The Function And Mechanism Of MiR-17-92 Gene Cluster In The Tumor Microenvironment Of Prostate Cancer

BackgroundsThe prostate cancer is a malignant tumor which affects health of men all over the world severely.The incidence and mortality of prostate cancer have been increased in recent years due to the changes in life style and living environment in China.Most of the prostate cancer patients are already at advanced stage and have distant metastasis when they are diagnosed,making the disease very difficult to treat.A good understanding of the molecular mechanism of the progression of prostate cancer will make a stride in both diagnostics and treatment.The tumor microenvironment(TME)is composed of stroma and variety of cells,such as cancer cells,fibroblasts and cancer associated fibroblasts(CAFs).CAFs have the characteristics of smooth muscle cells and normal fibroblasts.As the activated fibroblasts,CAFs can influence the growth,proliferation,metastasis and invasion of tumor cells by secreting many growth factors to participate in signal transductions.It has been confirmed that mi R-17-92 increases the proliferation,migration and invasion abilities of prostate cancer cells.But the function of mi R-17-92 gene cluster in the tumor microenvironment is rarely reported.Therefore,the study of mi R-17-92 gene cluster in in the tumor microenvironment of prostate cancer would provide some new therapy target for the treatment of prostate cancer.AimsThis study is designed to investigate the effect of mi R-17-92 gene cluster on the cell biological behavior of prostate cancer cells and fibroblasts in the tumor microenvironment of prostate cancer and to present a clearly understanding of mir-17-92 gene cluster in the tumor microenvironment.Part Ⅰ The role and mechanisms of mir-17-92 gene clusters in prostate cancer cells effects on the cellular biological of normal prostate fibroblastsMethods Normal prostate fibroblast cell line with G418 resistance was established.We treated WPMY-1 prostate fibroblast cells with DU145-contorl conditioned medium(CM)and DU145-mi R-17-92 conditioned medium for 48 h respectively.At the same time,we mixed WPMY-1 cells with DU145-contorl and DU145-mi R-17-92 cells for 48 h respectively.The cell growth,migration and invasion abilities were measured by a real-time x Celligence RTCA system.The proliferation and apoptosis of WPMY-1 cells were measured by CCK8 and flow cytometry.The characterizations of WPMY-1 and CAFs on the basis of cellular proteins were examined by immunohistochemistry(IHC).Expression of proteins related to the ability of proliferation,apoptosis,migration and invasion were examined by Western blotting.The cytokines were tested by ELISA.Results Normal prostate fibroblast cell line with G418 resistance was established successfully.The WPMY-1 cells treated with CM collected from tumor cells grew faster than the cells without the CM and dead much less.The proliferation of WPMY-1 cells treated with CM from tumor cells was much higher than that of the control cells.The WPMY-1 cells treated with CM collected from tumor cells migrated faster than that of the control cells.But the invasion ability of WPMY-1 was obtained only from the condition medium of DU145-mir-17-92 cell culture.The expression of desmin and FAP were much higher in the WPMY-1 cells cultured with DU145-mir-17-92 cell culture medium in IHC.The proteins expression of AKT and ERK signaling pathway,Desmin,HGF,ITGB1 and Kreatin8/18 were up regulated in WPMY-1 cells treated with CM collected from tumor cells.In ELISA test,the expression of TGF-β1and CXCL-12/SDF-1 was much higher in in WPMY-1 cells treated with CM collected from DU145-mir-17-92 cells than DU145-control.WPMY-1 mixed with DU145-control cells dead,but alive with DU145-mir-17-92 cells after 48 h cell-cell contact co-culture.The cell growth and proliferation of WPMY-1 cells co-cultured with DU145-mir-17-92 cells were inhibited compared with the cells without co-culture.The percentages of apoptosis WPMY-1 cells in the co-cultured group were lower than the other group.There were no significant differences in the abilities of migration and invasion.The proteins expression of desmin,Bcl-xl,ITGB1 and p-AKT-473 were up regulated in WPMY-1 cells co-cultured with DU145-mi R-17-92 cells.The proteins expression of ERK1/2,PTEN and Cyclin D1 were downregulated.In ELISA test,the expression of TGF-β1,CXCL-8/IL-8,CXCL-12/SDF-1 and CXCL-13/BCL/BCA-1 was much higher in WPMY-1 cells mixed with DU145-mir-17-92 cells than that with RPMI1640 only.Part Ⅱ The role and mechanisms of mir-17-92 gene clusters in effects on the cellular biological of prostate cancer cellsMethods We treated DU145-contorl cells and DU145-mi R-17-92 cells with WPMY-1 prostate fibroblast cells conditioned medium for 48 h respectively.At the same time,we mixed DU145-contorl and DU145-mi R-17-92 cells with WPMY-1 cells for 48 h respectively.The cell growth,migration and invasion abilities were measured by a real-time x Celligence RTCA system.The proliferation and apoptosis of DU145-contorl and DU145-mi R-17-92 cells were measured by CCK8 and flow cytometry.Expression of proteins related to the ability of proliferation,apoptosis,migration and invasion were examined by Western blotting.The cytokines were tested by ELISA.Results The cell growth and proliferation of prostate cancer cells treated with CM collected from WPMY-1 cells were decreased gradually than the cells without CM.And the CM promoted the apoptosis of both cancer cells.The cancer cells treated with CM collected from WPMY-1 migrated faster than control cells.But There were no significant differences in the ability of invasion.The proteins expression of PTEN and Cyclin D1 were up regulated in DU145-mi R-17-92 cells cultured with CM.The proteins expression of Bcl-2 and Bcl-xl were down regulated.The proteins expression of ITGB1、Kreatin8/18 and MMP9 were much stronger in DU145-mi R-17-92 cells than in DU145-control cells.The expressions of TGF-β1,CXCL-8/IL-8,CXCL-12/SDF-1 and CXCL-13/BCL/BCA-1 in ELISA were much lower in both cancer cell lines.There were no significant differences in the abilities of proliferation,migration and invasion in the DU145 cells mixed with WPMY-1.The percentage of apoptosis was up regulated in DU145-control cells with down regulation of the expression of Bcl-2 protein.But it is opposite in DU145-mi R-17-92 cells.The expressions of p-AKT-437 and ITGB1 were up regulated and p-ERK1/2 was down regulated.In ELISA test,the expressions of TGF-β1and CXCL-8/IL-8 were increased in both of the two cancer cell lines,but no difference in CXCL-13/BCL/BCA-1.the expression of CXCL-12/SDF-1 was up regulated in DU145-mi R-17-92 cells only.Part Ⅲ The effects of mir-17-92 gene clusters on the proteins of exosomes from prostate cancer cellsMethods We extracted exosomes from DU145 prostate cancer cells with Exoeasy Maxi Kit and examined the specific protein expression of the exosomes.The proteomic identification of exosomes was performed using chromatography mass spectrometry.The differential proteins of exosome were analyzed by heat map,Venn diagram analysis,GO analysis,metabolic pathways and protein-protein interaction network analysis.The expressions of mi R-17-92 gene clusters were examined by q-RT-PCR.The exosome uptake assay was performed with PKH-26 kit.Results The specific protein Flotillin in exosomes was examined successfully.The results of proteomic identification of exosomes showed that there were 1,677 proteins in the exosome of tumor cells,in which 1407 proteins were included in Exocarta,and the exosomes were extracted with high purity.The proteins of exosomes were mainly involved in the protein metabolism,transport,cell metabolism,cell growth and energy metabolism process.The difference proteins mainly had catalytic activity,transporter activity,ligase activity,heat shock protein activity,transcriptional regulation activity.The results of q-RT-PCR showed that there was a significant expression of mir-17-92 genes in the exosomes of the DU145-mir-17-92 cells.Exosome could be taken by normal fibroblasts.Conclusions1.mi R-17-92 gene clusters can significantly promote the abilities of cell growth,proliferation and migration of fibroblasts and inhibit apoptosis by activating the AKT and ERK signaling pathways.mi R-17-92 gene clusters can significantly promote the transformation of fibroblasts into fibroblasts.mi R-17-92 gene cluster can maintain fibroblasts alive by cell-cell direct contact and inhibit the apoptosis of fibroblast.mi R-17-92 gene cluster can promote the transformation of fibroblasts into the cancer associated fibroblasts.2.Normal prostate fibroblasts can inhibit the abilities of growth,proliferation of prostate cancer cells and reduce the secretion of bioactive factors of them by secreting cytokines.Normal prostate fibroblasts can promote apoptosis and enhance the ability of invasion of tumor cells through AKT signaling pathway.Fibroblasts can also activate the ERK pathway in the prostate cancer cells with mir-17-92 gene clusters to inhibit their apoptosis and promote the secretion of bioactive factors of tumor cells.3.The exosome extracted from mir-17-92 gene cluster high expression cells is rich in cellular active substances.3.The exosome contains high expression of mir-17-92 genes.The exosomes of tumor cells are likely to transfer mir-17-92 gene clusters through intracellular feeding into the normal cells to make its functions.