| Objective: Prostate cancer(PCa)is the most common tumor in men and is second only to lung cancer in cancer-related deaths in western countries.The incidence of prostate cancer is increasing rapidly in China.Symptom of initial stage of prostate cancer is not apparent,easy miss cure opportunity.Androgen deprivation therapy(ADT)is the treatment of choice for hormone-sensitive prostate cancer in patients with metastatic PCa or advanced prostate cancer.The median duration of hormone sensitivity is 18 to 36 months,and most patients will progress to metastatic castration-resistant prostate cancer(m CRPC).m CRPC patients are treated with chemotherapy drugs,androgen receptor axis targeting(ARAT)drugs,Radium 223 radiation therapy,and immunotherapy.However,even with some improvements and new treatment strategies,the clinical prognosis and outcomes of m CRPC are poor.Today,serum prostate-specific antigen(PSA)and digital rectal examination are the early screening methods for prostate cancer,and prostate biopsy(biopsy)remains the gold standard.Prostate puncture biopsy is an invasive examination with many postoperative complications.Specific,sensitive and non-invasive precise biomarkers are urgently needed in clinical practice.Of this study is to through the analysis of prostate cancer treatment group and castration resistant prostate cancer patients group of plasma source secrete body protein expression spectrum difference,screening the secrete of castration resistant prostate cancer related differences in protein,further to enzyme-linked immunosorbent(ELISA)method to detect and cytological experiments to test and verify the effect of differences in protein,It provides experimental basis for exploring new biomarkers and therapeutic targets for prostate cancer.Methods: 1)5ml of whole blood from 22 untreated prostate cancer patients and22 castration-resistant prostate cancer patients were collected with EDTA anticoagulant tubes,respectively.Plasma was separated,plasma exosomes were extracted and cryopreserved.Proteomic detection was performed in 3 cases in each group.Exosomes were detected by electron microscopy,and the isolated vesicles were confirmed to be exosomes.Their concentration was detected,particle size was analyzed,exosomes were lysed,and proteins were enzymatically hydrolyzed.Unlabeled quantitative liquid chromatography-mass spectrometry was used for detection and quantitative analysis of differential proteins.Further bioinformatics analysis of differential proteins;2)The detected differential proteins were screened with good selection reliability.Protein interaction analysis suggested that protein α-1B glycoprotein(corresponding gene A1BG)had good correlation with other proteins for subsequent experiments.Two groups of patients all outside the plasma cryopreserved secrete body specimen,enzyme-linked immunosorbent experiments(ELSA),at the same time all outside secrete specimens with enzyme-linked immunosorbent experiment(ELSA)examined CD9,CD9 biomarkers for outside secrete body,whether can confirm samples for outside body secretion,and at the same time outside the detection of two groups of patients with plasma body protein secretion of α-1B glycoprotein concentrations,The differences between the two groups were statistically compared.3)Four kinds of prostate cancer cells were cultured in culture medium with excluded secretions,including PC-3,22RV1,DU-145 and LNCap.PC-3 and DU-145 were castration-resistant prostate cancer cells,and 22RV1 and LNCap were hormone-sensitive cells.Westernblot was used to detect the α-1B glycoprotein concentration in the exosomes of four cell lines.The results showed that the α-1B glycoprotein concentration in the exosomes of the castration-resistant prostate cancer cell lines PC-3 was higher than that in the castration-resistant prostate cancer cell lines DU-145.Therefore,PC-3 cell lines with higher concentration of exosome α-1B glycoprotein were selected for subsequent experiments.PC-3 cells were transfected with plasmid and A1 BG gene was silenced by RNAi technique.CCK8 and Transwell methods were used to detect and compare the changes of proliferation and invasiveness of prostate cancer cells before and after RNA interference.The changes of cell growth and invasiveness after A1 BG protein expression inhibition were observed.Results: 1)2564peptides and 259 proteins were identified by label-free method.There were A total of 22 proteins with P<0.05 conforming to the expression differential multiple of more than1.5times,among which 4 proteins were up-regulated(B/A>1.5times)and 18 proteins were down-regulated(A/B>1.5times).There were 6 differentially expressed proteins with multiple of difference greater than 2.0,of which 3 were up-regulated and 3 were down-regulated.In addition,7 proteins with null values were detected in group B,and 4proteins with null values were detected in group A.The enrichment analysis of KEGG pathway showed that there was no significant enrichment of KEGG pathway.The specific location of the proteins or expressed products in the cells suggested that 87.5%of the differential proteins were mainly distributed outside the cells,and 6.2% of the proteins were located in mitochondria and solutes respectively.The results of structural domain annotation and enrichment analysis indicated that there was no significant enrichment of the structural domain;2)CD9 is a biomarker for exosomes.ELISA detection of CD9 concentration first confirmed that the detected exosomes were exosomes.Secondly,the expression of CD9 in untreated prostate cancer group was significantly higher than that in CRPC group(P<0.01),suggesting that the concentration of exosomes in untreated prostate cancer group was higher than that in CRPC group.The concentration of plasma exosome protein A1 BG in CRPC group was significantly higher than that in untreated prostate cancer group(P<0.01),suggesting a high correlation between α-1B glycoprotein and CRPC.3)the expression of A1 BG gene was silenced by RNAi technology,and the expression of A1 BG significantly decreased after RNA interference was verified by Westernblot.Comparative observation showed that the growth trend and invasiveness of PC3 cells were significantly decreased after A1 BG gene silencing.Conclusion: Label-free technique can analyze the difference of plasma exosome protein expression between untreated prostate cancer patients and CRPC patients.Label-free technology is a good platform for screening meaningful biomarkers from differential proteins.In this study,the differentially expressed protein obtained by label-free analysis of prostate cancer was reliable,and the differentially expressed proteinα-1B glycoprotein(A1BG)was screened out to further expand the number of cases for comparison and verification.There was significant difference between the two groups.The concentration of α-1B glycoprotein in the plasma of castration-resistant prostate cancer patients was significantly higher than that of untreated prostate cancer patients.These results suggest that α-1B glycoprotein may be a potential biomarker of castration-resistant prostate cancer.In vitro cultured prostate cancer PC3 cells,A1 BG was silenced,and the α-1B glycoprotein was significantly decreased by Westernblot after RNA interference.The growth and invasivity of prostate cancer cell PC-3 were decreased after RNA interference,suggesting that A1 BG may be a potential therapeutic target of CRPC.This study provides a new experimental basis for the diagnosis and treatment of CRPC,and the sample size can be expanded to verify the conclusions of this study in the future. |
PDF Full Text Request