| Objective:Breast cancer is one of the most common malignant tumors in women,and its incidence ranks first among female malignant tumors.According to data from the International Cancer Research Center of the World Health Organization,1.4 million women develop breast cancer and 460,000 women die of breast cancer worldwide each year.Surgical adjuvant chemotherapy and radiotherapy methods have significantly improved the efficacy of breast cancer.However,after a certain course of chemotherapy,patients often develop resistance to chemotherapy and the therapeutic effect is greatly reduced.The prognosis of breast cancer patients is still poor.Searching for new biomarkers can provide more possibilities to improve the overall survival of breast cancer patients.Ajuba is a member of the LIM family,which is characterized by tandem LIM protein in the C-termini.Ajuba can act as a tumor promoter in various cancers such as cervical cancer,esophageal squamous cell carcinoma,colorectal cancer,gastric cancer and pancreatic cancer.The regulatory network of Ajuba in tumor cells is complicated.In this research,we will explore the Ajuba expression and its clinicopathological significance in breast cancer.Also,the effect of Ajuba on the biological functions of breast cancer cells,especially on paclitaxel chemotherapy resistance will be investigated.Methods:1.Patients and Specimens93 cases of invasive breast cancer tissues and 15 cases of normal breast tissues were gained from patients undergoing breast cancer surgical resection in the First Affiliated Hospital of China Medical University.2.ImmunohistochemistryThe breast cancer tissues were embedded in paraffin and 4μm sections were prepared.Endogenousperoxidase was blocked by H2O2solution.All sections were incubated in citrate buffer.After non-specific blocking and incubation with Ajuba antibody（1:100）,the sections were treated with Elivision plus Polymer HRP IHC Kit.The sections were stained by DAB kit and counterstained with hematoxylin.The sections were deparaffinized,antigen retrieval,non-specific blocked and antibody incubation.The sections were treated with Elivision plus Polymer HRP（Mouse/Rabbit）IHC Kit（Maixin Biotech,Fuzhou,China）.The sections were stained by DAB kit and counterstained with hematoxylin3.Cell cultureHuman breast cancer cell lines（MDA-MB-453,MDA-MB-468,BT474,BT549,T47D,MCF7 and SK-BR-3）and the human normal breast cell line（MCF-10A）were obtained from American Type Culture Collection.All these cell lines were incubated in PRIM-1640 medium supplied with 10%（v/v）fetal bovine serum（Gibco,USA）,streptomycin and Penicillin.The cells were cultured in 5%CO2 at 37°C.4.Cell transfectionThe Ajuba plasmid and the corresponding negative control plasmid（p CMV6）were obtained from Origene company.Transfection was performed using Lipofectamine 3000.Ajuba small interfering si RNA was purchased from Dharmacon.Dharmafect1 reagent was used for si RNA transfection.5.Western blotTotal protein lysate was harvested using RIPA cell lysis buffer with protease inhibitors.Proteins were separated by SDS-PAGE and transferred to PVDF membranes.The PVDF membranes were incubated with primary antibodies Ajuba（1:800）,Survivin,GLUT3,TAZ,DDK,GAPDH（1:1000）.Protein visualization was performed using a chemiluminescence ECL Detection kit.6.Quantitative PCRTotal RNA was extracted using TRIzol reagent.RNAwas reverse transcribed using Prime Script RT Master Mix Kit.SYBR select master Mix Kitwas used for q PCR.Relative expression of TRIM66 was normalized to GAPDH and calculated according to2-△△ct.7.Cell proliferation assay（1）CCK8:cells were firstly plated into 96-well plates（3000 cells/well）.10μl of CCK-8reagent was added into each well.After incubation,absorbance was examined using a microplate reader（wavelength:450 nm）.（2）Colony formation assays:cells were seeded into plates and cultured for about 2 weeks.The cells were then stained using Giemsa and counted under a microscope.8.Cell cylce assayThe modified cells were treated with trypsin and washed with PBS butter.Cells were fixed in paraformaldehyde and stained with 5 mg/ml propidium iodide.The cell solution was incubated in the dark for 15 minutes,and flow cytometry was used to detect the fluorescence.9.Transwell invasion assayThe invasion assay was performed using 24-well transwell chambers,and the chambers were coated with matrigel.Cell mixture without serum was plated in the upper chamber,and medium supplied with 10%（v/v）FBS was added in the lower chamber.Subsequently,plates were incubated at 37℃for 24 hours.Then cells at the bottom of the transwell were fixed and stained with 0.1%hematoxylin.The number of invading cells was counted under a microscope.10.Apoptosis assayThe modified cells were treated with trypsin and washed with PBS butter.Cells were stained with 5 mg/ml propidium iodide together with Annexin V/FITC.The cell solution was incubated in the dark for 15 minutes,and flow cytometry was used to detect the fluorescence.11.Glucose uptake assayCells were trypsinizated from the plate and washed with PBS buffer.The 2-NBDG was added to each cell suspension at a final concentration of 10μM and incubated for 30minutes.The fluorescence intensity was measured using Flow Cytometer.12.Paclitaxel resistance assaysMCF-7 and BT549 cells were transfected with Ajuba plasmid and si RNA,then were treated with paclitaxel.CCK-8 assays were used to examine the effect of Ajuba on cell viability.Annexin V/PI staining assays were used to examine the effect of Ajuba on cell apoptosis.13.Chromatin immunoprecipitation（ChIP）assayThe binding sites of TEAD4 and GLUT3/Survivin promoter were predicted via JASPAR.Chromatin immunoprecipitation（ChIP）assay was performed using the Magna ChIP A/G Assay Kit.The precipitated DNA fragments were quantified using PCR analysis with SYBR Green Master Mix.14.Statistical analysisAll statistical analyses were performed using the software package SPSS 22.0.Aχ2 test was used to analyze the possible associations between Ajuba expression and clinicopathological parameters.Overall survival was visualized using Kaplan-Meier curves,and the differences were compared by Log-rank test.Differences in gene expression,proliferation capacity,invasion capacity,and apoptosis rate were assessed using the Student’s t-test.P<0.05 was considered to indicate statistical significance.Results:1.Ajuba is upregulated in breast cancerAjuba is highly expressed in human breast cancer tissues and cells.Normal breast tissues showed negative or weak Ajuba staining.In 93 cases examined,51（54.8%）cases showed high Ajuba expression.There was a significant association between Ajuba expression and poor patient survival.Endogenous expression of Ajuba was relatively higher in breast cancer cell lines compared to that in MCF-10A,especially in triple-negative cell lines.2.Ajuba promotes proliferation,invasion and cell cycle transition in breast cancer cells CCK-8 assays demonstrated that Ajubadepletion downregulated the growth rate while Ajuba plasmid transfection increased the cellular growth rate.Colony formation assays showed that Ajuba knockdown decreased colony formation ability while Ajuba transfection upregulated colony formation ability.Transwell invasion assays showed that Ajuba depletion inhibited invasion while Ajuba overexpression increased invading ability in breast cancer cells.Ajuba overexpression significantly increased the S phase percentage and decreased the G1 phase percentage,while Ajuba depletion decreased the S phase percentage and increased the G1 phase percentage in breast cancer cells.3.Ajubapromotesglucose uptake in breast cancer cellsCancer cells adopt glucose metabolism to produce energy.2-NBDG uptake assay showed that Ajuba knockdown inhibited the level of glucose uptake while Ajuba overexpression upregulated glucose uptake4.Ajuba enhances paclitaxel resistance of breast cancer cells MCF-7 and BT549 cells were treated with paclitaxel.CCK-8 assays showed that Ajubaknockdown increased the paclitaxel inhibition rate while Ajuba overexpression decreased it after 24 and 48 h of treatment.Ajuba overexpression decreased the apoptosis rate,while Ajuba depletion showed the opposite results.5.Ajubapositivelyregulates TAZ-GLUT3/Survivin signalingAjuba overexpression promotes expression of GLUT3,Survivin and TAZ,while Ajuba depletion inhibits GLUT3,Survivin and TAZ expression.We used TAZ si RNA in MCF7cells co-transfected with the Ajuba plasmid.TAZ knockdown significantly suppressed the protein and m RNA expression of GLUT3 and Survivin.TAZ depletion also ameliorated the effect of Ajuba overexpression on GLUT3/Survivin at both the protein and m RNA levels.TAZhas been reported as a transcription co-activator of TEAD4,TEAD4 could bind to the promoter regions of both GLUT3 and Survivin in BT549 cells.Conclusion:Ajuba is upregulated in human breast cancer and is negatively correlated with the overall survival of patients.Ajuba promotes breast cancer cell proliferation,invasion,and cell cycle transition.Ajuba overexpression upregulates GLUT3 expression,enhancesglucose uptake and promotes glucose metabolism of breast cancer cells.Ajuba inhibits paclitaxelinduced apoptosis and enhances paclitaxel resistance of breast cancer cells.Ajuba upregulates the expression of the apoptosis-inhibiting protein Survivin.Ajuba promotes the transcriptional expression of Hippo signaling target genes GLUT3and Survivinvia upregulating the expression of TAZ,which is transcriptional co-factor of TEAD4. |
