AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal c...AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P 【 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.展开更多
Objective:To vexplore expression of HSP90,SIRT3 in liver cancer tissue and its effect on liver cancer cell invasion ability.Methods:Moderate expression of HSP90 in SMMC-7721,HepG2,LO2 and Hep-3B cell lines were screen...Objective:To vexplore expression of HSP90,SIRT3 in liver cancer tissue and its effect on liver cancer cell invasion ability.Methods:Moderate expression of HSP90 in SMMC-7721,HepG2,LO2 and Hep-3B cell lines were screened,which was validated by RT-PCR.Overexpression of HSP90 cell line and lentivirus packaging HSP90-RNAi were established,which was validated by RT-PCR and western blot.The level of epithelial-mesenchymal transition(EMT)related gene was detected by western blot.The percentage of cancer stem cells was assayed by flow cytometry.Results:RT-PCR demonstrated the highest expression of HSP90mRNA in SMMC-7721 cells,the lowest expression of HSP90 mRNA in Hep3B and LO2 and the moderate expression of HSP90 mRNA in Hep-G2.Therefore,HepG2 was selected as a follow-up experiment cell lines.Compared with the blank control group,expression of HSP90in HSP overexpression group was increased obviously,and expression of HSP90 in HSP90shRNA group was significantly decreased,which indicated successful establishment of HSP overexpression and shRNA group.The apoptotic cell in hsp-siRNA group was higher than the blank control group,while the HSP overexpression group showed opposite results.Western blot results showed transfection HSP promoted cells EMT transformation,up-regulated the level of E-cadherin,and down-regulated the level of Vimentin;meanwhile,shRNA group showed opposite results.Conclusions:Carcinoma HepG2 cell transfeeted high expression of HSP can promote the transformation of EMT,improve the expression of Vimentin,reduce the expression of E-cadherin,and inhibit apoptosis of cancer stem cells,which improve the invasive ability of cancer of the liver cells.While hsp-siRNA group presents opposite results.In summary,the expression of HSP is closely related to the occurrence,development and invasion of cancer of the liver tissue.展开更多
Lymphatic metastasis(LM)emerges as an independent prognostic marker for hypopharyngeal squamous cell carcinoma(HSPSCC),chiefly contributing to treatment inefficacy.This study aimed to scrutinize the prognostic relevan...Lymphatic metastasis(LM)emerges as an independent prognostic marker for hypopharyngeal squamous cell carcinoma(HSPSCC),chiefly contributing to treatment inefficacy.This study aimed to scrutinize the prognostic relevance of HSP90AA1 and its potential regulatory mechanism of concerning LM in HPSCC.Methods:In a preceding investigation,HSP90AA1,a differential gene,was discovered through transcriptome sequencing of HPSCC tissues,considering both the presence and absence of LM.Validation of HSP90AA1 expression was accomplished via qRT-PCR,western-blotting(WB),and immunohistochemistry(IHC),while its prognostic significance was assessed employing Kaplan–Meier survival analysis(KMSA),log-rank test(LR),and Cox’s regression analysis(CRA).Bioinformatics techniques facilitated the prediction and analysis of its plausible mechanisms in LM,further substantiated by in vitro and in vivo experiments utilizing FaDu cell lines.Results:HSP90AA1 is substantially upregulated in HPSCC with LM and is identified as an independent prognostic risk determinant.The down-regulation of HSP90AA1 can achieve inhibition of tumor cell proliferation,migration and invasion.Both in vivo experiments and Bioinformatics exploration hint at promoting LM by Epithelial-mesenchymal transition(EMT),regulated by HSP90AA1.Conclusions:HSP90AA1,by controlling EMT,can foster LM in HPSCC.This finding sets the foundation for delving into new therapeutic targets for HPSCC.展开更多
AIM:To investigate the effects of transplantation of insulin-producing cells(IPCs) in the treatment of diabetic rats after 90% pancreatectomy.METHODS:Human umbilical cord mesenchymal stem cells(UCMSCs) were isolated a...AIM:To investigate the effects of transplantation of insulin-producing cells(IPCs) in the treatment of diabetic rats after 90% pancreatectomy.METHODS:Human umbilical cord mesenchymal stem cells(UCMSCs) were isolated and induced into IPCs using differentiation medium.Differentiated cells were examined by dithizone(DTZ) staining,reverse transcription-polymerase chain reaction(RT-PCR),and real-time RT-PCR.C-peptide release,both spontaneously and after glucose challenge,was measured by ELISA.IPCs were then transplanted into Sprague-Dawley rats after 90% pancreatectomy and blood glucose levels and body weight were measured.RESULTS:The differentiated cells were positive for DTZ staining and expressed pancreatic β-cell related genes.C-peptide release by the differentiated cells increased after glucose challenge(380.6 ± 15.32 pmol/L vs 272.4 ± 15.32 pmol/L,P < 0.05).Further,in the cell transplantation group,blood sugar levels were significantly lower than in the sham group 2 wk after transplantation(18.7 ± 2.5 mmol/L vs 25.8 ± 1.25 mmol/L,P < 0.05).Glucose tolerance tests showed that 45 min after intraperitoneal glucose injection,blood glucose levels were significantly lower on day 56 after transplantation of IPCs(12.5 ± 4.7 mmol/L vs 42.2 ± 9.3 mmol/L,P < 0.05).CONCLUSION:Our results show that UCMSCs can differentiate into islet-like cells in vitro under certain conditions,which can function as IPCs both in vivo and in vitro.展开更多
Objective:To examine the role of heat shock protein 90(Hsp90) in the maintenance of actin cytoskeleton in human neuroblastoma tumor cells.Methods:Co-precipitation experiments were performed to examine Hsp90 interactio...Objective:To examine the role of heat shock protein 90(Hsp90) in the maintenance of actin cytoskeleton in human neuroblastoma tumor cells.Methods:Co-precipitation experiments were performed to examine Hsp90 interaction with actin.Hsp90 and actin interactions were evaluated by protein refolding and acto-myosin motility assays.17-(AUylamino)-17- demethoxygeldanamycin(17AAG) induced actin-cytoskeleton re-organization was examined by laser scanning confocal microcopy.Results:It was shown that inhibition of Hsp90 by 17AAC accelerates detergent induced cell lysis of neuroblastoma tumor cells through destabilization of actin cytoskeleton.The in vitro co-precipitation experiments showed that functional but not mutant Hsp90 binds with F-actin.Among biochemical modifications,phopshorylation and oligomerization enhanced Hsp90 binding with F-actin.F-actin binding to Hsp90 interfered with Hsp90 chaperone activity in protein refolding assays,and Hsp90 binding to F-actin interfered with actin motility on myosin coated flow cell.In the combination treatment,17AAG irreversibly augmented the effect of cytochalasin D,an inhibitor of actin polymerization.Conclusions:It can be concluded that Hsp90 binds to F-actin in tumor cells and maintains the cellular integrity. The results display a novel element of Hsp90 inhibition in destabilizing the actin cytoskeleton of tumor cells,therefore suggest that 17AAG combination with cytoskeletal disruptor may be effective in combating cancer.展开更多
AIM:To identify circulating CD90 + CD73 + CD45 cells and evaluate their in vitro proliferating abilities.METHODS:Patients with cirrhosis(n=43),and healthy volunteers(n=40)were recruited to the study.Mononuclear cells ...AIM:To identify circulating CD90 + CD73 + CD45 cells and evaluate their in vitro proliferating abilities.METHODS:Patients with cirrhosis(n=43),and healthy volunteers(n=40)were recruited to the study.Mononuclear cells were isolated and cultured from the peripheral blood of controls and cirrhosis patients.Fibroblast-like cells that appeared in cultures were analyzed for morphological features,enumerated by flow cytometry and confirmed by immunocytochemistry(ICC).Colony forming efficiency(CFE)of these cells was assessed and expressed as a percentage.RESULTS:In comparison to healthy volunteers,cells obtained from cirrhotic patients showed a significantincrease(P<0.001)in the percentage of CD90+CD73+ CD45 cells in culture.Cultured cells also showed 10 fold increases in CFE.Flow cytometry and ICC confirmed that the proliferating cells expressed CD90 + CD73 + in the cultures from cirrhosis patients.CONCLUSION:These results indicate the presence of circulating CD90 + CD73 + CD45 cells in patients with liver cirrhosis that have the potential to proliferate at a higher rate.展开更多
Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanism...Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.展开更多
建立了单粒子多位翻转的测试方法和数据处理方法,在此基础上开展了体硅90nm SRAM重离子单粒子多位翻转的实验研究。通过分析单粒子多位翻转百分比、均值、尺寸等参数随线性能量转移(linear energy transfer,LET)的变化关系,表明了纳米...建立了单粒子多位翻转的测试方法和数据处理方法,在此基础上开展了体硅90nm SRAM重离子单粒子多位翻转的实验研究。通过分析单粒子多位翻转百分比、均值、尺寸等参数随线性能量转移(linear energy transfer,LET)的变化关系,表明了纳米尺度下器件单粒子多位翻转的严重性,指出了单粒子多位翻转对现有重离子单粒子效应实验方法和预估方法带来的影响。构建了包含多个存储单元的全三维器件模型,数值模拟研究了不同阱接触布放位置对单粒子多位翻转电荷收集的影响机制,表明阱电势扰动触发多单元双极放大机制是导致单粒子多位翻转的主要因素,减小阱接触与存储单元之间的距离是降低单粒子多位翻转的有效方法。展开更多
基金Project supported by the National Natural Science Foundation of China,No.39570806National Excel1ent Youth Scientific Foundation,No.3952020.
文摘AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P 【 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.
基金supported by Department of Scientific Research Projects in Anhui Province(No.09020304021)
文摘Objective:To vexplore expression of HSP90,SIRT3 in liver cancer tissue and its effect on liver cancer cell invasion ability.Methods:Moderate expression of HSP90 in SMMC-7721,HepG2,LO2 and Hep-3B cell lines were screened,which was validated by RT-PCR.Overexpression of HSP90 cell line and lentivirus packaging HSP90-RNAi were established,which was validated by RT-PCR and western blot.The level of epithelial-mesenchymal transition(EMT)related gene was detected by western blot.The percentage of cancer stem cells was assayed by flow cytometry.Results:RT-PCR demonstrated the highest expression of HSP90mRNA in SMMC-7721 cells,the lowest expression of HSP90 mRNA in Hep3B and LO2 and the moderate expression of HSP90 mRNA in Hep-G2.Therefore,HepG2 was selected as a follow-up experiment cell lines.Compared with the blank control group,expression of HSP90in HSP overexpression group was increased obviously,and expression of HSP90 in HSP90shRNA group was significantly decreased,which indicated successful establishment of HSP overexpression and shRNA group.The apoptotic cell in hsp-siRNA group was higher than the blank control group,while the HSP overexpression group showed opposite results.Western blot results showed transfection HSP promoted cells EMT transformation,up-regulated the level of E-cadherin,and down-regulated the level of Vimentin;meanwhile,shRNA group showed opposite results.Conclusions:Carcinoma HepG2 cell transfeeted high expression of HSP can promote the transformation of EMT,improve the expression of Vimentin,reduce the expression of E-cadherin,and inhibit apoptosis of cancer stem cells,which improve the invasive ability of cancer of the liver cells.While hsp-siRNA group presents opposite results.In summary,the expression of HSP is closely related to the occurrence,development and invasion of cancer of the liver tissue.
基金supported by the National Natural Science Foundation of China(Grant No.82173303)Natural Science Foundation of Chongqing,China(Grant No.cstc2021ycjh-bgzxm0149).
文摘Lymphatic metastasis(LM)emerges as an independent prognostic marker for hypopharyngeal squamous cell carcinoma(HSPSCC),chiefly contributing to treatment inefficacy.This study aimed to scrutinize the prognostic relevance of HSP90AA1 and its potential regulatory mechanism of concerning LM in HPSCC.Methods:In a preceding investigation,HSP90AA1,a differential gene,was discovered through transcriptome sequencing of HPSCC tissues,considering both the presence and absence of LM.Validation of HSP90AA1 expression was accomplished via qRT-PCR,western-blotting(WB),and immunohistochemistry(IHC),while its prognostic significance was assessed employing Kaplan–Meier survival analysis(KMSA),log-rank test(LR),and Cox’s regression analysis(CRA).Bioinformatics techniques facilitated the prediction and analysis of its plausible mechanisms in LM,further substantiated by in vitro and in vivo experiments utilizing FaDu cell lines.Results:HSP90AA1 is substantially upregulated in HPSCC with LM and is identified as an independent prognostic risk determinant.The down-regulation of HSP90AA1 can achieve inhibition of tumor cell proliferation,migration and invasion.Both in vivo experiments and Bioinformatics exploration hint at promoting LM by Epithelial-mesenchymal transition(EMT),regulated by HSP90AA1.Conclusions:HSP90AA1,by controlling EMT,can foster LM in HPSCC.This finding sets the foundation for delving into new therapeutic targets for HPSCC.
文摘AIM:To investigate the effects of transplantation of insulin-producing cells(IPCs) in the treatment of diabetic rats after 90% pancreatectomy.METHODS:Human umbilical cord mesenchymal stem cells(UCMSCs) were isolated and induced into IPCs using differentiation medium.Differentiated cells were examined by dithizone(DTZ) staining,reverse transcription-polymerase chain reaction(RT-PCR),and real-time RT-PCR.C-peptide release,both spontaneously and after glucose challenge,was measured by ELISA.IPCs were then transplanted into Sprague-Dawley rats after 90% pancreatectomy and blood glucose levels and body weight were measured.RESULTS:The differentiated cells were positive for DTZ staining and expressed pancreatic β-cell related genes.C-peptide release by the differentiated cells increased after glucose challenge(380.6 ± 15.32 pmol/L vs 272.4 ± 15.32 pmol/L,P < 0.05).Further,in the cell transplantation group,blood sugar levels were significantly lower than in the sham group 2 wk after transplantation(18.7 ± 2.5 mmol/L vs 25.8 ± 1.25 mmol/L,P < 0.05).Glucose tolerance tests showed that 45 min after intraperitoneal glucose injection,blood glucose levels were significantly lower on day 56 after transplantation of IPCs(12.5 ± 4.7 mmol/L vs 42.2 ± 9.3 mmol/L,P < 0.05).CONCLUSION:Our results show that UCMSCs can differentiate into islet-like cells in vitro under certain conditions,which can function as IPCs both in vivo and in vitro.
基金supported by Department of Biotechnology,Department of Science and Technology,Government of India
文摘Objective:To examine the role of heat shock protein 90(Hsp90) in the maintenance of actin cytoskeleton in human neuroblastoma tumor cells.Methods:Co-precipitation experiments were performed to examine Hsp90 interaction with actin.Hsp90 and actin interactions were evaluated by protein refolding and acto-myosin motility assays.17-(AUylamino)-17- demethoxygeldanamycin(17AAG) induced actin-cytoskeleton re-organization was examined by laser scanning confocal microcopy.Results:It was shown that inhibition of Hsp90 by 17AAC accelerates detergent induced cell lysis of neuroblastoma tumor cells through destabilization of actin cytoskeleton.The in vitro co-precipitation experiments showed that functional but not mutant Hsp90 binds with F-actin.Among biochemical modifications,phopshorylation and oligomerization enhanced Hsp90 binding with F-actin.F-actin binding to Hsp90 interfered with Hsp90 chaperone activity in protein refolding assays,and Hsp90 binding to F-actin interfered with actin motility on myosin coated flow cell.In the combination treatment,17AAG irreversibly augmented the effect of cytochalasin D,an inhibitor of actin polymerization.Conclusions:It can be concluded that Hsp90 binds to F-actin in tumor cells and maintains the cellular integrity. The results display a novel element of Hsp90 inhibition in destabilizing the actin cytoskeleton of tumor cells,therefore suggest that 17AAG combination with cytoskeletal disruptor may be effective in combating cancer.
文摘AIM:To identify circulating CD90 + CD73 + CD45 cells and evaluate their in vitro proliferating abilities.METHODS:Patients with cirrhosis(n=43),and healthy volunteers(n=40)were recruited to the study.Mononuclear cells were isolated and cultured from the peripheral blood of controls and cirrhosis patients.Fibroblast-like cells that appeared in cultures were analyzed for morphological features,enumerated by flow cytometry and confirmed by immunocytochemistry(ICC).Colony forming efficiency(CFE)of these cells was assessed and expressed as a percentage.RESULTS:In comparison to healthy volunteers,cells obtained from cirrhotic patients showed a significantincrease(P<0.001)in the percentage of CD90+CD73+ CD45 cells in culture.Cultured cells also showed 10 fold increases in CFE.Flow cytometry and ICC confirmed that the proliferating cells expressed CD90 + CD73 + in the cultures from cirrhosis patients.CONCLUSION:These results indicate the presence of circulating CD90 + CD73 + CD45 cells in patients with liver cirrhosis that have the potential to proliferate at a higher rate.
基金This work was supported by grants from the NSFC Shandong Joint Fund(Grant No.U1606403)the National Natural Science Foundation of China(Grant No.81673450)+4 种基金the State Key Program of the National Natural Science Foundation of China(Grant No.82030074)the NSFC-Shandong Joint Fund(Grant No.U1906212)the Qingdao National Laboratory for Marine Science and Technology(Grant No.2015ASKJ02)the National Science and Technology Major Project for Significant New Drugs Development(Grant No.2018ZX09735-004)the Shandong Provincial Natural Science Foundation(major basic research projects,Grant No.ZR2019ZD18).
文摘Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.
文摘建立了单粒子多位翻转的测试方法和数据处理方法,在此基础上开展了体硅90nm SRAM重离子单粒子多位翻转的实验研究。通过分析单粒子多位翻转百分比、均值、尺寸等参数随线性能量转移(linear energy transfer,LET)的变化关系,表明了纳米尺度下器件单粒子多位翻转的严重性,指出了单粒子多位翻转对现有重离子单粒子效应实验方法和预估方法带来的影响。构建了包含多个存储单元的全三维器件模型,数值模拟研究了不同阱接触布放位置对单粒子多位翻转电荷收集的影响机制,表明阱电势扰动触发多单元双极放大机制是导致单粒子多位翻转的主要因素,减小阱接触与存储单元之间的距离是降低单粒子多位翻转的有效方法。
