The Mechanism Of C-Myc-GPC5/HDGF Signaling For The Progression Of Prostate Cancer

ObjectiveProstate cancer is one of the most common urogenital cancers among men.Its morbidity and mortality are increasing year by year in our country.Our previous study showed that the the abnormal expression of c-myc and GPC5 were closely related to the progression of prostate cancer(PCa)and c-Myc could supress the expression of GPC5 with no certain mechanism.In addition,HDGF expressed unusually high in PCa and our study also showed GPC5 could supress HGDF targetedly also without certain mechanism.This article aims at exploring the role of c-Myc-GPC5/HDGF signaling pathway in the development and progression of PCa by bioinformatics and molecular biology methods.We hope to reveal the development of PCa and provide scientific basis for effective and targeted diagnosis and treatment.Methods1.We analyzed the promoter region of GPC5 by bioinformatics technology and found that there were two special E-Box sequence on the upstream of GPC5 transcription initiation site.We then constructed pGL3 plasmid containing the E-Box sequence to verify and confirmed the specific combining site with luciferase assay and Chip experiment.MTT assays was used to verify that c-Myc can regulate cell proliferation by inhibiting GPC5.2.DNA methylation is also one of the important transcriptional regulation mechanism by c-Myc.Through the network prediction database about CpG,we found there were two CpG islands on GPC5 promoter and the first exon region.We applied DNA methylation inhibitors and qMSP experiment to verify the specific methylation of GPC5.We then constructed an overexpression c-Myc plasmid and designed SiRNAs targeting DNMT1,3a,3b and its negative control sequence.We changed the expression of c-Myc and DNA methyltransferase in prostate cancer cells by exogenous transfection technique and tested how c-Myc regulated GPC5 with DNA methylation by qRT-PCR?Western blot?qMSP and CO-IP.3.c-Myc can transcriptionally regulate abnormal expression of miR-17-92 cluster.By network database,we found that 3`UTR of GPC5 had acting site of miR-19b-1and thus we assumed miR-17-92 cluster might also play an important role in regulating GPC5 abnormal expression.We tested the abnormal expression of miR-17-92 cluster in PCa with Affymetrix miRNA chip,verified the expression of miR-19b-1 in tissues and PCa cells with RT-qPCR,and showed the suppression of miR-19b-1 for GPC5 with luciferase assay.4.Finally we made primary discussion about the downstream of GPC5.Early we found GPC5 could inhibit the expression of HDGF and especially in the necleus of PCa by confocal,nuclear mass separation,western blot experiments after transfecting plasmid with GPC5 overexpression into PC3 cells exogenously.As a kind of membrane protein,how GPC5 influenced the expression of HDGF? We introduced an important role kind of in a transport of organelles: Exosome.Firstly,we collected the exosome from PCa cells by ultra-high speed centrifugation and then identified it by transmission electron microscopy and NTA.By using western blot,we aimed to verify whether HDGF was contained in exosome.We handled PC3 cells with exosome to verify whether exosome could transport HDGF into nucleus through confocal,nuclear mass separation and western blot.Finally we used overexpression GPC5 plasmid or added low molecular heparin(HS simulation)to show whether GPC5 could inhibit HDGF transportion with its HS chain structure.Results1.c-Myc can suppress the expression of GPC5 by binding the two E-box binding sites in the promoter region of GPC5 and then influenced the cell proliferation of PCa cells.2.c-Myc can change GPC5 methylation status by recruiting DNMT3 b,thus inhibit the expression of GPC5.3.miR-19b-1 which regulated by c-Myc is highly expressed in PCa and could inhibit the expression of GPC5 through targeting its' 3' UTR.4.Exosome can carry HDGF into cells and nucleus and GPC5 can inhibit this process with its special HS chain structure.ConclusionIn PCa,c-Myc can inhibit the expression of GPC5 through direct or indirect way and GPC5 can suppress the transport of exosome for HDGF with its HS chain.For this reason,c-Myc-GPC5/HDGF signaling pathway may be an excellent direction in controlling the development of PCa.