate Eg5 protein expression levels.The above results indicate that TRAF4 relies on the its zinc finger domain to interact with Eg5,and depends on both its zinc finger domain and its RING domain to up-regulate Eg5 expression.5.TRAF4 up-regulates the expression of Eg5 by inhibiting its ubiquitination.Because the RING domain of TRAF4 has ubiquitin E3 ligase activity,we tested whether TRAF4 affects the ubiquitination of Eg5.We overexpreseed TRAF4 and detected the ubiquitination of Eg5 through ubiquitination experiments,and found the ubiquitination level of Eg5 was decreased.Similarly,after interfering with the expression of TRAF4,the ubiquitination of Eg5 is enhanced.We further found through proteasome inhibition experiments that found after blocking the proteasome pathway with MG132,the regulation of Eg5 by TRAF4 was weakened,further verifying that the up-regulation of Eg5 by TRAF4 depends on the ubiquitin-proteasome pathway.Then we synchronized the cells to the G2/M phase and added cycloheximide to test the half-life of the Eg5 protein,and found that after interfering with the expression of TRAF4,the half-life of the Eg5 protein in the mitotic phase was shorter.These results suggest that TRAF4 improves the stability of Eg5 protein during mitosis by inhibiting the ubiquitination of Eg5.Finally,we transfected the full-length TRAF4 plasmid or the RING domain deleted TRAF4 into MCF-7 cells,and carried out ubiquitination experiments,and found that the inhibitory effect of TRAF4 on the ubiquitination of Eg5 depends on its RING domain.6.The inhibitory effect of TRAF4 on Eg5 ubiquitination is mediated by Smurf2.It is reported in the literature that TRAF4 interacts with Smad Specific E3 Ubiquitin Protein Ligase 2,Smurf2,and down-regulates its expression by promoting the ubiquitination of Smurf2.By screening the biogrid database,we found that Smurf 1,the homologous protein of Smurf2,may interact with Eg5.Therefore,we speculate that Smurf2 may be involved in mediating the regulation of Eg5 by TRAF4.We first verified the combination of TRAF4 and Smurf2 through co-immunoprecipitation experiments and ubiquitination experiments,and found TRAF4 down-regulated its expression by promoting the ubiquitination of Smurf2.Subsequently,we detected that Smurf2 and Eg5 can interact with each other through the co-immunoprecipitation experiment.Interference with Smurf2,the expression of Eg5 protein increased.Because Smurf2 has E3 ubiquitin ligase activity,we tested whether Smurf2 regulates the ubiquitination of Eg5 through ubiquitination experiments.The results showed that when Smurf2 was down-regulated,the ubiquitination of Eg5 protein was weakened,indicating that Smurf2 down-regulated the expression of Eg5 by promoting its ubiquitination.Therefore,we speculate whether TRAF4 stabilizes Eg5 by inhibiting the ubiquitination of Eg5 by Smurf2,thereby up-regulating its expression?We first tested whether the presence of TRAF4 could affect the binding of Smurf2 and Eg5.The co-immunoprecipitation results showed that after TRAF4 was down-regulated,the interaction between Smurf2 and Eg5 increased significantly,indicating that TRAF4 inhibits the interaction between Smurf2 and Eg5.We further co-transfected TRAF4-siRNA and Smurf2-siRNA,and detected the expression of Eg5 protein by western blot.The results show that interference with the expression of Smurf2 can weaken the down-regulation of Eg5 protein levels caused by TRAF4 depletion.Ubiquitination experiments also showed that interference with the expression of Smurf2 can weaken the ubiquitination of Eg5 protein caused by TRAF4 depletion.The above results indicate that TRAF4 inhibits the expression of Smurf2 protein by promoting the ubiquitination of Smurf2,thereby inhibiting the ubiquitination of Eg5 by Smurf2,thereby up-regulating the expression of Eg5 protein.7.TRAF4 inhibits breast cancer apoptosis and promotes breast cancer proliferation by up-regulating the expression of Eg5.We examined how TRAF4 and Eg5 regulate the biological behavior of tumor cells.We detected cell apoptosis by flow cytometry,and the results showed that the number of apoptosis in cells transfected with TRAF4 was reduced,and when we co-transfected Eg5-siRNA the inhibition of cell apoptosis caused by overexpression of TRAF4 was weakened.We have also detected some apoptosis-related proteins,and the results showed that overexpression of TRAF4 leaded to a decrease in the expression of Cleaved Caspase-3 and the pro-apoptotic protein Bax,while the expression of the anti-apoptotic protein Bcl-2 increased,and when we when we co-transfected Eg5-siRNA,the expression levels of all kinds of proteins recovered.The above results indicate that TRAF4 inhibits the apoptosis of breast cancer cells by up-regulating the expression of Eg5.Then we tested the effects of TRAF4 and Eg5 on the proliferation of breast cancer cells through MTT and clone formation experiments.We found that overexpression of TRAF4 promoted the proliferation of breast cancer cells,while co-transfecting TRAF4 plasmid and Eg5 si-RNA significantly reduced the TRAF4-induced proliferation of breast cancer cells,indicating that TRAF4 promotes the proliferation of breast cancer cells by up-regulating the expression of Eg5.Finally,we verified the results in vivo.We transplanted breast tumours developed from MCF-7 cells,which were stably transfected with empty plasmid or TRAF4 overexpressed pasmid or both TRAF4 overexpressed pasmid and Eg5 sh-RNA into nude mice and measured the tumors size every three days and draw growth curves.The results showed that the growth of tumors over-expressing TRAF4 was significantly accelerated,and when Eg5 was depleted at the same time,the promotion effect of TRAF4 on tumor growth was inhibited,verifying that TRAF4 promotes the proliferation of breast cancer cells by up-regulating the expression of Eg5.Conclusion:1.TRAF4 and Eg5 are highly expressed in breast cancer,and the expression levels of these proteins are positively correlated.2.TRAF4 and Eg5 co-localize in the cytoplasm of breast cancer cells,TRAF4 interacts with Eg5 and up-regulates the expression of Eg5.3.The expression level of TRAF4 in mitosis is higher than that in interphase,and it co-localizes with Eg5 during every stage of mitosis.Interference with TRAF4 mainly caused the down-regulation of Eg5 expression during mitosis.TRAF4 participates in the assembly of mitotic spindles by up-regulating the expression of Eg5,and interference with the expression of TRAF4 leads to the formation of monopolar spindles during mitosis..4.TRAF4 relies on the Zinc finger domain to interact with Eg5,and depends on its Zinc finger domain and the RING domain to up-regulate Eg5 expression.5.TRAF4 inhibits the ubiquitination of Eg5,therefore improves the stability of Eg5 protein during mitosis,and up-regulates the expression of Eg5.6.Smurf2 interacts with Eg5 and down-regulates the expression of Eg5 by promoting its ubiquitination.TRAF4 down-regulates the expression of Smurf2 by promoting its ubiquitination,and inhibits the interaction between Smurf2 and Eg5,thereby inhibiting the ubiquitination of Eg5 by Smurf2,thereby up-regulating Eg5 expression.7.TRAF4 inhibits breast cancer cells apoptosis and promotes breast cancer cells proliferation by up-regulating the expression of Eg5. |
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