| Objectives:Many evidences show that inflammation regulates the growth,progression and metastasis of cancer by releasing inflammatory cytokines and growth factors,and plays a key role in the pathogenesis of prostate cancer.Inflammatory cytokines have diverse functions in the regulation of growth,metastasis and differentiation of many human cancers,including prostate cancer.Increased levels of inflammatory biomarkers were found in clinical specimens of patients with prostate cancer and in the serum of patients with advanced prostate cancer resistant to treatment.In addition,interleukin overexpression can promote the growth and metastasis of prostate cancer by regulating cell proliferation,invasion,migration and epithelial-mesenchymal transition(EMT).However,the molecular mechanisms of interleukin induced EMT and cancer metastasis have not been thoroughly studied.GCN5 is the first histone lysine acetyltransferase of the HAT superfamily identified.GCN5 is involved in a wide range of cellular processes,including gene transcription,differentiation,DNA repair,nuclear body assembly and cell cycle regulation.However,the role and mechanism of GCN5 in EMT and metastasis of prostate cancer remain unclear.The aim of this study was to find differentially expressed genes related to inflammation using biological information analysis,to analyze the expression of differential genes in serum and tissues of patients with clinical prostate hyperplasia and prostate cancer,to evaluate the role of GCN5 in inflammation-induced metastasis of prostate cancer and to further explore the possible molecular mechanisms.Methods:This research is divided into three parts.The first part is bioinformatics analysis,by analyzing the gene expression profile data of prostate cancer and BPH in GEO database,the differentially expressed genes were found in prostate tissue samples.GO biological enrichment analysis and KEGG pathway enrichment analysis were performed on the screened differentially expressed genes using David database,to investigate the enrichment of these genes in cell composition(CC),molecular function(MF),biological process(BP)and KEGG pathway.In the protein level,the PPI map of protein interaction network was constructed using the STRING database.The Cytohubba plugin of Cytoscape software was used to screen the top 10 hub genes in PPI network.Then,the top-10 hub genes were retrieved from the Oncomine database for Meta-analysis.Finally,the key genes with significantly different expression were obtained and annotated respectively.The abnormal expression gene associated with prostate tumors was screened out,and then the obtained key gene for inflammation was used for subsequent experimental studies.The second part is the retrospective analysis of clinical data.In this study,a total of 116 patients who were admitted to the Department of Urology in our hospital with complete clinical data meeting the admission and discharge criteria were followed up,including 34 patients with prostatic hyperplasia,82 patients with prostate cancer,and 15 patients with CRPC.They were divided into BPH group,G1(Gleason?6)group,G2(Gleason=7)group,G3-4(Gleason?8)group and CRPC group.The expressions of PSA and IL-6 in serum of patients in the above groups were detected,and the correlation between IL-6 and blood PSA and Gleason grading was analyzed.HE and immunohistochemical staining were performed on the obtained case data to detect the expression of IL-6 in prostate hyperplasia and prostate cancer tissues of different Gleason grades.Statistical analysis was performed on the above data to explore whether IL-6 could be used as a diagnostic and prognostic indicator for tumors.The third part is mechanism experimental study in vitro.Human prostate cancer cell lines were first treated with IL-6,the expression of GCN5 in cell lysate was studied by RT-PCR and Western blot to determine the effect of IL-6 on the expression of GCN5 in prostate cancer cells.Next,MTT,Martigel invasion and Transwell assay were used to study the effect of GCN5 gene silencing on the proliferation of LNCaP cells.Western blot was used to detect Vimentin,N-cadherin,E-cadherin and?-catenin proteins,to determine the effect of siGCN5 on EMT process.Next,we examined whether GCN5 could affect the cell biological function through the expression of Egr-1.IL-6 treated cells with siGCN5 and pcEgr-1 was used to detect cell proliferation,metastasis,invasion and EMT ability.Finally,the effects of GCN5 on PI3K/PTEN/Akt signaling pathway were studied.siGCN5,pcEgr-1 and pcGCN5 were transfected into cells respectively,Akt phosphorylation level and PTEN protein content were detected,and PI3K/Akt activation level was detected when cells interfered with GCN5 and over-expressed Egr-1 at the same time.In addition,the cells were pretreated with LY294002,a specific inhibitor of PI3K/Akt signaling pathway,and then overexpressed GCN5 in the cells.The ability of cell proliferation and infection was detected to determine whether GCN5 affected cell phenotype through PI3K/Akt signaling pathway.ResultsIn part one,by analyzing the gene expression profile data of metastatic prostate cancer,clinically localized prostate cancer and prostate hyperplasia in GEO database,101 genes were found to be differentially expressed in prostate tissue samples,including 29 up-regulated genes and 72 down-regulated genes.DAVID database was used to filter of 101 differentially expressed genes GO biological enrichment and KEGG pathway enrichment analysis,we found that these genes are mainly distributed in the extracellular space,the cell membrane and the extracellular matrix,such as biological processes focused on the serine phosphorylation,cell division and apoptosis regulation,and other functions,to participate in the mother cell meiosis,PI3K signaling pathways,cell cycle,HIF-1 signal pathway,P53 signaling pathway and so on.At the protein expression level,we used the STRING database to construct the PPI map of the protein interaction network,and then used Cytohubba plugin in Cytoscape software to screen the top 10 Hub genes in the PPI network,which were MKI67,Myc,AURKA,CCNB1,UBE2C,TOP2A,MELK,IL6,BIRC5,and BUB1.Meta-analysis was performed by literature search of Oncomine database,and three key genes with high expression were identified respectively as IL-6,MYC and TOP2A.This study will explore the relationship between inflammation and prostate tumors,so IL-6 was selected as the key gene for the study.Prognostic information of prostate cancer patients was obtained from MSKCC and TCGA databases,and survival analysis was conducted.The results showed that changes in IL-6 level had clinical significance in influencing the overall survival rate of prostate cancer patients.In part two,Clinical data were retrospectively analyzed to study the expression of IL-6 in serum and prostate tissue of patients with BPH and prostate cancer.Clinical data analysis showed that the expression level of IL-6 in serum and tissues of the prostate cancer group was higher than that of the prostate hyperplasia group,and the difference was statistically significant.IL-6 was positively correlated with blood PSA and Gleason grade.In the prostate cancer group,the expression level of IL-6 in blood increased with the increase of Gleason grade.However,when the low-risk prostate cancer group(Gleason?6)was compared with the BPH group,it was found that there was no significant difference in the expression of IL-6 both in blood and tissues(P<0.05).This indicates that the serum IL-6 level is not correlated with the occurrence and risk of prostate cancer,and cannot be used as an indicator to predict the occurrence of prostate cancer.However,the higher the level of serum IL-6 in tumor patients,the higher the degree of malignancy of prostate cancer,and IL-6 can be used as an indicator to judge the prognosis of tumor.In part three,PCR and western blot results showed that the expression of GCN5 in all prostate cancer cell lines stimulated by IL-6 was significantly higher than that before treatment(P<0.05),especially in LNCaP cell lines.LNCaP cell line was selected for the following functional experiments.IL-6 stimulation significantly promoted cell proliferation,and enhanced the ability of cell invasion,migration and EMT(P<0.05).Interference with GCN5 expression could inhibit the proliferation,invasion and migration of LNCaP cells induced by IL-6.IL-6 stimulated the expression of Egr-1 in LNCaP cells,while siGCN5 significantly inhibited the expression of Egr-1.Further study of the effect of Egr-1 overexpression on prostate cancer cells after GCN5 interference showed that,compared with cells transfected with siGCN5 alone,overexpression of Egr-1 gene in cells could prevent the decrease of cell proliferation induced by GCN5 interference to a certain extent(P<0.05).In addition,the overexpression of exogenous Egr-1 in cells also weakened the inhibitory effect of siGCN5 on cell invasion,migration and EMT.These results indicate that Egr-1 mediates the biological function of cells as a downstream regulatory protein of siGCN5.Western blot showed that overexpression of GCN5 in cells could increase the phosphorylation of Akt and decrease the expression of PTEN,while siGCN5 could significantly decrease the expression of p-Akt and increase the expression of PTEN,which was contrary to the result of overexpression of Egr-1(P<0.05).At the same time,the expression level of p-Akt and PTEN in pcEgr-1 and siGCN5 transfected cells was significantly higher than that in cells interfering with GCN5 expression alone,and the expression level of PTEN was down-regulated.These results suggest that siGCN5 inhibits Akt phosphorylation and activates PTEN,thereby inhibiting PI3K/Akt signaling pathway,which can be reversed by Egr-1 overexpression.MTT analysis showed that overexpression of GCN5 alone could promote cell proliferation,but LY294002 could inhibit cell proliferation induced by pcGCN5.Transwell assay was consistent with MTT results.LY294002 could inhibit cell invasion caused by overexpression of GCN5.These results further suggest that GCN5 influences the proliferation and invasion of LNCaP by altering the activity of PI3K/Akt signaling pathway.Conclusion:In conclusion,in this study,the differentially expressed key gene IL-6 related to inflammation was obtained through the bioinformation analysis of tumor database.Clinical studies have shown that the expression level of IL-6 is increased in both serum and tissues of prostate cancer,and increases with the increase of tumor malignancy.However,there is no significant difference in the expression level of low-risk PCA and BPH,indicating that IL-6 is a possible prognostic indicator for prostate cancer,but not a diagnostic indicator.Our experiments confirm that GCN5 can alter the proliferation,migration,invasion and EMT of prostate cancer cells mediated by IL-6 by regulating the expression of Egr-1 protein and the activation of downstream PI3K/PTEN/Akt signaling pathway.In this study,we investigated the molecular mechanism of the role of the inflammation-related gene IL-6 in the development of prostate cancer,and provided new ideas for targeted therapy and drug development for prostate cancer patients. |
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