| Objective1.The expression of PLC? and HIF-1? are differences beweent prostate cancer tissues and benign prostatic hyperplasia.And the expressions have relevance with clinicopathological characteristics of PCa patients.The level of HIF-1? in serum has a correlation with various clinicopathological characteristics.2.PLC? regulates HIF-1? targets and metabolic activity in PCa cells.3.Energy metabolism regulated by PLC? elevates the migration of PCa cell lines.4.The mechanism of HIF-1? regulated by PLC?.Methods1.We examined PLC? and HIF-1? expression in 42 PCa tissue samples and 42 BPH tissue samples by IHC.HIF-1? expression in 56 PCa serum was determined by ELISA.Pearson analysis was used to analyze the correlation with clinicopathological characteristics.2.Genetically altered cell lines were constructed by infecting with PLC? shRNA(sh-PLC?)lentivirus particles.Glucose consumption andlactate production assay were applied to detect the metabolic activity in PCa cell lines.The mRNA and protein expression of HIF-1? and all target genes(PKM2,GLUT1,LDHA and HK2)were detected by qRT-PCR and western blot in PCa cell lines.3.Cell migration was observed through transwell assay.The expressions of N-cadherin,E-cadherin,Vimentin,Slug,Snail,MMP2 and MMP9 were detected by western blot.4.The luciferase reporter assay was applied to validate the transcriptional activity of HIF-1?.After SCH772984 and Honokiol treated with PCa cell lines,the expression of HIF-1? and all target genes(PKM2,GLUT1,LDHA and HK2)were detected by qRT-PCR and western blot.Immunofluorescence analysis was used to observe the HIF-1? nuclear translocation.After using MG-132 and si-pVHL treatment with PC3 cell,the expression of HIF-1? was detected by western blot.Results1.Our findings showed that high expression of PLC? and HIF-1? in clinical PCa samples by IHC.In addition,Pearson correlation analysis for these values showed that a positive association with PLC? activation level and HIF-1? aberrant expression(r=0.3285,p=0.0337).We observed highly significant differences between the mean of HIF-1? measurements in sera of PCa patients and healthy controls.High HIF-1? serum level was a statistically significant correlation with multiple clinicopathologicalfeatures: N stage(p=0.036),M stage(p=0.006),Gleason grade(p= 0.023),D'Amico grade(p=0.008).2.We observed the level of glucose consumption and lactate production remarkably decreased of after PLC? knockdown in PCa cells as compared to their respective control group.The date indicated that PLC?knockdown significantly reduced the mRNA and protein expression of HIF-1? and all target genes(PKM2,GLUT1,LDHA and HK2)in PCa cell lines compared to control group.We found that knock down HIF-1? by siRNA reduced the expression of PKM2,GLUT1,LDHA,glucose consumption and lactate production(p<0.01)in PC3 and DU145 cells on hypoxic condition.3.The results demonstrate that PCa cells transfected with sh-PLC? or si-HIF-1? displayed weaker migratory capacity under hypoxia by transwell assay.After combined treatment with sh-PLC? and si-HIF-1? in PC3 cells,cell migration ability was notably reduced.The protein expression of EMT(N-cadherin and Vimentin)and other migration-associated genes(Slug,Snail,MMP2 and MMP9)were roughly decreased in PCa cell lines with sh-PLC? or si-HIF-1?.These protein expressions were conspicuously reduced after combined treatment with sh-PLC? and si-HIF-1? in PC3 cells.However,expression of E-cadherin was enhanced compared with control in PCa cells.4.We observed that PLC? knockdown markedly impaired p-mTOR,p-S6 K,p-4EBP,p-MEK and p-ERK expression compared with control group in PC3 and LNCap cell lines.The protein expression of p-ERK,HIF-1?,PKM2,GLUT1,LDHA and HK2 was significantly reduced in PC3 and LNCap cells with combined treatment of sh-PLC? and SCH772984 compared with that in cells treated with sh-PLC? or SCH772984 alone.Sh-PLC? weakened the up-regulation effect of p-ERK,HIF-1?,PKM2,GLUT1,LDHA and HK2 by Honokiol in PCa cells.The results demonstrated that sh-PLC? decreased the HIF-1? HRE luciferase activity in comparison to control group,which weakened by Honokiol and fortified by SCH772984,under hypoxic condition.The results showed that PLC? knockdown prevented HIF-1? nucleus translocation,whereas no significant alteration was observed in the control group by immunofluorescence analysis.Western blot analysis was used to assess the distribution of HIF-1? in the nucleus and cytoplasm.We found that HIF-1?expression was impaired in the nucleus after PLC? knockdown.Sh-PLC?-mediated inhibition of HIF-1? protein levels could be reversed by treatment with MG132 or si-pVHL,indicating that PLC? regulation of HIF-1? was proteasome dependent.Conclusions1.PLC? status correlates well with the HIF-1? expression in human prostate cancer.HIF-1? may be a prognostic marker for PCa.2.PLC? regulates HIF-1? targets and metabolic activity in PCa cells.3.Energy metabolism regulated by PLC? elevates the migration of PCa cell lines.4.HIF-1? is regulated by PLC? via mTOR and ERK signaling in PCa cells.PLC? knockdown blocks HIF-1? nuclear translocation.PLC?knockdown regulates HIF-1? proteasomal degradation in a VHL-dependent manner. |
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