| Background and purpose:Glioma is the most common intracranial malignant tumor,which is characterized by high incidence,strong invasiveness,high recurrence rate,easy resistance to radiotherapy and chemotherapy,and poor prognosis.The prognosis of the patients was still poor even after multimodal treatment,including surgical resection,radiotherapy and chemotherapy.At present,the mechanism of malignant progression of glioma is unclear.It is important to explore the underlying molecular mechanism of glioma and identify novel therapeutic targets.In recent years,the ubiquitin-proteasome system(UPS)is involved in the cellular processes.E3 ubiquitin ligase F-box /WD40 domain protein 7(FBXW7)is one of the important tumor suppressors.However,its effect on glioma cell metabolism is rarely reported.Warburg effect is the most important way of energy supply for cancer cells,which produce amounts of lactic acid.Abnormal accumulation of intracellular and extracellular lactic acid is closely related to biological performances,such as tumor metastasis,immune escape,angiogenesis and drug resistance.What is worth exploring is how to regulate the metabolic process to block the malignant progress of glioma.Therefore,this study will explore the clinical significance and biological functions of FBXW7 in glioma,reveal the mechanism of its regulation of lactic acid metabolism in glioma cells,and clarify its key role in the regulation of glioma metabolism and progression,so as to provide new ideas for the treatment of glioma.Methods:1.The m RNA expression data of FBXW7 and LDHA were extracted from the public database to analyze the expression pattern of FBXW7 in glioma.The relationship between FBXW7,LDHA and prognosis of patients was analyzed by survival curves.2.The expression level of FBXW7 was explored by immunohistochemical staining and RT-q PCR in the glioma tissues.The relationship between FBXW7 and clinical parameters was analyzed.The relationship between FBXW7,LDHA and clinical prognosis was analyzed.3.Cell lines with stable overexpression or knockdown of FBXW7 and LDHA were constructed by lentivirus transfection,which were detected by RT-q PCR and Western blot.The migration,invasion,proliferation and cell cycle of glioma cells were detected by scratch assay,Transwell migration assay,Transwell invasion assay,CCK-8 assay,cell clonal formation assay and flow cytometry.Molecular markers associated with epithelial-mesenchymal transformation(EMT)were detected by Western blot.4.Intracranial in situ tumorigenesis model was established in nude mice,and detected by HE staining.The effect of FBXW7 on the survival of nude mice was analyzed.5.Immunoprecipitation combined with mass spectrometry was used to analyze FBXW7 interacting proteins,and functional enrichment analysis was used to identify the underlying mechanism of FBXW7 in glioma.6.FBXW7 was knocked out or overexpressed to detect the expression of LDHA by Western blot.The interaction between FBXW7 and LDHA was confirmed by Co-immunoprecipitation.The co-localization was observed by immunofluorescence.7.The production of ATP,lactate and pyruvate,and the LDHA activity were detected after FBXW7 was knocked down or overexpressed in glioma cells.8.Protein half-life assay and ubiquitination assay were used to detect the degradation of LDHA by ubiquitination of FBXW7.9.The production of ATP,lactate and pyruvate,and the LDHA activity in glioma cells were detected after co-expression of FBXW7 and LDHA or co-inhibition of FBXW7 and LDHA.The effects of co-expression of FBXW7 and LDHA or co-inhibition of FBXW7 and LDHA on cell migration and proliferation were detected by scratch assay,Transwell invasion assay,migration assay,CCK-8 assay and cell clonal formation assay.Results:1.Bioinformatics analysis,immunohistochemistry and RT-q PCR showed that FBXW7 was down-expressed in glioma tissues,and low expression of FBXW7 was associated with glioma malignant phenotype and indicated poor prognosis.2.Stable overexpression or knockdown FBXW7 cell lines were constructed in U251,T98 G,SHG44 and HS683 cells,respectively.3.CCK8,cell clonal formation assay,scratch assay,Transwell invasion assay,Transwell migration assay and flow cytometry analysis showed that the proliferation,invasion and migration of glioma cells were reduced after overexpression of FBXW7,and the cell cycle was arrested in G1/S phase.After FBXW7 knockdown,the proliferation,invasion and migration of glioma cells were enhanced,and the G1/S phase was shortened.4.Intracranial glioma model showed that nude mice with over-expression FBXW7 formed smaller gliomas and extended the survival time.On the contrary,nude mice with knockdown FBXW7 formed larger gliomas and shortened the survival time.5.Immunoprecipitation-mass spectrometry analysis showed that proteins interacting with FBXW7 were enriched in glycolytic metabolism-related signaling pathways.6.Co-immunoprecipitation and immunofluorescence analysis showed that FBXW7 and LDHA interacted with each other and partially co-located in the nucleus.7.The regulation of LDHA protein level by FBXW7 depends on its ubiquitination activity,and interference of FBXW7 significantly prolongs the half-life of LDHA degradation.8.After FBXW7 was knocking down in glioma cells,the production of ATP and lactic acid and the activity of LDHA was increased,while the production of pyruvate was decreased.On the contrary,the production of ATP and lactic acid and the activity of LDHA was decreased,while the production of pyruvate increased,when FBXW7 was over-expressed.9.Successive interference of FBXW7 and LDHA,partially rescued the enhanced proliferation,invasion and migration ability of glioma cells.Overexpression of FBXW7 and LDHA resulted in partial recovery of the reduced proliferation,invasion and migration capacity of glioma cells.10.Overexpression of LDHA partially rescued the effects of overexpression of FBXW7 on the production of ATP,lactic acid and pyruvate and the activity of LDHA in glioma cells.Similarly,the effects of FBXW7 interference on the production of ATP,lactate and pyruvate and LDHA activity of glioma cells were partially recovered by interfering LDHA expression.Conclusions:1.The down-regulated FBXW7 was negatively correlated with glioma malignant phenotype and predicted a poor prognosis.2.Abnormal expression of FBXW7 negatively regulates the proliferation,invasion,migration and cell cycle of glioma cells,and affects the survival time of nude mice.3.FBXW7 degrades the expression of LDHA through ubiquitination,to inhibit the proliferation,invasion,migration and lactic acid metabolism of glioma cells,and ultimately block the malignant progression of glioma.43 figures,18 tables and 127 references... |
PDF Full Text Request