PLCε Regulates Glutaminase-mediated Glutamine Metabolism And Contributes To Tumorigenesis In Bladder Cancer

Purpose Glutamine is the most abundant amino acid in the blood and plays a particularly important role in cell growth.One of the hallmarks of tumor metabolism is glutamine addiction.The purpose of this study was to investigate whether there are metabolic abnormalities in bladder cancer and the effect of glutamine on the growth of bladder cancer,as well as the mechanism of glutamine addiction during the growth of bladder cancer cells.The correlation between PLCε and glutamine metabolism was analyzed by gene microarray and mass spectrum,and the mechanism of whether PLCε could regulate the autophagy and apoptosis of bladder cancer cells by regulating GLS activity mediated by PLCε,so as to promote the tumorigenesis of bladder cancer cells was further explored.Method(1)The expression of PLCε and GLS in bladder cancer was analyzed by database.Then the total RNA was extracted from the sh PLCε and sh NC group.The total RNA was detected by Aligent4 × 4K whole-genome microarray,and the KEGG and GO analysis were performed.The contents of glutamine and glutamate in the sh PLCε and sh NC group were analyzed by LC-MS/MS and GC-MS.Q-PCR and WB confirmed the expression of PLCε and GLS in SV-HUC-1,T24 and EJ.The silencing PLCε was verified by q-PCR and WB assay and its effect on GLS expression.At the same time,the effects of silencing PLCε on intracellular glutaminase activity and glutamate content were analyzed by GLS kit and mass spectrometry.(2)The expressions of PLCε and GLS in non-bladder cancer group(n=17)and bladder cancer tissue(n=39)were detected by Q-PCR.The expression of PLCε and GLS in non-bladder cancer tissues(n=18)and bladder cancer tissues(n=48)was analyzed by IHC staining,and the correlation between PLCε and GLS and clinical parameters was analyzed.(3)The effects of glutamine deprivation on the proliferation and autophagy of bladder cancer cells were detected by colony formation and immunofluorescence.Q-PCR and WB was used to detect the effects of glutamine deprivation on the expression of PLCε,GLS,PCNA,LC3 and p62.(4)Transmission Electron Microscope(TEM),Immunofluorescence and flow cytometry were used to detect the changes in the number of autophagosomes and the apoptosis rate after silencing PLCε.Q-PCR and WB was used to detect the effects of knockdown PLCε on autophagy molecules LC3,p62,and apoptosis molecules Bax.(5)Flow cytometry and immunofluorescence to detect the effect of GLS overexpression on the apoptosis and autophagy of PLCε-silenced cells in T24/EJ.WB detect the protein expression of PLCε,GLS,Cleaved-Caspase3,LC3,p62.Western blot detected the effects of specific knockdown of GLS on apoptosis and autophagy proteins.The database analyzes the correlation between JNK/c-JUN and GLS.MTT detected the appropriate concentration of SP600125.The influence of PLCε silence and SP600125,alone or in combination,on the activity of GLS enzymes.Western blot detected the effects of PLCε silencing and SP600125,alone or in combination on JNK,p-JNK,c-JUN,p-c-JUN and GLS proteins.Results(1)Database analysis PLCε and GLS are highly expressed in bladder cancer;KEGG and GO analysis results show that PLCε is closely related to glutamate and aspartic acid metabolism;compared with healthy control group.Plasma glutamine content(Gln)was lower in patients with BCa than in healthy individuals,while levels of alanine(Ala),glutamate(Glu),aspartate(Asp),serine(Ser),and glycine(Gly)were higher.Compared with SV-HUC-1,PLCε and GLS expressions are higher in T24 and EJ;knocking down PLCε inhibits the expression of GLS,and reduces the intracellular glutamate content and glutaminase GLS activity.(2)Q-PCR and IHC results showed that compared with non-bladder cancer tissues,PLCε and GLS are highly expressed and positively correlated in bladder cancer tissues.And the expression of PLCε and GLS is significantly related to clinical stage.(3)The colony formation results showed that the proliferation of bladder cancer cells was inhibited after deprivation of glutamine,and the proliferation was further inhibited after silencing PLCε.The immunofluorescence results showed glutamine deprivation promoted autophagy in bladder cancer cells,and PLCε-silencing further promote autophagy.At the same time,q-PCR and WB results showed that deprivation of glutamine reduced the expression of GLS,PCNA,and p62,increased the expression of LC3,and did not change PLCε level.(4)TEM results show that PLCε silence increased the number of autophagosomes.And the results of immunofluorescence and flow cytometry showed that knockdown PLCε increased the number of LC3 puncta and the cell apoptosis rate.Both q-PCR and WB results showed that knockdown PLCε increases the expression of LC3,Bax,and decreases the expression of p62.(5)Compared with the Control group,after adding overexpression of GLS,the apoptosis rate and the number of autophagosomes decreased.Compared with the sh PLCε group,the addition of overexpression of GLS reversed the expression of the Cleaved-Caspase3,LC3,and p62 in T24/EJ,but PLCε did not change significantly;knockdown GLS alone made the expression of Cleaved-Caspase3,Bax,LC3 increased,and the expression of p62 decreased.Compared with the sh PLCε group,after adding the JNK inhibitor SP600125,the expression of JNK,c-JUN,GLS and enzyme activity in T24 and EJ will be further reduced.Conclusion Abnormal glutamine metabolism exists in bladder cancer.PLCε is related to glutamine metabolism.Knockdown of PLCε inhibits glutamine metabolism;PLCε and GLS were highly expressed and positively correlated with one another in BCa tissues;glutamine promotes proliferation and inhibits autophagy by regulating GLS expression;PLCεregulates the glutamine metabolism of bladder cancer cells via GLS,and inhibits autophagy and apoptosis,thereby promoting tumorigenesis;at the same time,PLCε can regulate GLS expression through JNK/c-JUN.