TRIM66 Promots Glucose Metabolism And Reduces Temozolomide Sensitivity Via MYC/GLUT3 Signaling In Glioma

Objective:Glioma is one of the most common malignant tumors in the brain,and its incidence accounts for about 80%of central nervous system malignant tumors.At present,the treatment of glioma is still based on surgical resection,supplemented by radiotherapy and chemotherapy.In recent years,some improvementshave been made in early diagnosis,radical surgery,radiotherapy,and chemotherapy of glioma.However,glioma is characterized as highly aggressive and resistant to radiation and chemotherapy.The prognosisis still poor,and the median survival time of glioma patients is less than oneyear.The malignant development of glioma is a complex pathological process involving multiple steps,multiple stages,and multiple genes.Finding and identifying key target genescan provide the necessary scientific basis for the clinical diagnosis and treatment of glioma.TRIMs(Tripartite Motif)family proteins have three typical domains,including zinc finger domain,B-box domain and coiled coil domain.TRIM superfamily proteins,most of which have E3 ubiquitin ligase activities,exhibit various cellular functions and biological processes including transcription regulation,intracellular signaling,cell growth,apoptosis,innate immunity,autophagy,developmental processes,and carcinogenesis.Tripartite motif 66(TRIM66)was a member of the TRIM family which possesses potential functions as a transcriptional repressor.TRIM66 has emerged as an oncogenic protein that is closely related to multiple malignant tumors,including hepatocellular carcinoma,prostate cancer,colorectal cancer,non-small cell lung cancer and osteosarcoma.However,its expression pattern and biological effects in glioma have not been investigated.Here,we investigated the expression pattern,clinicopathologic significance,biological effects and underlying mechanism of TRIM66 in glioma.Methods:1.ImmunohistochemistryNinety-five cases of glioma tissues were taken from patients who underwent surgical resection in The Fourth Affiliated Hospital of China Medical University from 2013 to2019.4?m sections were firstly prepared fromthe paraffin tissues and then the sections were deparaffinized,hydrated,antigen retrieval,antibody incubation and color development.Staining intensity and percentage were combined to evaluate TRIM66expression levels.2.Bioinformatics analysisTRIM66 mRNA expression data in glioma and normal brain tissues was downloaded from Oncomine database(https://www.oncomine.org/resource/login.html),and the differences of TRIM66 expression between tumor tissues and normal tissues were statistically analyzed.Data of TRIM66 mRNA expression and the overall survival of glioma patients were downloaded from the TCGA database.The correlation between TRIM66 mRNA levels and the overall survival was statistically analyzed.3.Cell culture and tranfectionHuman normal astrocytes SVG p12 and glioma cell lines A172,U87MG,U251 were used in this experiment.Cells were cultured with DMEM and EMEM mediums supplied with 10%fetal bovine serum(FBS)at the condition of 37?,CO₂ and saturated humidity.TRIM66 overexpression plasmid and negative control plasmid were transfected into U251 cells using Lipofectamine 3000.TRIM66 knockdown small interfering RNA and the corresponding non-targeting si RNA were transfected into A172 cells using Dharmafect1 reagent.Transfected cells were colleted,and the transfection efficiency was estimated through western blot and qRT-PCR.4.Western blotTotal protein was isolated by SDS-PAGE and transferred to the PVDF membrane.Nonspecific blocking is performed using BSA.Antibodies used were:TRIM66,GAPDH,MYC,GLUT3.The secondary antibody used is horseradish peroxidase coupled secondary antibody.The PVDF membrane was placed in the dark of the gel imaging system for protein visualization.5.Real-time fluorescent quantitative PCRTotal RNA solution of glioma cells was prepared using RNAiso reagent.After quantification,reverse transcription was performed using Prime Script RT Kit.SYBR select master Mix Kit was used for qPCR.Relative expression of TRIM66 was normalized to GAPDH and calculated according to 2^-??ct.6.Cell proliferation,invasion and cell cycle transition assaysTRIM66 plasmid and si RNA transfected cells were applied to CCK8,colony formation,transwell invasion and PI staining assays to determine the effect of TRIM66 on cell proliferation,invasion and cell cycle transition.For CCK8 assay,cells were plated in96-well plates and incubated for 1,2,3,4,5 days respectively.For quantitation of cell proliferation rate,optical density was measured using a microplate reader.For colony formation assay,cells were seeded into dishes(about 1000 cells),incubated for 2 weeks,and then washed and stained with Giemsa.Colonies were counted using a microscope.For transwell invasion assay,cells in 100?l of serum-free DMEM medium were seeded on the upper chamber,and DMEM medium with 10%FBS was added into the bottom chamber.Then the cells on the bottom surface were stained and counted using a microscope.For cell cycle transition assay,cells were harvested and fixed in 75%ethanol.Cells were stained with 50?g/ml propidium iodide and a flow Cytometer was used to analyze the cell cycle distribution.7.Temozolomide resistance assayTRIM66 plasmid and siRNA transfected cells were treated with temozolomide.Cells were stained with Annexin V/PI and JC-1 respectively.A flow Cytometer was used to analyze the apoptosis rate and mitochondrial membrane potential.8.Glucose metabolism assaysFor glucose uptake assay,cells were treated with 2-NBDG and incubated for 30 minutes at 37?C.The fluorescence intensity was measured using Flow Cytometer.For glucose consumption,the supernatants were collected and the glucose consumption was measured using the Glucose Oxidase Assay Kit.For ATP level measurement,cells were lysed in ATP Assay Buffer.100?l ATP reaction solution was added into a 96-well plate and 20?l samples were added to the ATP reaction solution.A standard curve was performed and the fluorescence was measured using a microplate reader.9.Chromatin immunoprecipitation(Ch IP)Cells were cross-linked with formaldehyde and the lysates were sonicated to yield 300–1,000?bp DNA fragments.The DNA-protein complex was incubated with Cmycantibody and Ig G antibody in the presence of protein A/G beads.q PCR was performed by using primers for the promoter of the SLC2A3 gene.10.Statistical analysisThe data was analyzed using SPSS 22.0 software for Windows(SPSS,Chicago,IL,USA).Data of TRIM66 expression was downloaded from the Oncomine database and statistically analyzed by Mann-Whitney U-test.Overall survival was visualized using Kaplan-Meier curves,and the differences were compared by Log-rank test.Comparisons of biological experiments between different groups were performed using the Student?s t-test.P<0.05 was considered a statistically significant difference.Results:1.The expression pattern and pathological significance of TRIM66 in gliomaTRIM66 is overexpressed in glioma tissues.Among 95 cases of glioma tissues,TRIM66 high levels were present in 52 cases(54.7%).TRIM66 was overexpressed in glioma cell lines.TRIM66 expression was significantly correlated with Glioma grade(p=0.0284).Glioma patients with high TRIM66 expression have shorter overall survival time compared with those with low TRIM66 expression,although the p-value did not reach a statistical significance.2.TRIM66 promotes proliferation,invasion and cell cycle transitionTRIM66 expression was firstly exogenous regulated through TRIM66 plasmid and si RNA transfection in glioma cell lines.TRIM66 overexpression increased cell growth rate,colony formation number and invading cell number.In addition,TRIM66overexpression significantly increased the S phase percentage and decreased the G1phase percentage.TRIM66 depletion showed the opposite results.3.TRIM66 enhances temozolomide resistance of glioma cellsTemozolomide was used to treat glioma cells after TRIM66 plasmid and si RNA transfection.Annexin V/PI staining was used to determine the change of apoptosis.TRIM66 overexpression reduced the apoptosis rate,while TRIM66 depletion upregulated the levels of TMZ-induced apoptosis.JC-1 staining results showed that TRIM66 si RNA increased the percentage of green fluorescence while TRIM66overexpression decreased green fluorescence.4.TRIM66 promotes glucose metabolism of glioma cellsCancer cells use glucose metabolism to produce ATP,which is essential for the growth and development of chemoresistance.TRIM66 overexpression upregulated gluocose uptake,glucose consumption and ATP levels in glioma cells,while TRIM66knockdown showed the opposite results.5.TRIM66 promotes expression of transcription factor MYC and its target gene GLUT3TRIM66 positively regulated both MYC and GLUT3 protein expression.MYC knockdown downregulated protein and mRNA levels of GLUT3,suggesting that MYC depletion abolished the effect of TRIM66 on GLUT3 upregulation.Ch IP results showed that MYC regulated GLUT3 by binding to its promoter in glioma cells.Conclusion:TRIM66 expression is increased in glioma tissues and cell lines.TRIM66high level is significantly correlated with the grade of glioma,and is negatively correlated with the overall survival time of patients.TRIM66 promotes proliferation,invasion,cell cycle transition,and enhances temozolomide chemotherapy resistance of glioma cells.TRIM66 activates the transcription factor MYC related to glucose metabolism,up-regulating the expression level of its target gene GLUT3,and promotes glucose metabolism in glioma cells to supply ATP needed for maintaining the growth of tumor cells.