Prostate Cancer Biomarker Identification From Exosome The Function And Mechanism Of EYA1 Protein In Melanoma

Background:Prostate cancer(PCa)has now become the second largest killer of men’s health in the world,while clinically used biomarkers have serious limitations in sensitivity and specificity,such as prostate-specific antigens(PSA).Therefore,we need new biomarkers,but the complexity of body fluids often hampers the discovery of biomarkers.Recent studies on exosomes isolated from human body fluids indicated their potential in the discovery of improved biomarkers for PCa.The exosomes are small vesicles between 30-150 nm in diameter,which contain proteins that are specific to the tissue from which they derived and can therefore considered as treasure troves of disease-specific biomarkers.Materials and methods:Exosomes were extracted from clinical blood samples by using a commercial kit.The samples were divided into four groups:Normal,Low-risk,High-risk and Metastatic groups according to the pathological parameters and clinical diagnosis.After trypsin digestion of proteins in exosomes,proteins were identified by nano liquid chromatography and Q-Exactive Orbitrap Mass Spectrometry series(LC-MS/MS),and quantified in a label-free manner via the Normalized Spectral Abundance Factor(NSAF).These proteins were further excavated by bioinformatics to identify candidate biomarkers and validated with Western blot and PRM-MS techniques.Results:When the detection rate of each group was 50%,these samples could be classified into 4 groups very well after clustering analysis,which was almost the same as the initial grouped.After the depth screening of bioinformatics,10 candidate internal reference proteins,20 candidate detected differential proteins and 14 candidate expression differential proteins were identified.After the Western blot and LC-PRM-MS technology verification,it is determined that the three high expression and stable proteins of CD5L,Clusterin and GPX3 are suitable as internal reference proteins in the urinary system.At the same time,the differential protein was preliminarily verified.Conclusions:The use of ultra-high resolution liquid series mass spectrometry can be a good test detect the exosome proteins from the blood.The screening of three proteins CD5L,Clusterin,GPX3 can be serve as the internal reference proteins of plasma exosome in the urinary system.In order to screen differential proteins with specificity and sensitivity,we need further validation.Background:Cutaneous melanoma is a highly aggressive malignancy that has a high mortality.Despite the recent maj or progresses in developing new therapies,the maj ority of metastatic melanoma patients still succumb to this disease,indicating that there is an urgent need for more effective melanoma treatments.Recently we discovered a previously unknown phenomenon that melanoma biopsies contain constitutive aberrant expression of EYA1 gene over the normal controls.The Eyes Absent(EYA)proteins,first described in the context of fly eye development,have important physiological functions in cell proliferation,differentiation,apoptosis,cell cycle,innate immunity and DNA damage repair,and are also involved in the development of various tumors.However,the relationship of EYA and melanoma is unknown.Materials and Methods:Tissue microarray was used to measure the EYA1 expression in different stages of melanomas and normal skin controls.The EYA1 overexpression or knockdown A375 cells was used to measure cell proliferation,migration and invasion.Luciferase expressed B16 cells was used to investigate the tumor formation in vivo.Flow cytometry technique was used to examine the cell cycle progression and immunofluorescence staining was used for sub-cellular localization of EYA 1 protein.Comet assay was used to investigate the role of EYA 1 in DNA damage repair.Bio-ID technology was used to find the proteinsthat interact with EYA1.Results:Tissue microarray analysis results showed that EYAl was highly expressed in melanoma and increased with disease progression.And immunoflurecsence staining results showed that EYA1 protein was localized mostly in the nucleus.In vitro MTS assay and in vivo mouse tumor formation experiments shoewed that high expression of EYA1 promoted melanoma cell proliferation.Wound healing assay data showed that EYA1 enhanced the melanoma cell migration and invasion.Western blotting and flow cytometry results showed that EYA1 protein not only promoted the cell cycle from G2/M to G1 phase,but also increased the cyclin D1 expression and promote the cell cycle from G1 to S phase.Conclusion:EYA1 is acausative important factor in the pathogenesis and progression of melanoma,and it promoted the cell proliferation and melanoma formation through accelerated cell cycle,therefore,it could serve as a novel promising therapeutic target for melanoma.