Background:p53 and DIRAS3 are tumor suppressors that are frequently silenced in tumors.In this study,we sought to determine whether the concurrent re-expression of p53 and DIRAS3 could effectively induce head and neck...Background:p53 and DIRAS3 are tumor suppressors that are frequently silenced in tumors.In this study,we sought to determine whether the concurrent re-expression of p53 and DIRAS3 could effectively induce head and neck squamous cell carcinoma(HNSCC)cell death.Methods:CAL-27 and SCC-25 cells were treated with Ad-DIRAS3 and rAd-p53 to induce re-expression of DIRAS3 and p53 respectively.The effects of DIRAS3 and p53 re-expression on the growth and apoptosis of HNSCC cells were examined by TUNEL assay,flow cytometric analysis and MTT.The effects of DIRAS3 and p53 re-expression on Akt phosphorylation,oncogene expression,and the interaction of 4 E-BP1 with eIF4 E were determined by real-time PCR,Western blotting and immunoprecipitation analysis.The ability of DIRAS3 and p53 re-expression to induce autophagy was evaluated by transmission electron microscopy,LC3 fluorescence microscopy and Western blotting.The effects of DIRAS3 and p53 re-expression on HNSCC growth were evaluated by using an orthotopic xenograft mouse model.Results:TUNEL assay and flow cytometric analysis showed that the concurrent re-expression of DIRAS3 and p53 significantly induced apoptosis(P<0.001).MTT and flow cytometric analysis revealed that DIRAS3 and p53 reexpression significantly inhibited proliferation and induced cell cycle arrest(P<0.001).Mechanistically,the concurrent re-expression of DIRAS3 and p53 down-regulated signal transducer and activation of transcription 3(STAT3)and upregulated p21WAF1/CIP1 and Bax(P<0.001).DIRAS3 and p53 re-expression also inhibited Akt phosphorylation,increased the interaction of eIF4 E with 4 E-BP1,and reduced the expression of c-Myc,cyclin D1,vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF),epidermal growth factor receptor(EGFR)and Bcl-2(P<0.001).Moreover,the concurrent re-expression of DIRAS3 and p53 increased the percentage of cells with GFP-LC3 puncta compared with that in cells treated with control adenovirus(50.00%±4.55%vs.4.67%±1.25%,P<0.001).LC3 fluorescence microscopy and Western blotting further showed that DIRAS3 and p53 re-expression significantly promoted autophagic activity but also inhibited autophagic flux,resulting in overall impaired autophagy.Finally,the concurrent re-expression of DIRAS3 and p53 significantly decreased the tumor volume compared with the control group in a HNSCC xenograft mouse model[(3.12±0.75)mm^(3) vs.(189.02±17.54)mm^(3),P<0.001].Conclusions:The concurrent re-expression of DIRAS3 and p53 is a more effective approach to HNSCC treatment than current treatment strategies.展开更多
目的探讨DIRAS家族GTP酶3(DIRAS3)在胶质瘤中的表达及其预后预测价值。方法使用阿拉巴马大学伯明翰分校癌症数据分析门户(The University of ALabama at Birmingham Cancer data analysis Portal,UALCAN)、基因表达谱交互分析2.0(Gene E...目的探讨DIRAS家族GTP酶3(DIRAS3)在胶质瘤中的表达及其预后预测价值。方法使用阿拉巴马大学伯明翰分校癌症数据分析门户(The University of ALabama at Birmingham Cancer data analysis Portal,UALCAN)、基因表达谱交互分析2.0(Gene Expression Profiling Interactive Analys,GEPIA 2.0)和人类蛋白质图谱(Human Protein Atlas,HPA)数据库评估DIRAS3在癌症基因组图谱计划(The Cancer Genome Atlas Program,TCGA)数据库中低级别胶质瘤(low-grade glioma,LGG)和胶质母细胞瘤(glioblastoma,GBM)中的表达及对临床预后的预测价值,并利用反转录实时定量PCR(real-time quantitative reverse tranion PCR,qRT-PCR)和蛋白印迹(western blotting,WB)验证DIRAS3在人星型胶质细胞和胶质瘤细胞中的表达。基于cBioPortal数据库分析GBM中与DIRAS3正相关的基因,利用STRING数据库进行聚类,选择包含DIRAS3的Cluster进行基因本体论(gene ontology,GO)和京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析。利用泛癌药物敏感性的综合分析(comprehensive analysis of drug sensitivity in pancancer,CADSP)数据库进行药物敏感性分析。结果DIRAS3在胶质瘤中高表达并与世界卫生组织(World Health Organization,WHO)分级、异柠檬酸脱氢酶(isocitrate dehydrogenase,IDH)状态、1p/19q共缺失状态、年龄以及较差的预后相关。此外,DIRAS3被预测参与细胞运动正向调节、焦点粘附、血管发育,以及癌症中的转录失调,并对多种抗肿瘤药物敏感。结论DIRAS3在胶质瘤中具有致癌作用,是一种新的、预后相关的候选基因,并且是胶质瘤的潜在诊断标志物和有希望的治疗靶点。展开更多
基金supported by Funding for the Basic Research of the Ministry of Sciences and Technology,Sichuan Province(2015JY0090)the National Natural Science Foundation of China(81972546,81602373)。
文摘Background:p53 and DIRAS3 are tumor suppressors that are frequently silenced in tumors.In this study,we sought to determine whether the concurrent re-expression of p53 and DIRAS3 could effectively induce head and neck squamous cell carcinoma(HNSCC)cell death.Methods:CAL-27 and SCC-25 cells were treated with Ad-DIRAS3 and rAd-p53 to induce re-expression of DIRAS3 and p53 respectively.The effects of DIRAS3 and p53 re-expression on the growth and apoptosis of HNSCC cells were examined by TUNEL assay,flow cytometric analysis and MTT.The effects of DIRAS3 and p53 re-expression on Akt phosphorylation,oncogene expression,and the interaction of 4 E-BP1 with eIF4 E were determined by real-time PCR,Western blotting and immunoprecipitation analysis.The ability of DIRAS3 and p53 re-expression to induce autophagy was evaluated by transmission electron microscopy,LC3 fluorescence microscopy and Western blotting.The effects of DIRAS3 and p53 re-expression on HNSCC growth were evaluated by using an orthotopic xenograft mouse model.Results:TUNEL assay and flow cytometric analysis showed that the concurrent re-expression of DIRAS3 and p53 significantly induced apoptosis(P<0.001).MTT and flow cytometric analysis revealed that DIRAS3 and p53 reexpression significantly inhibited proliferation and induced cell cycle arrest(P<0.001).Mechanistically,the concurrent re-expression of DIRAS3 and p53 down-regulated signal transducer and activation of transcription 3(STAT3)and upregulated p21WAF1/CIP1 and Bax(P<0.001).DIRAS3 and p53 re-expression also inhibited Akt phosphorylation,increased the interaction of eIF4 E with 4 E-BP1,and reduced the expression of c-Myc,cyclin D1,vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF),epidermal growth factor receptor(EGFR)and Bcl-2(P<0.001).Moreover,the concurrent re-expression of DIRAS3 and p53 increased the percentage of cells with GFP-LC3 puncta compared with that in cells treated with control adenovirus(50.00%±4.55%vs.4.67%±1.25%,P<0.001).LC3 fluorescence microscopy and Western blotting further showed that DIRAS3 and p53 re-expression significantly promoted autophagic activity but also inhibited autophagic flux,resulting in overall impaired autophagy.Finally,the concurrent re-expression of DIRAS3 and p53 significantly decreased the tumor volume compared with the control group in a HNSCC xenograft mouse model[(3.12±0.75)mm^(3) vs.(189.02±17.54)mm^(3),P<0.001].Conclusions:The concurrent re-expression of DIRAS3 and p53 is a more effective approach to HNSCC treatment than current treatment strategies.
文摘目的探讨DIRAS家族GTP酶3(DIRAS3)在胶质瘤中的表达及其预后预测价值。方法使用阿拉巴马大学伯明翰分校癌症数据分析门户(The University of ALabama at Birmingham Cancer data analysis Portal,UALCAN)、基因表达谱交互分析2.0(Gene Expression Profiling Interactive Analys,GEPIA 2.0)和人类蛋白质图谱(Human Protein Atlas,HPA)数据库评估DIRAS3在癌症基因组图谱计划(The Cancer Genome Atlas Program,TCGA)数据库中低级别胶质瘤(low-grade glioma,LGG)和胶质母细胞瘤(glioblastoma,GBM)中的表达及对临床预后的预测价值,并利用反转录实时定量PCR(real-time quantitative reverse tranion PCR,qRT-PCR)和蛋白印迹(western blotting,WB)验证DIRAS3在人星型胶质细胞和胶质瘤细胞中的表达。基于cBioPortal数据库分析GBM中与DIRAS3正相关的基因,利用STRING数据库进行聚类,选择包含DIRAS3的Cluster进行基因本体论(gene ontology,GO)和京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析。利用泛癌药物敏感性的综合分析(comprehensive analysis of drug sensitivity in pancancer,CADSP)数据库进行药物敏感性分析。结果DIRAS3在胶质瘤中高表达并与世界卫生组织(World Health Organization,WHO)分级、异柠檬酸脱氢酶(isocitrate dehydrogenase,IDH)状态、1p/19q共缺失状态、年龄以及较差的预后相关。此外,DIRAS3被预测参与细胞运动正向调节、焦点粘附、血管发育,以及癌症中的转录失调,并对多种抗肿瘤药物敏感。结论DIRAS3在胶质瘤中具有致癌作用,是一种新的、预后相关的候选基因,并且是胶质瘤的潜在诊断标志物和有希望的治疗靶点。
