Meta Analysis Of PI3K/Akt Signaling Pathway Gene Polymorphism On Prostate Cancer And Prostate Cancer-derived Exosomes Alter Macrophage Polarization Via PI3K/Akt Signaling

Background and Objective: Increasing evidence suggested that polymorphisms in genes of PI3K/Akt pathway were closely related to prostate cancer(PCa)risk.Nevertheless,these results are controversial.Here,we conducted a comprehensive updated meta-analysis to precisely illustrate the association between polymorphisms in genes of PI3K/Akt signaling pathway and PCa risk.Materials and Methods: Relevant studies were identified by the systematically researching on PubMed,Web of Science and Google Scholar databases up to October 1,2017.The gene set of PI3K/Akt pathway was referenced from the Kyoto Encyclopedia of Genes and Genomes(KEGG)website.The odds ratios(ORs)with a corresponding95% confidential intervals(95%CIs)were applied to test their associations.All the analyses were conducted by using Stata 12.0.The random effect model was used for meta-analysis.We used Begg's and Egger's method to detect publication bias.Results: Finally,38 articles comprising 62 case-control studies were enrolled for 13 polymorphisms in genes of PI3K/Akt pathway.However,overall results failed to present a positive association between polymorphisms in genes of PI3K/Akt pathway and PCa risk.Nevertheless,in the subgroup analysis by ethnicity,we identified that IL-6-rs1800795 polymorphism was associated with an increased risk of PCa for Caucasian individuals in dominant model(MM + MW vs.WW: OR = 1.245,95% CI =1.176-1.318,P < 0.001).Conclusion: Our work suggests that polymorphisms in genes of PI3K/Akt Signaling Pathway are not risk factor for PCa.Further well-designed studies with larger samples and precise designs are demanded to corroborate our findings.Background: prostate cancer(PCa)is a malignant tumor which seriously threatens male health.In recent years,the incidence rate and mortality rate of PCa are on the rise.Therefore,it is urgent to explore the potential mechanism of PCa and find the potential treatment methods.Tumor and its surrounding stromal cells constitute the tumor microenvironment(TME),which provides opportunities for the interaction among cancer,fibroblasts,inflammatory cells and capillaries.TME contains that tumor induced cytokines,growth factors and a variety of immune cells(including macrophages),which play an important role in immunosuppression.Macrophages have high functional plasticity and heterogeneity.The phenotype and functional transfer of macrophages in TME often affect the occurrence and development of diseases.Prostate cancer is rich in tumor associated macrophages(TAMs),which could be cytotoxic M1 or tumorigenic M2.M2 macrophages are associated with wound repair,neovascularization,immunosuppression and tumor enhancement,and promote Th2 immune response;while M1 macrophages are characterized by releasing cytotoxic free radicals,inhibiting tumor,and promoting T helper(Th)1 response.For example,hypoxia,ischemia and low p H TME could induce endoplasmic reticulum stress(ERS)in tumor cells,thus enhancing the immunosuppressive ability of TAMs and promoting the infiltration and polarization of macrophages.Exosomes are extracellular vesicles with a diameter of 30-120 nm.They have a double membrane structure and carry a variety of biological functions,such as miRNAs,mRNAs,lncRNA proteins,lipids and virus particles.Exosomes are released by exocytosis of polyvesicles,and the substances in the vesicles could transfer and change the signaling pathway of receptor cells.Exosomes are related to tumor growth and progression to a large extent,but they can also play an anti-tumor function,which depends on cell type and microenvironment.For example,cancer cells secrete more exosomes under hypoxia and other stress conditions.Therefore,it is of great clinical significance to further study how tumor cells transmit ERS signals to macrophages in tumor microenvironment so as to change their polarization function.Objective(1)To observe the expression of PD-L1 and related inflammatory factors in macrophages.(2)To investigate the effect of exosomes secreted by prostate cancer cells under ERS on THP-1 macrophage polarization?(3)Is there a significant association between PI3 K / Akt and its phosphorylation signaling pathway and prostate cancer risk?Methods(1)Different concentrations of TG were used to treat prostate cancer cells for 24 hours,and the same concentration of TG was used to treat prostate cancer cells for different times.(2)Western blot was used to detect the changes of ers marker proteins GRP78 and CHOP.(3)Isolation and identification of exosomes from prostate cancer cells: PC-3 prostate cancer cells were co-cultured with 3 ?M TG for 24 hours,and Exo Quick-TC kit was used to extract exosomes from conditioned medium,named as non stimulation group(EXO-CON)and stimulation group(EXO-TG).(4)The morphology of exosomes was observed by transmission electron microscope after phosphotungstic acid staining.(5)Western blot was used to detect the expression of specific molecules(CD63 and TSG101)and chaperone protein calnexin on exosomes(EXO-CON and EXO-TG).(6)THP-1 macrophages were cultured and induced(50 ng / ml PMA for 24 hours)into macrophages(M?).The expression of CD68 was detected by flow cytometry.(7)The exosomes labeled with green fluorescent PKH67 were added into macrophages,and the absorption efficiency of exosomes was observed by laser confocal microscopy.(8)Exosomes from different sources(EXO-CON and EXO-TG)were co-cultured with macrophages for 24 hours.The cells were collected and totalRNA was extracted.The expressions of IL-6,IL-10,TGF-?,IL-1 ?,IL-12 and TNF-? related inflammatory factors in EXO-CON and EXO-TG groups were detected by q RT-PCR.(9)Exosomes from different sources(EXO-CON and EXO-TG)were co-cultured with macrophages for 24 hours.The supernatant of co-culture was collected.The levels of IL-10,IL-1 ?,IL-6,IL-8,TNF-? and IL-12 were detected by CBA cytokine kit.(10)Exosomes from different sources(EXO-CON and EXO-TG)were co-cultured with macrophages for 24 hours.The expression of M1 and M2 macrophage marker proteins was detected by flow cytometry: the expression of M1 macrophage marker proteins(CD11c and CD192);whether the expression abundance of M2 macrophage marker proteins(CD206,CD16 and CD274)increased?(11)Exosomes from different sources(EXO-CON and EXO-TG)were co-cultured with macrophages for 24 hours.The cells were collected and the total proteins were extracted.The expression levels of PI3 K,Akt and their phosphorylated proteins were detected by Western blot.Results(1)Western blot analysis of ERS marker protein GRP78 and CHOP showed that the optimal time point and concentration of TG were 24 hours and 3 ?M,respectively.(2)The exosomes extracted by Exo Quick-TC kit were stained with phosphotungstic acid and observed by transmission electron microscope.The exosomes were round or quasi round membranous vesicle like structures,with a diameter of about 30-120 nm,which conformed to the typical characteristics of exosomes.(3)Western blot analysis showed that exosomes expressed their specific molecules(CD63 and TSG101),but did not express endoplasmic reticulum chaperone protein calnexin,which indicated that exosomes extracted did not contain cell components;exosome protein concentration in EXO-TG group was higher than that in EXO-CON group.(4)The results of flow cytometry showed that the positive rate of CD68 was more than 90%,which indicated that the purity of macrophages was high.There was a significant difference before and after induction,which indicated that M? could be successfully obtained for subsequent experiments.(5)Laser confocal microscopy showed that exosomes labeled with green fluorescent PKH67(EXO-CON and EXO-TG)were absorbed by macrophages.(6)Compared with EXO-CON group,EXO-TG group significantly increased the levels of IL-6,IL-10 and TGF-? related inflammatory factors,while decreased the levels of IL-1 ?,IL-12 and TNF-? related inflammatory factors.(7)Exosomes from different sources(EXO-CON and EXO-TG)and macrophages were co-cultured for 24 hours.The supernatant of co-culture was collected,and the inflammatory factors were detected by CBA cytokine kit.The results showed that compared with EXO-CON group,the levels of IL-6 and IL-10 related inflammatory factors in EXO-TG group were significantly increased,while the levels of IL-1?,IL-8 and TNF-? related inflammatory factors in EXO-TG group were significantly decreased.(8)Exosomes from different sources(EXO-CON and EXO-TG)were co-cultured with macrophages for 24 hours.The expression of M1 and M2 macrophage marker proteins was detected by flow cytometry: The expression of M1 macrophage marker proteins(CD11c and CD192)was significantly up-regulated;the expression abundance of M2 macrophage marker proteins(CD206,CD16 and CD274)was significantly increased.(9)Western blot results showed that: exosomes from different sources(EXO-CON and EXO-TG)and macrophages were co-cultured for 24 hours,cells were collected,and total protein was extracted.Compared with EXO-CON group,the expression levels of PI3 K,Akt and their phosphorylated proteins in EXO-TG group were significantly increased.Conclusion(1)The endoplasmic reticulum of prostate cancer cells secretes more exosomes under stress.(2)Exosomes released from prostate cancer cells under endoplasmic reticulum stress can be effectively absorbed by macrophages,and affect the polarization of macrophages by up regulating the expression of PD-L1 and inflammatory factors.(3)Exosomes act as a bridge of intercellular information transmission.PI3 K / Akt signaling pathway is significantly associated with the risk of prostate cancer.(4)This is contrary to the conclusion of the first part of the meta-analysis,so we need to expand the sample size and use subgroup analysis to conduct a new meta-analysis.