PLC? Regulates Prostate Cancer Mitochondrial Oxidative Metabolism And Migration Via Upregulation Of Twist1

Objective1.To explore the differential expression of PLC? and Twist1 in prostate cancer(PCa)and benign prostatic hyperplasia(BPH)tissues,and their relationship with clinical pathological parameters.Detect the expression of Twist1 in serum of patients with PCa and BPH,and its relationship with clinical pathological parameters.2.Whether Twist1 can regulate mitochondrial metabolism in PCa cells.3.To explore whether PLC? regulates the mitochondrial metabolism and migration of PCa cells through Twist1.4.Further explore the mechanism of Twist1 regulated by PLC?.Methods1.GEO,TCGA and GTEX database were used to determine Twist1 and PLC? m RNA levels in PCa tissues and their non-tumor counterparts,and the survival rates of PLC? and Twist1.GTEX database was used to analyze the correlation between them.2.55 PCa and 48 BPH tissue samples weretested for the presence of PLC? and Twist1 immunohistochemically.An association between PLC?and Twist1 was determined by Pearson's correlation analysis.q RT-PCR was used to detect the expression of Twist1 in serum.3.PLC? was knocked down with a lentiviral short hairpin RNA(sh-PLC?).Mitochondrial activity was assessed by measuring the oxygen consumption rate.western blot analyses and q RT-PCR were used to measure levels of PLC?,Twist1,PGC-1?,CPT1 B,ERR?,UCP-1 and ACADM.4.Knockdown or overexpression of Twist1 plasmid,then mitochondrial activity was evaluated by measuring oxygen consumption rate.western blot analyses and q RT-PCR were used to measure levels of PLC?,Twist1,PGC-1?,CPT1 B,ERR?,UCP-1 and ACADM.5.The expression of p-Twist1(ser68)was detected by q RT-PCR and Western blot.The m RNA and protein expressions of PLC?,Twist1,p-MEK,t-MEK,p-ERK,t-ERK,p-JNK,t-JNK,p-P38,P38 and B-Raf were detected by q RT-PCR and western blot after the prostate cancer cell lines were treated with trametinib,JNK-In-8 and SB203580.6.The luciferase reporter assay was applied to validate the transcriptional activity of Twist1.Immunofluorescence analysis was used to observe the PPAR? nuclear translocation.After GW501516 and GSK3787 treated with PCa cell lines,the expression of Twist1 and migration related molecules(N-cadherin ? E-cadherin ? Vimentin)were detected by q RT-PCR and western blot.Transwell assay was used to detect the migration ability.7.Tumorigenesis experiment in nude mice to explore the effect of knockdown PLC? and Twist1 on the proliferation of tumor cells in vivo.Results1.The expression level of Twist1 in PCa was significantly higher than that in non prostate cancer tissues,and it was also significantly higher in metastatic prostate cancer than that in primary prostate cancer.In TCGA database,the high expression of PLC? was related to high Gleason scores.Patients with high PLC? and Twist1 expression have shorter survival time.GTEX database showed a positive correlation between PLC? and Twist1 expression in PCa.2.IHC analyses showed high expression of PLC? and Twist1 in PCa samples.In addition,Pearson correlation analysis for these values showed that a positive association with PLC? activation level and Twist1 aberrant expression(r=0.8037,p<0.001).Both are highly expressed in high Gleason scores(r = 0.007,P = 0.037).We observed high Twist1 serum level was correlated with tumor metastasis.3.PLC? knockdown promoted mitochondrial oxidative activity and the expression of PGC-1?,CPT1 B,ERR?,UCP-1 and ACADM in PCa cells.4.sh-Twist1 promotes mitochondrial oxidative activity and expression of related molecules in PCa cell lines.Vector-Twist1 inhibited mitochondrial oxidative activity and expression of related molecules.5.PLC? knockdown inhibited the phosphorylation of Twist1 through MAPK signaling pathways,thereby inhibiting Twist1 protein level.All three inhibitors effectively antagonized their corresponding targets and decreased Twist1 protein levels in the absence or presence of PLC? but had little effect on Twist1 m RNA levels.6.The dual luciferase report experiment showed that PLC?knockdown inhibited Twist1 transcription through PPAR?.Immunofluorescence experiments and western blot results showed that sh-PLC? could inhibit the nuclear translocation of PPAR?.Treatment of sh-PLC? PCa cells with GW501516 prevented the PLC?depletion-mediated migration ability,and the expression of related molecules,while GSK3787 has the opposite effect.7.Knockdown of PLC? or Twist1 can inhibit the proliferation of PC3 cells in nude mice.Conclusions1.There is a positive correlation between PLC? and Twist1 expression in PCa.Twist1 may be a potential marker to evaluate the metastasis of prostate cancer.PLC? and Twist1 are expected to be targets for the treatment of prostate cancer.2.PLC? regulates mitochondrial metabolism and migration of PCa cells through Twist1.3.PLC? regulates the expression of Twist1 mainly through the following ways: PLC? regulates the degradation of Twist1 protein through three MAPK signaling pathways,thereby affecting its protein level;PLC?regulates the nuclear translocation of PPAR?,thereby affecting the transcription of Twist1.