The Effect And Mechanism Of Phosphoribosyl Pyrophosphate Synthetase 2 On The Phenotype Of Prostate Cancer Cells

Background and purposePhosphoribosyl pyrophosphate synthases 2(PRPS2)is a key rate-limiting enzyme in the purine biosynthesis pathway.It catalyzes 5-phosphoribosyl to phosphoribosyl pyrophosphate(PRPP)With the help of Mg2+-ATP,and it is an irreversible reaction.It has been reported that PRPS2 abnormal expression leading to diseases from the aspects of nucleotide biosynthesis and cell metabolism control.Our previous studies have shown that the expression of PRPS2 is regulated by androgen receptors,but its functions and mechanisms in prostate cancer(PCa)remain unclear.The purpose of this study was to explore the role of PRPS2 in PCa cells in order to elucidate the effect of PRPS2 on the phenotype of PCa cells and the molecular mechanisms inside.MethodsWe first used immunohistochemistry(IHC)staining to determine the expression difference of PRPS2 protein in human PCa samples and normal prostate tissues and the clinical significance.Then real-time quantitative polymerase chain reaction(RT-qPCR)and western blotting(WB)were used to discover the expression level of PRPS2 between human PCa cell lines and normal prostatic epithelial cell line.The next step we used lentiviral vectors to silent PRPS2 in PCa cell lines,so that stable silent cell lines and control cell lines were constructed.After that the effects of PRPS2 loss of function on PCa cell proliferation were detected by cck-8,cell cloning and 5-ethynyl-20-deoxyuridine(EdU)incorporation assay,and cell cycle and apoptosis was analyzed by flow cytometry.In vivo experiments were conducted to investigate the effect of silencing PRPS2 on PCa cell growth by subcutaneous tumor formation in male nude mice,apoptosis of subcutaneous tumor tissues was detected by TdT-mediated dUTP Nick-End Labeling(TUNEL).Subcutaneous PCa tissues routinely used the IHC test for ki-67 antigen to assess cell proliferation activity.Finally,the downstream key molecules related to the phenotype were screened to clarify parts of the molecular mechanisms of PRPS2 in PCa,providing evidences for further understanding the important role of PRPS2 in the carcinogenic development of PCa and exploring its clinical value.Results1.PRPS2 expression was higher in human PCa tissues than in normal or adjacent prostate tissues,and it was higher in DU 145 and PC-3 cell lines(RWPE-1 as control).2.After PRPS2 knocked down,the proliferation of DU 145 and PC-3 cells were observably inhibited,and PRPS2 silencing induced the increase of prostate cancer cells in G0/G1 phase and the decrease of cells in S phase.What's more,PRPS2 loss of function also induced increased apoptosis of DU 145 and PC3 cells.3.In vivo experiments showed that PRPS2 silencing inhibited subcutaneous transplanted tumor growth of PC-3 cells in male nude mice,and increased tumor cell apoptosis.Tumor tissues of shPRPS2 group was manifested with a lower percentage of Ki-67 antigen positive.4.PRPS2 silencing could up-regulate the expression of pro-apoptotic protein Bax whereas inhibit the expression of apoptotic inhibiting protein Bcl-2.Cascade cleaved and activated caspase-9 and caspase-3,lead to PARP activation and induced cell apoptosis.On the other hand,knocked down of PRPS2 inhibited the formation of cyclinD1/CDK4/6 complex by inhibiting the expression of cyclinD1,and upregulated the expression of p27kipl to enhance the G1/S checkpoint restriction,thus inhibited the proliferation of PCa cells.ConclusionOur study has showed that overexpression of PRPS2 protein is associated with the clinical characteristics of PCa.Silencing PRPS2 can effectively inhibit the proliferation of PCa cells,induce cell cycle arrest in G0/G1 phase and induce mitochondrial apoptosis.These results revealed that PRPS2 may play a fatal role in the development of PCa.Treatment strategies that target PRPS2 may have certain therapeutic value for PCa patients with overexpression of PRPS2.