The Role And Mechanism Of The Deubiquitinating Enzymes UCH-L5 And Poh1 In The Development Of Glioma

Glioma is the most common primary brain tumor,accounting for more than 36% of primary central nervous system tumors,with high morbidity and mortality.The treatment of glioma is mainly based on traditional therapies such as surgical resection,radiotherapy,chemotherapy and drug combination.However,the prognosis of patients after treatment is poor and they are prone to relapse.The pathogenesis of glioma is still unclear,and in-depth study of key protein molecules and their regulatory mechanisms in the development of glioma is of great significance for finding new therapeutic targets and effective therapeutic drugs for glioma.The ubiquitin proteasome system is composed of ubiquitin activating enzyme E1,ubiquitin binding enzyme E2,ubiquitin ligase E3,deubiquitinating enzyme and 26 S proteasome system,which is one of the main ways for eukaryotes to maintain protein homeostasis.As a member of the ubiquitin proteasome system,deubiquitinating enzyme can specifically remove single ubiquitin molecules or polyubiquitin chains from substrate proteins modified by ubiquitination,thereby deubiquitinating substrate proteins.It plays an important role in the regulation of protein homeostasis.UCH-L5 and POH1 belong to two different deubiquitinating enzyme families,UCH-L5 belongs to the UCHs family,and POH1 belongs to the JAMMs family.Compared with normal brain tissue,we found that UCH-L5 was low-expressed,and POH1 was highly expressed in glioma tissue,suggesting that UCH-L5 and POH1 might be involved in the occurrence and development of glioma.However,the roles and molecular mechanism of UCH-L5 and POH1 in glioma cell proliferation and tumorigenesis need to be further elucidated.Using EDU analysis and clone formation experiments to detect cell proliferation,we found that knockdown of UCH-L5 expression promoted the proliferation of glioma cells U251.Previous studies in our laboratory have demonstrated that U251 cells stably knocking down UCH-L5 promoted glioma growth in nude mice,while knocking down UCH-L5 has no effect on the proliferation of glioma cells U87 MG cells,but the mechanism is unclear.In combination with literature reports of PTEN deletion in U87 MG cells,we speculate that PTEN may be a molecular target for UCH-L5.The UCH-L5 expression in U251 cells was interfered by specific si RNA.And Western blot detection of protein expression showed that the levels of PTEN and phosphorylated PTEN proteins in U251 were significantly reduced,while RT q PCR results showed no change in the m RNA level of PTEN.In the lentivirus-mediated UCH-L5 stable knockdown U251 cell line,PTEN and phosphorylated PTEN protein levels were significantly down-regulated.U251 cells were treated with b-AP15 inhibitor that inhibited the activity of UCH-L5 deubiquitinating enzyme,and the expression of PTEN protein in U251 cells was down-regulated.Interfering with UCH-L5 expression in glioma cell line U251 can promote the degradation of PTEN protein,which can be reversed by proteasome inhibitor MG132,while the lysosomal inhibitor CQ has no effect.IP experimental results confirmed that interference with UCH-L5 expression in glioma cell line U251 could increase the ubiquitination of PTEN.In HEK293 cells,overexpression of UCH-L5 promoted deubiquitination of PTEN.Therefore,UCH-L5 may inhibit the proliferation of U251 glioma cells by regulating the stability of PTEN.TCGA database information and brain tissue chip immunohistochemistry results showed that POH1 was highly expressed in glioma.CCK8 results showed that knockdown of POH1 inhibited the proliferation of A172 glioma cells and knockdown of POH1 promoted apoptosis of A172 glioma cells by Flow cytometry.Transwell and scratch experiments showed that knockdown of POH1 inhibited the migration and invasion of A172 glioma cells.Through literature review and experimental verification,we further explored the molecular mechanism of POH1 regulating the occurrence of glioma.Western blot was used to detect the expression of related proteins.Knockdown of POH1 inhibited the activation of TGF-β /Smads signaling pathway,and down-regulated the expression of downstream transcription factor ZEB1 protein in TGF-TGF /Smads signaling pathway.RT q PCR results showed that knockdown of POH1 did not affect ZEB1 m RNA level.Treatment of A172 cells with the protein synthesis inhibitor CHX reduced the stability of ZEB1 protein by knocking down POH1.Interfering with POH1 expression in glioma cell line A172 can promote the degradation of ZEB1 protein,which can be reversed by proteasome inhibitor MG132,while lysosomal inhibitor CQ has no effect.TCGA database information shows that in low-grade glioma patients,POH1 is positively correlated with ZEB1 expression.In this study,we found that UCH-L5 could inhibit U251 glioma cell proliferation by regulating PTEN protein stability.POH1 promoted the proliferation,migration,invasion of A172 glioma cells,and inhibits the apoptosis of A172 glioma cells.The related mechanism might be related to the regulation of POH1 on ZEB1 stability.These results suggest that UCHL5 and POH1 can be potential targets for glioma treatment,and provide ideas and experimental basis for finding new ways of glioma treatment.