Phospholipase C? Regulates Prostate Cancer Lipid Metabolism And Proliferation Through Targeting AMPK/SREBP-1 Signalling Pathway

OBJECTIVE:Metabolic reprogramming is a common characteristic for numerous kinds of tumors,involving prostate cancer(PCa).Tumor metabolism such as lipid metabolism can not only provide sufficient lipids for tumor cell's division and rapid growing but also be a vital source for the composition of new cellular membranes,moreover,it can aslo enhance the signaling pathways of the tumor.Phospholipase C?(PLC?),as an oncogene,can drive proliferation,progression and lipid metabolism of tumors,but its effect in lipid metabolism of PCa is not fully clear.The study aim to screen some diagnostic biomarkers associated with lipid metabolism for prostate cancer and to explore the role of PLC? in lipid metabolism and proliferation of prostate cancer.METHODS:(1)The fasting serum samples of 60 PCa patients hospitalized in the second ward of Urology Department and the fasting serum samples of physical examination of 30 normal healthy adult male in the Health Management Center were collected from August 2018 to August 2019 in the First Affiliated Hospital of Chongqing Medical University.And timely made of dry filter paper blood tablets stored in a dry environment of 4° C standby.Finally,the contents of lipid metabolites(lipid acyl carnitine)inthe serum of PCa patients and healthy subjects were analyzed by liquid chromatography tandem mass spectrometry(LC-MS/MS).Moreover,the feasibility of the selected fatty acyl carnitine as a diagnostic marker of PCa was determined by the receiver operating curve(ROC)and the area under the curve(AUC).(2)Postoperative histopathological specimens of 60 patients with PCa and 60 patients with BPH hospitalized in the second ward of urology department of the First Affiliated Hospital of Chongqing Medical University from March 2015 to August 2019 were collected.Immunohistochemistry(IHC)was used to detect the protein expression of related factors in PLC? and AMPK/ SREBP-1 signaling pathway.Meanwhile,Spearman's correlation analysis was used to reveal the correlation between the expression of PLC? and SREBP-1,FASN.Progression-free survival(PFS)between the two(High-PLC??Low-PLC?)was compared by Kaplan? Meier survival curve analysis.Finally,the relationship between the expression of PLC? and the demographic and clinicopathological characteristics of PCa patients was analyzed retrospectively.(3)At the cellular level,the lipid droplet contents of PCa cells(PC3LNCaP)and human normal prostatic epithelial cells(RWPE-1)were first compared by Nile Red staining and Oil Red O staining.Meanwhile,Western blot and RT-qPCR were used to compare the expression of lipid metabolism-related factors in RWPE-1 and PCa cells.Then LNCaP and PC3 cells were transfected with lentivirus short hairpin RNA that interfered with PLC?(LV-shPLC?#1/#2/#3)and lentivirus short hairpin RNA that interfered with no load(i.e.negative control lentivirus,LV-shNC).Subsequently,the PCa cell lines with stable transfection with knockdownPLC? lentivirus(LV-shPLC?)and negative control lentivirus(LV-shNC)were obtained by screening and verification.After the successful construction of shPLC? cell lines,the protein and mRNA of PCa cells were extracted,and the expression of downstream factors related to metabolism and proliferation was detected.At the same time,the lipid droplets content of PCa cells after shPLC? was determined.And the proliferation of PCa cells after shPLC? was detected by Ki-67 fluorescence staining and colony formation test.(4)To further investigate the molecular mechanism of PLC?regulating PCa lipid metabolism,the PC3 and LNCaP cells after shPLC?were selected and the distribution of SREBP-1 protein in the two cell lines was detected by cell immunofluorescence.Then the nucleoprotein and cytoplasm protein of the two cell lines were extracted,and the expression of SREBP-1 in nucleus and cytoplasm was analyzed by western blot.Then,the negative control group(NC),shPLC? group,shPLC? +AMPK inhibitor(Dorsomorphin)group and NC+ Dorsomorphin group were respectively set in the two cell lines.Total proteins were extracted from treated cells,and the changes of total-AMPK,p-AMPK and SREBP-1 proteins were analyzed by Western blot.Changes in lipid metabolism of PCa cells were detected by Nile Red staining,and proliferation of PCa cells was detected by ki-67 fluorescence staining and colony formation test.(5)In vivo,six weeks old nude mice were divided into two groups(NC group and shPLC? group),with 5 mice in each group,PC3+NC cells and PC3+shPLC? cells were injected into the right flank subcutaneous tissue of nude mice in each group(2x106 cells were injected into each group).Tumor weight and volume were measured weekly.After 6 weeks,the nude mice were sacrificed,the tumor tissue samples were taken and measured for their volume and weight.Finally,IHC was used to detect thedifferences in the expression of related proteins in the two groups of tumor tissues.RESULTS:(1)The results of LC-MS /MS test showed that in the serum of more than 30 kinds of acylcarnitine of PCa patients,the level of C0,C2,C4 DC,C5,C5 DC,C5OH,C8,C8,C10,C10,C12 and C16 was higher than that of the normal control group.Among them,further ROC analysis revealed that C0,C5,C5 DC,C5OH,C8,C8:1,C10,C10:1 and C12 may become a potential biomarker for the diagnosis of prostate cancer and be used for PCa screening.(2)IHC test showed that the expression of PLC?,SREBP-1 and FASN in PCa tissues was higher than that in BPH tissues.Spearman's correlation analysis revealed that PLC? was positively correlated with SREBP-1 and FASN.By analyzing the demographic and clinicopathological parameters of PCa patients,it was found that the high expression of PLC? was positively correlated with the histological staging,bone and visceral metastasis of the patients.Kaplan-Meier survival analysis showed that patients with high PLC? expression(median PFS: 23 months)had shorter progression-free survival than patients with low PLC? expression(median PFS: 29 months).(3)The results of Nile Red and Oil Red O staining showed that the lipid droplets content in PCa cell lines(PC3 LNCaP)was higher than that in RWPE-1 cells.Western blot and RT-qPCR results showed that the protein and mRNA expression levels of PLC?,SREBP-1 and FASN in PC3 and LNCaP were higher than those in RWPE-1 cells.The results of PC3 and LNCaP cells transfected with lentivirus were showed that,compared with the negative control group,the protein and mRNA levels of lentivirus short hairpin RNA 3 interfering with PLC? were significantly decreased inthe two cell lines.The shPLC? cell lines were successfully constructed for subsequent experiments,and the results were showed that the protein and mRNA levels of the lipid metabolism-related factors(SREBP-1,FASN,ACACA,ACLY and SCD1)and the proliferation-related factors(PCNA and Cylin D1)were down-regulated,and the lipid droplets content in PC3 and LNCaP cells were decreased when the PLC? was knocked down.Ki-67 fluorescence staining,colony formation test and CCK-8 assay also showed that the proliferation of PCa cells decreased significantly when the PLC?was knocked down.(4)Immunofluorescence assay showed that silencing PLC? in PC3 and LNCaP cells significantly reduced the distribution of SREBP-1 protein in the nucleus.Western blot analysis of nuclear proteins and plasma proteins of the two cell lines showed that the expression of SREBP-1 in the nucleus was significantly down-regulated by silencing PLC?,but the expression in the cytoplasm was not significantly changed.Interestingly,when PLC? was silenced,the phosphorylated AMPK(p-AMPK)protein levels were up-regulated,while the total AMPK(total-AMPK)did not change significantly.However,when the PLC? was silenced and Dorsomorphin(inhibit AMPK phosphorylation)was added,the protein expressions of p-AMPK and SREBP-1 were reversed.The same results were obtained in the subsequent lipid metabolism function test(Nile Red staining)and cell proliferation tests(Ki-67 fluorescence and clonal formation).(5)In vivo experiments,IHC results of tumor tissues in nude mice showed that,compared with the control group,the expression level of SREBP-1 and PLC? was decreased,the expression of p-AMPK was increased,and the positive cell rate of Ki-67 staining was decreased.Meanwhile,the weight and volume of subcutaneous tumor in posteriornude mice were significantly reduced by silence PLC?.CONCLUSION:This study illustrates that partial lipid acylcarnitine may be a potential hematological biomarker for the diagnosis of PCa.Overexpression of PLC?may indicate low PFS in PCa and be connected with the metastasis of PCa.Moreover,our finding pointed to a signaling network,PLC?/AMPK/SREBP-1,which can drive the progression of PCa through lipid metabolism in vivo and vitro.In conclusion,this study first uncovers the lethal role of PLC? in lipid metabolism and malignant behavior of PCa for detailed comprehending PCa occurrence and progression.