PLCε Knockdown Inhibits Serine/Glycine Metabolism And Proliferation Of Prostate Cancer Via YAP Signaling Pathway

Objective: To investigate the expression and correlation of phosphatidylinositol lipid phospholipase Cε(phospholipase Cε,PLCε)and related proteins(yes-related protein,YAP)in prostate tissues,and their correlation with clinical pathological parameters;to investigate the serine/glycine concentration levels in blood samples;to study the specific mechanism of PLCε on serine/glycine metabolism and cell proliferation in prostate cancer(prostate cancer,PCa)cells.Methods: Liquid chromatography-tandem mass spectrometry(LC-MS/MS)and gas chromatography-mass spectrometry(GC-MS)techniques were used to detect concentration levels in blood samples;Immunohistochemistry staining(immunohistochemistry,IHC)and H&E techniques were used to detect PLCε and YAP expression levels in prostate hyperplasia(BPH)and PCa tissue samples;PCa cells were transfected with lentivirus and plasmids,mRNA and protein expression of PLCε,YAP,serine/glycine generating enzymes and proliferation-related genes were detected by q-PCR and Western blot,respectively.Clone formation assay to detect the colony formation of PCa cells;MTT test to detect cell proliferation activity;Immunofluorescence(IF)was used to detect YAP cell localization and distribution;Western blot was used to exam YAP expression in nucleus and p-YAP in cytoplasm.The key enzymes in the production pathway include three of them: phosphoserine converting aminoase 1(PSAT1),phosphoserine phosphatase(PSPH),serine hydroxymethyltransferase 2(SHMT2);Two genes related to reproduction include: cyclin D1(CyclinD1),proliferating cell nuclear antigen(PCNA).Results: Serine/glycine concentrations in the blood of PCa patients were significantly higher than those in normal people.At the same time,compared with BPH tissues,the PLCε and YAP staining intensity in PCa tissues were significantly increased,and both were related to clinical stage and Gleason score;(1)Infection with LV-shPLCε can significantly down-regulate the expression of PLCε,YAP,serine/glycine key enzymes and proliferation-related genes,and can also inhibit the colony-forming ability and proliferation activity of PCa cells;(2)The addition of the overexpression plasmid vector-YAP can significantly reverse the down-regulation of YAP,PSAT1,PSPH,SHMT2 and proliferation-related genes caused by lentiviral interference with PLCε,but the interference effect was most obvious when sh-PLCε added with the interference plasmid sh-YAP,and the addition of plasmids had no significant effect on PLCε;(3)After knocking down PLCε,the fluorescence intensity of YAP in the nucleus of PCa cells decreased significantly;(4)Interference with PLCε significantly decreased the expression of YAP in the nucleus of the cells,whereas p-YAP level in the cytoplasm was significantly increased.Conclusion: Serine/glycine concentrations in the blood of PCa patients are abnormally elevated;PLCε and YAP are both highly expressed in PCa tissues and have significant correlations,both of which are closely related to clinical stage and Gleason score;Interference with PLCε can block the serine/glycine production and cell proliferation,and PLCε achieves this pathway by alternating YAP expression;In addition,the specific mechanism by which PLCε affects YAP is to change its nuclear translocation and dephosphorylation.