| Objective:Incidence of prostate cancer is increasing in China during these years[1].Patients with prostate cancer have no obvious symptoms in the early stage.There is no efficient method for early diagnosis of prostate cancer.A few patients with early-stage prostate cancer were diagnosed during physical examination.Most of patients with prostate cancer were diagnosed in the advanced stage and they missed the opportunity for prostatectomy[2-4].Therefore,it is important for patients to diagnose prostate cancer in the early stage.Detection of biomarkers for early diagnosis of malignant tumors is still an important content of clinical research in recent years.Eyes absent homolog 2?Eya2?belongs to the the eyes absent?EYA?family proteins,which contain a highly conserved Eya domain and function as transcriptional cofactors[5].Recent years,some studies have reported that Eya2 is involved in the progression of cancer.The abnormal expression of Eya2 was observed in some malignant tumors including lung cancer,head and neck cancer,cervical cancer and astrocytoma[6-9].In this study,we evaluated expression pattern and clinical significance of Eya2 in prostate cancer.Then,we explored the biological function of Eya2 in prostate cancer cells and the potential mechanism.The research results may provided a basis for whether Eya2 may use as a biomarker for early diagnosis of prostate cancer.Methods:1.Tissue samples98 cases of human prostate cancer tissue and 16 cases of normal human prostate tissue were obtained from patients treated in Shengjing Hospital of China Medical University between 2013 and 2016.2.ImmunohistochemistryFormalin-fixed,paraffin-embedded tissues were used for immunohistochemistry staining.After deparaffinized and rehydrated,they were boiled?2 min in 0.01 M citrate buffer pH6.0?for antigen retrieval and blocking with BSA.Then,the first antibody Eya2 was added and incubated overnight.Biotin-labeled secondary antibodies were added to continue incubation.After the antibody incubation,the staining was carried out by DAB kit,and the nucleus was stained with hematoxylin.The staining intensity and positive percentage were observed to calculate the expression of Eya2 in prostate tissues.3.Cell cultureProstate cancer cell lines including DU145,LNCaP,PC-3 and normal cell line RWPE-1and the 293 cell were obtained from Shanghai Cell Bank,Chinese Academy of Sciences.Cells were cultured in medium containing 10%fetal bovine serum,100U/ml penicillin and streptomycin,in constant temperature 37?and saturated humidity.4.Western BlotProteins were subjected to SDS-PAGE.The membrane were incubated with the following primary antibodies at 4°C overnight:Eya2,cleaved caspase3,cleaved PARP,caspase3,PRAP,AKT,p-AKT,bcl-2,MMP7 and actin.This was followed by secondary antibody incubation with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody,and imaging on the imaging system using ECL kit.5.Realtime PCRCellular RNA was isolated using TRIZOL?Life technology?.For quantitative PCR cDNA was synthesized using the iScriptTM Reverse Transcription Supermix.The samples were analyzed using SYBR Green MasterMix on an ABI 7500 Realtime PCR system.?-actin was used as the endogenous calibrator control.Fold change of target gene was calculated according to 2-??Ct method.6.TransfectionEya2 shRNA,pMD2.G and pspax2 were purchased from Addgene,and lentivirus transfection was carried out by Lipofectamine 3000.Infected cells were selected with puromycin for one week.Eya2 plasmid was purchased from Addgene and plasmid was transfected with Lipofectamine 3000.7.CCK8 and colony formationCCK8 assay:Cells were seeded into 96-well plates at about 3000 cells per well,and CCK8 was used to detect the cell proliferation at 24h,48h,72h and 96h,respectively.A polyline diagram was plotted to observe changes of cell proliferation.Colony formation assay:Cells were seeded into 6cm dishes,cultured for 2 weeks and then stained with Giemsa.We collected images of dishes and counted the colony numbers.Then we analyzed changes caused by our treatment.8.Matrigel invasion assaysCells were seeded into a matrigel-coated transwell chamber.Cells which suspended in medium with 3%serum were seeded into the upper chamber.Medium contained 13%serum were added into the lower chamber.ALL chambers were incubated at 37?for18h.After incubation,erased the matrigel upon the membrane and fixed the cells on the membrane.Then we stained cells with hematoxylin.Cutting the membrane and sealed.We collected images of cells and counted the cell numbers.Then we analyzed changes caused by our treatment.9.Cell apoptosisAnnexin V/PI kit?BD bioscience,USA?was used for analysis of apoptosis.The assays were performed on ACEA Flow and the data was analyzed by NovoExpress software.10.Mitochondrial membrane potential?jc-1?Cells were suspended in serum-free medium.Positive control sample prestained with CCCP for 5min.And then all samples were incubated with jc-1 at 37?for 30min.Then stained cells were washed with PBS and analyzed using a flow cytometer.11.Statistic analysisWe carried out statistical analysis using SPSS 17 software.We obtained data of Eya2mRNA expression?gene chip?from Oncomine,which contains 52 prostate cancers with50 normal prostate tissues.Eya2 RNA-seq data containing 497 cases of prostate cancers was obtained from The Cancer Genome Atlas?TCGA?.Mann-Whitney U test was used to compare Eya2 mRNA in normal/cancer tissues and cancers with/without nodal metastasis.Kruskal-Wallis H test was used to compare Eya2 mRNA in cancers with different Gleason scores and T stages.A?2 test was used to examine possible correlations between Eya2 expression and clinicopathologic factors.Student's t-test was used to compare data obtained from biological experiments.p<0.05 was regarded as statistical significance.Res?lts:1.Eya2 was overexpressed in human prostate cancer tissues.5 out of 16?31.3%?cases of normal prostate specimens showed positive Eya2 nuclear staining.While 66 out of 98?67.3%?cases of prostate cancer specimens showed positive Eya2 nuclear staining.Overexpression of Eya2 was significantly correlated with higher Gleason score,TNM stage and T stage in prostate cancer.2.CCK8 assay results showed that Eya2 promoted prostate cancer cells proliferation;Colony formation results showed that Eya2 increased colony numbers of prostate cancer cells;Matrilgel invasion results suggested that Eya2 induced invasion of prostate cancer cells.3.Eya2 regulated Docetaxel sensitivity of prostate cancer cells.Eya2 overexpression enhanced cell vability in prostate cancer cells treated with Docetaxel.On the contrary,Eya2 depletion decresed cell vability.Eya2 overexpression inhibited the docetaxel-induced apoptosis.Conversely,Eya2 depletion increased apoptosis rate induced by docetaxel.4.Eya2 regulated expression of proteins related to cell proliferation,invasion and apoptosis.Eya2 overexpression increased expression of PARP and caspase3,and reduced expression of cleaved-caspase 3 and cleaved-PARP.Conversely,Eya2downregulation inhibited PARP and caspase3 expression,and promoted cleaved-caspase3 and cleaved-PARP expression.5.Eya2 regulated the mitochondrial membrane potential of prostate cancer cells.When Eya2 overexpression,the proportion of red fluorescence increased and the proportion of green fluorescence decreased.On the contrary,the red fluorescence decreased and the green fluorescence increased after Eya2 depletion.6.Eya2 activated AKT/bcl-2 signaling pathwy in prostate cancer cells.Overexpression of Eya2 upregulated the expression of bcl-2 and MMP7.Overexpression of Eya2 also promoted AKT phosphorylation.There was no significant change of Bcl2 expression when Eya2 overexpressed cell treated with AKT inhibitor.Conclusion:Eya2 was overexpressed in human prostate cancer.Eya2 overexpression was significantly correlated with higher Gleason score,adanced TNM stage and T stage in prostate cancer.Eya2 promoted proliferation and colony fomation in prostate cancer cells.Eya2 facilitated invasion and enhanced expression of MMP7.Eya2 inhibited cell apoptosis and upregulated bcl-2 level.Eya2 enhanced resistance of docetaxel in prostate cancer.Eya2 regulated the mitochondrial apoptosis pathway of prostate cancer cells through activating the AKT/Bcl-2 signaling pathway. |
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