目的：探讨胶质瘤细胞接受联合X线辐射及T M Z化疗干预后其干细胞标志物的变化情况。方法将51例人脑胶质瘤标本作为观察对象，将标本分为22例脑胶质瘤Ⅰ～Ⅱ级的低级别组及29例脑胶质瘤Ⅲ～Ⅳ级的高级别组，所有入选标本均给予联合X线...目的：探讨胶质瘤细胞接受联合X线辐射及T M Z化疗干预后其干细胞标志物的变化情况。方法将51例人脑胶质瘤标本作为观察对象，将标本分为22例脑胶质瘤Ⅰ～Ⅱ级的低级别组及29例脑胶质瘤Ⅲ～Ⅳ级的高级别组，所有入选标本均给予联合X线辐射及 TMZ 化疗干预。对比分析2组干预前后胶质细胞中CD133及 ABCG2的表达情况，并对CD133及ABCG2改善情况与细胞恶性程度的相关性进行分析。结果2组间CD133及ABCG2的表达均存在显著差异，高级别组表达程度均明显高于低级别组（P＜0.05）。2组在接受联合X线辐射及TMZ化疗干预后，CD133及ABCG2均明显改善，以低级别组改善更为明显（ P＜0.05），且治疗前后CD133及ABCG2改善度与细胞恶性程度存在负向直线相关性（ P＜0.05）。结论不同恶性程度的胶质瘤细胞中CD133及ABCG2的表达存在显著差异。联合X线辐射及TMZ化疗可有效改善CD133及ABCG2表达，且与细胞恶性程度存在负向直线相关性。展开更多
Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standa...Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.展开更多
Temozolomide(TMZ)represents a standard-of-care chemotherapeutic agent in glioblastoma(GBM).However,the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma.Although spec...Temozolomide(TMZ)represents a standard-of-care chemotherapeutic agent in glioblastoma(GBM).However,the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma.Although specific innovative approaches,such as immunotherapy,have shown favorable clinical outcomes,the inherent invasiveness of most gliomas continues to make them challenging to treat.Consequently,there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development.This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors.A total of 648 proteins exhibiting significant differential expression were identified.Gene Set Enrichment Analysis(GSEA)unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups.Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate(IP3)kinase B(ITPKB)in the recurrence group,correlating with poor survival in glioma patients.In TMZ-resistant cells,the depletion of ITPKB led to an increase in reactive oxygen species(ROS)related to NADPH oxidase(NOX)activity and restored cell sensitivity to TMz.Mechanistically,the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination.This,in turn,elevated ITPKB stability and impaired ROS production.Furthermore,ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model.These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target forTMZ-resistant GBM.展开更多
Background:High-grade glioma(HGG)is a fatal human cancer.Bortezomib,a proteasome inhibitor,has been approved for the treatment of multiple myeloma but its use in glioma awaits further investigation.This study aimed to...Background:High-grade glioma(HGG)is a fatal human cancer.Bortezomib,a proteasome inhibitor,has been approved for the treatment of multiple myeloma but its use in glioma awaits further investigation.This study aimed to explore the chemotherapeutic effect and the underlying mechanism of bortezomib on gliomas.Methods:U251 and U87 cell viability and proliferation were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay,tumor cell spheroid growth,and colony formation assay.Cell apoptosis and cell cycle were detected by flow cytometry.Temozolomide(TMZ)-insensitive cell lines were induced by long-term TMZ treatment,and cells with stem cell characteristics were enriched with stem cell culture medium.The mRNA levels of interested genes were measured via reverse transcription-quantitative polymerase chain reaction,and protein levels were determined via Western blotting/immunofluorescent staining in cell lines and immunohistochemical staining in paraffin-embedded sections.Via inoculating U87 cells subcutaneously,glioma xenograft models in nude mice were established for drug experiments.Patient survival data were analyzed using the Kaplan-Meier method.Results:Bortezomib inhibited the viability and proliferation of U251 and U87 cells in a dose-and time-dependent manner by inducing apoptosis and cell cycle arrest.Bortezomib also significantly inhibited the spheroid growth,colony formation,and stem-like cell proliferation of U251 and U87 cells.When administrated in combination,bortezomib showed synergistic effect with TMZ in vitro and sensitized glioma to TMZ treatment both in vitro and in vivo.Bortezomib reduced both the mRNA and protein levels of Forkhead Box M1(FOXM1)and its target gene Survivin.The FOXM1-Survivin axis was markedly up-regulated in established TMZ-insensitive glioma cell lines and HGG patients.Expression levels of FOXM1 and Survivin were positively correlated with each other and both related to poor progno-sis in glioma patients.Conclusions:Bortezomib was found to inhibit glioma growth and improved TMZ chemotherapy efficacy,probably via down-regulating the FOXM1-Survivin axis.Bortezomib might be a promising agent for treating malignant glioma,alone or in combination with TMZ.展开更多
Glioblastoma multiform(GBM)is the most lethal intracranial tumor in adults.Glioblastoma stem-like cells(GSCs)are responsible for tumorigenesis and chemotherapy resistance.BMPs are known to increase temozolomide(TMZ)re...Glioblastoma multiform(GBM)is the most lethal intracranial tumor in adults.Glioblastoma stem-like cells(GSCs)are responsible for tumorigenesis and chemotherapy resistance.BMPs are known to increase temozolomide(TMZ)response in GSCs,however,the intracellular molecular mechanism remains largely unknown.In this study,we built a GSC cell model called U87S,and performed RNA sequencing to identify differentially expressed(DE)miRNA profiles in U87S cells treated with BMP2,TMZ or combined BMP2 and TMZ respectively.Bioinformatics analysis revealed that most DE miRNAs were involved in the cancer pathways,suggesting their crucial roles in gliomagenesis.Eight miRNAs from RNA-seq were validated.Four out of these miRNAs(has-miR-199a-3p,hsa-miR-374b-5p,hsa-miR-320d,and hsa-miR-339-5p)were found significantly up-regulated in GBM tumor tissues.One of them,hsa-miR-199a-3p,was significantly correlated with the survival of GBM patients,and differentially expressed in U87S cells.Expression of hsa-miR-199a-3p was up-regulated by BMP.Overexpression of hsa-miR-199a-3p in U87S cells inhibited cell viability and enhanced the cytotoxicity of TMZ.And activation of BMP boosted the effect of hsa-miR-199a-3p on cell viability and TMZmediated cytotoxicity.Besides,expressions of five predicted targets of hsa-miR-199a-3p were evaluated.Four of them were differentially expressed in GBM tumors.And one of them,SLC22A18,was associated with the survival of GBM patients.In the end,a hsa-miR-199a-3pmediated ceRNA network was constructed for the convenience of future study.Together,our data provided DE miRNA expression profiles associated with BMP2 and TMZ in GSCs,which might lead to finding out miRNA-based target therapies that specially target GSCs.展开更多
基金supported by the Science and Technology Project of Tianjin Municipal Health Commission(Grant Nos.TJWJ2022MS003 and TJWJ2021ZD008)the Tianjin Science and Technology Plan Project(Grant Nos.21JCYBJC01520 and 20JCYBJC01070)。
文摘Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.
基金supported by grants from the National Natural Science Foundation of China (82272659,82030087)the Science and Technology Innovation Program of Hunan Province (2022RC1210,2021RC3029)the Hunan Provincial Natural Science Foundation Project (2022JJ30073,2021JC0002).
文摘Temozolomide(TMZ)represents a standard-of-care chemotherapeutic agent in glioblastoma(GBM).However,the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma.Although specific innovative approaches,such as immunotherapy,have shown favorable clinical outcomes,the inherent invasiveness of most gliomas continues to make them challenging to treat.Consequently,there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development.This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors.A total of 648 proteins exhibiting significant differential expression were identified.Gene Set Enrichment Analysis(GSEA)unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups.Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate(IP3)kinase B(ITPKB)in the recurrence group,correlating with poor survival in glioma patients.In TMZ-resistant cells,the depletion of ITPKB led to an increase in reactive oxygen species(ROS)related to NADPH oxidase(NOX)activity and restored cell sensitivity to TMz.Mechanistically,the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination.This,in turn,elevated ITPKB stability and impaired ROS production.Furthermore,ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model.These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target forTMZ-resistant GBM.
基金This work was primarily supported by Grants from the National Natural Science Foundation of China(NSFC-81972360)
文摘Background:High-grade glioma(HGG)is a fatal human cancer.Bortezomib,a proteasome inhibitor,has been approved for the treatment of multiple myeloma but its use in glioma awaits further investigation.This study aimed to explore the chemotherapeutic effect and the underlying mechanism of bortezomib on gliomas.Methods:U251 and U87 cell viability and proliferation were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay,tumor cell spheroid growth,and colony formation assay.Cell apoptosis and cell cycle were detected by flow cytometry.Temozolomide(TMZ)-insensitive cell lines were induced by long-term TMZ treatment,and cells with stem cell characteristics were enriched with stem cell culture medium.The mRNA levels of interested genes were measured via reverse transcription-quantitative polymerase chain reaction,and protein levels were determined via Western blotting/immunofluorescent staining in cell lines and immunohistochemical staining in paraffin-embedded sections.Via inoculating U87 cells subcutaneously,glioma xenograft models in nude mice were established for drug experiments.Patient survival data were analyzed using the Kaplan-Meier method.Results:Bortezomib inhibited the viability and proliferation of U251 and U87 cells in a dose-and time-dependent manner by inducing apoptosis and cell cycle arrest.Bortezomib also significantly inhibited the spheroid growth,colony formation,and stem-like cell proliferation of U251 and U87 cells.When administrated in combination,bortezomib showed synergistic effect with TMZ in vitro and sensitized glioma to TMZ treatment both in vitro and in vivo.Bortezomib reduced both the mRNA and protein levels of Forkhead Box M1(FOXM1)and its target gene Survivin.The FOXM1-Survivin axis was markedly up-regulated in established TMZ-insensitive glioma cell lines and HGG patients.Expression levels of FOXM1 and Survivin were positively correlated with each other and both related to poor progno-sis in glioma patients.Conclusions:Bortezomib was found to inhibit glioma growth and improved TMZ chemotherapy efficacy,probably via down-regulating the FOXM1-Survivin axis.Bortezomib might be a promising agent for treating malignant glioma,alone or in combination with TMZ.
基金We would like to thank Dr.Richard W.Padgett from University of Rutgers,U.S.A.for his meaningful advice on this studyThis work was partially supported by grants from the National Natural Science Foundation of China(31501039)+3 种基金Chongqing Yuzhong District Science and technology project(20140104)Chongqing Basic and Frontier Research Project(cstc2016jcyjA0305)Open Project of Key Laboratory of Oncology and Immunology of Ministry of Education(2012jszl10)to YL,the National Natural Science Foundation of China(81473284)to LW.
文摘Glioblastoma multiform(GBM)is the most lethal intracranial tumor in adults.Glioblastoma stem-like cells(GSCs)are responsible for tumorigenesis and chemotherapy resistance.BMPs are known to increase temozolomide(TMZ)response in GSCs,however,the intracellular molecular mechanism remains largely unknown.In this study,we built a GSC cell model called U87S,and performed RNA sequencing to identify differentially expressed(DE)miRNA profiles in U87S cells treated with BMP2,TMZ or combined BMP2 and TMZ respectively.Bioinformatics analysis revealed that most DE miRNAs were involved in the cancer pathways,suggesting their crucial roles in gliomagenesis.Eight miRNAs from RNA-seq were validated.Four out of these miRNAs(has-miR-199a-3p,hsa-miR-374b-5p,hsa-miR-320d,and hsa-miR-339-5p)were found significantly up-regulated in GBM tumor tissues.One of them,hsa-miR-199a-3p,was significantly correlated with the survival of GBM patients,and differentially expressed in U87S cells.Expression of hsa-miR-199a-3p was up-regulated by BMP.Overexpression of hsa-miR-199a-3p in U87S cells inhibited cell viability and enhanced the cytotoxicity of TMZ.And activation of BMP boosted the effect of hsa-miR-199a-3p on cell viability and TMZmediated cytotoxicity.Besides,expressions of five predicted targets of hsa-miR-199a-3p were evaluated.Four of them were differentially expressed in GBM tumors.And one of them,SLC22A18,was associated with the survival of GBM patients.In the end,a hsa-miR-199a-3pmediated ceRNA network was constructed for the convenience of future study.Together,our data provided DE miRNA expression profiles associated with BMP2 and TMZ in GSCs,which might lead to finding out miRNA-based target therapies that specially target GSCs.
