| Objective: Prostate cancer(PCa)is one of the commonest malignant solid tumors in the male urological system.Its incidence ranks the second place among male cancers worldwide,only second to lung cancer.There are significant regional differences in the distribution of PCa.In the United States,prostate cancer has the highest morbidity and the second highest mortality in men,accounting for 19% of the total incidence and 9% of mortality in male malignant tumors.With the social development of China,the gradually aging population,the westernization of dietary structure and lifestyle,the improvement of medical early detection and screening,the incidence of prostate cancer is increasing year by year and growing rapidly.According to the latest report released by the national cancer center in 2018,PCa has surpassed bladder cancer as the commonest malignant tumor in male urinary system in China.Our research team screened the differentially expressed proteins in the serum and urine from the patients with benign prostatic hyperplasia and different levels of PCa by proteomics study.We then conducted bioinformatics analysis to find the candidate protein PF4V1(platelet factor 4 variant 1),and verified that the PF4V1 level has the same trend in serum and urine from other 100 patients by ELISA experiment.PF4V1 is a chemokine that can significantly inhibit neovascularization and is secreted by platelets,smooth muscle cells and tumor cells.PF4V1 can inhibit endothelial cell migration and angiogenesis.In recent years,studies have found that PF4V1 is abnormally expressed in ovarian cancer,osteosarcoma and melanoma,compared with the normal control group.Over-expression of PF4V1 will inhibit the proliferation and metastasis of tumor cells.However,relevant studies are still in the preliminary stage.The signal pathways in which PF4V1 is involved among the tumor microenvironment,and the mechanisms of PF4V1 in the proliferation,invasion and metastasis in different tumors are not clear.At present,the function and mechanisms of PF4V1 in PCa have not been reported.Methods: In the first part,we used the clinical data with large samples from thepublic database(GEO,TCGA)to explore the expression difference and prognostic value of PF4V1 in PCa.The expression level of PF4V1 in prostate cancer cell lines was verified by western blot and PCR.Lentivirus vector was transfected to overexpress PF4V1.The effects of PF4V1 on the proliferation,migration,invasion and other biological behaviors of PCa cells were tested by CCK-8,clonal formation experiment,scratch wound experiment,transwell experiment,etc.We then explore the therapeutic effect of PF4V1 protein on PCa in xenograft tumor model of nude mice.In the second part,multiple bioinformatics softwares were used to predict the possible miRNAs that binding and regulating PF4V1.Mimics transfection was used to overexpress miRNA.We then observe whether a single miRNA had regulatory effect on PCa cells with high PF4V1 expression.Dual-luciferase reporter assay was used to prove the binding region of miRNA and the target mRNA.We also used multiple pathway enrichment analyses,such as GO,KEGG,PPI and INTERPRO,to find related signaling pathways,and explored the expression differences of downstream key proteins of by western blot.We wish to expand the understanding of the function and regulatory mechanisms of PF4V1 in PCa,and to provide molecular targets for potential therapeutic applications.Results: Part I: 1.We searched the prostate cancer gene expression in the GEO database and selected two datasets,GSE60329 and GSE55945.The GSE60329 dataset included 54 PCa samples and 14 normal prostate controls.The GSE55945 dataset included 13 PCa specimens and 8 normal prostate controls.The expression level of PF4V1 mRNA in PCa tissues in the two datasets above was lower than that in the control group of normal prostate tissues(P<0.05).2.In the TCGA database,the expression level of PF4V1 in 497 cases of PCa tissues was lower than that in 52 cases of normal prostate tissues,with a statistically significant difference(P<0.05).3.PF4V1 has prognostic ability in PCa.We analyzed the survival data of 490 patients with PCa,and found that PCa patients with high PF4V1/TP53 had a shorter disease-free survival time(P < 0.05)but a longer overall survival time(P < 0.05)through the Kaplan-Meier survival curve.4.Overexpression of PF4V1 inhibited PCa cell proliferation.We used puromycin to screen the stable transfected lentivirus PF4V1 DU145 and LNCaP cell lines.CCK-8 experiment showed that compared withthe control group transfected with empty virus,the OD value of PCa cell lines with stable overexpression of PF4V1 was significantly reduced at 48h(P<0.05).Compared with the control group,the number of clones of PCa cell lines with stable over expression of PF4V1 decreased significantly(P<0.05).5.Overexpression of PF4V1 inhibited PCa cell migration.Scratch wound test showed that the scratch width of PCa cell lines with stable overexpression of PF4V1 at 24 h was significantly higher than that of the control group(P<0.05).Transwell assay suggested that compared with the control group,PCa cell lines with stable overexpression of PF4V1 significantly reduced the number of cells passing through the compartment membrane(P<0.05).6.Overexpression of PF4V1 inhibited PCa cell invasion.The transwell assay using matrigel suggested that compared with the control group,the number of cells crossing the compartment membrane of PCa cell lines with stable overexpression of PF4V1 was significantly reduced(P<0.05).Part Ⅱ: We used 6 kinds of bioinformatics software,miRanda,miRDB,miRwalk,PICTAR,RNA22,Targetscan,to predict the miRNAs that might bind to PF4V1.We took the intersection from high to low rank according to the number of results,and obtained 8 candidates: miR-129-5p,miR-27 a,miR-374 a,miR-875-3p,miR-543,miR-27 b,miR-410,miR-1299.2.After transfecting the mimics to up-regulate these 8miRNAs in DU145 cell line,it was found that overexpression of miR-875-3p promoted the proliferation level of DU145 cells with PF4V1 stably up-regulation(P<0.05).3.In the mimics transfection group,the mRNA and protein expressions of PF4V1 were decreased after the up-regulation of mir-875-3p to DU145 cells with stably overexpression of PF4V1(P<0.05).4.The ability of proliferation,migration and invasion of DU145 and LNCaP cells with stably overexpression of PF4V1 was significantly higher in the transfecting miR-875-3p mimics group than in the transfecting null mimics group(P<0.05).5.The dual-luciferase reporter assay showed that the relative fluorescence intensity of the co-transfection of wild type reported plasmids and miR-875-3p mimics group was significantly lower than that of the mutant group and the control group(P<0.05).6.GO/KEGG/PPI/INTERPRO analyses showed that PF4V1 mainly regulates biological processes such as "inflammation,migration,stimulation,stress,immunity,wound and angiogenesis" through "chemokine signaling pathway" and "interaction between cytokines and their receptors".7.Western blot results showed that in DU145 and LNCaP cell lines overexpressing PF4V1,p-AKT/p-ERK/SRC-1 protein were decreased,Snail/Slug/N-cadherin protein were down-regulated and E-cadherin protein was up-regulated.Conclusions: 1.PF4V1 is lower expressed in PCa tissues and cell lines,and PF4V1/TP53 is significantly associated with disease-free survival and overall survival.2.Overexpression of PF4V1 significantly inhibits the proliferation,migration and invasion of PCa cells,and intratumoral injection of PF4V1 protein can significantly inhibit the growth of PCa in vivo.3.MiR-875-3p targets the 174-180 region in the3’-UTR of PF4V1,and the up-regulation of miR-875-3p partially reverses the inhibitory effects of PF4V1 on PCa proliferation,migration and invasion.4.PF4V1 plays an inhibitory role in PCa through AKT/ERK/EMT and other downstream pathways of the chemokine pathway. |
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