Human Neurobeachin-like 1(NBEAL1)gene was an important member of BEACH-WD40 domain family,which was confirmed to be overexpressed in I stage glioma.In this study,we extracted total RNAs from U251 cell line,acquired it...Human Neurobeachin-like 1(NBEAL1)gene was an important member of BEACH-WD40 domain family,which was confirmed to be overexpressed in I stage glioma.In this study,we extracted total RNAs from U251 cell line,acquired its cDNA sequence by RT-PCR,and cloned part of NBEAL1 cDNA fragments into the vector pGEX-KG.The recombinant expression vector achieved high expression in E.coli BL21 as a GST fusion protein.NBEAL1 recombinant protein was purified by affinity chromatography.Monoclonal antibody was prepared against the recombinant NBEAL1 protein.Its bioactivity was identified by Western Blotting analysis.Anti-NBEAL1 antibody was conjugated with CdTe quantum dots.Resultant anti-NBEAL1 antibody-conjugated nanoprobes were injected into mice via tail vessel.After 12h,it is clearly observed that prepared nanoprobes located in brain tissues of mice model with glioma by IVIS Imaging system.In conclusion,NBEAL1 protein was successfully expressed and its monoclonal antibody was successfully prepared.Anti-NBEAL1 antibody-conjugated quantum dots may be used to image glioma.These prepared nanoprobes have great potential in early detection of glioma.展开更多
Although temozolomide （TMZ） is the first-line chemotherapeutic agent for glioblastoma, it is often non-curative due to drug resistance. To overcome the resistance of glioblastoma cells to TMZ, it is imperative to id...Although temozolomide （TMZ） is the first-line chemotherapeutic agent for glioblastoma, it is often non-curative due to drug resistance. To overcome the resistance of glioblastoma cells to TMZ, it is imperative to identity prognostic markers for outcome prediction and to develop chemo-sensitizing agents. Here, the gene expression profiles of TMZ-resistant and TMZ-sensitive samples were compared by microarray analysis, and mitogen-activated protein kinase kinase 2 （MEK2） was upregulated specifically in resistant glioma cells but not in sensitive tumor cells or non-tumor tissues. Moreover, a comprehensive analysis of patient data revealed that the increased level of MEK2 expression correlated well with the advancement of glioma grade and worse prognosis in response to TMZ treatment. Furthermore, reducing the level of MEK2 in U251 glioma cell lines or xenografted glioma models through shRNA-mediated gene knockdown inhibited cell proliferation and enhanced the sensitivity of cells toward TMZ treatment. Further analysis of tumor samples from glioma patients by real-time PCR indicated that an increased MEK2 expression level was closely associated with the activation of many drug resistance genes. Finally, these resistance genes were downregulated after MEK2 was silenced in vitro, suggesting that the mechanism of MEK2-induced chemo-resistance could be mediated by the transcriptional activation of these resistance genes. Collectively, our data indicated that the expression level of MEK2 could serve as a prognostic marker for glioma chemotherapy and that MEK2 antagonists can be used as chemo-sensitizers to enhance the treatment efficacy of TMZ.展开更多
基金supported by China National 973 Project(No.2010CB933901)National 863 Hi-tech Project(Nos.2007AA022004 and 2005AA001070)National Natural Scientific Fund(No.30672147).
文摘Human Neurobeachin-like 1(NBEAL1)gene was an important member of BEACH-WD40 domain family,which was confirmed to be overexpressed in I stage glioma.In this study,we extracted total RNAs from U251 cell line,acquired its cDNA sequence by RT-PCR,and cloned part of NBEAL1 cDNA fragments into the vector pGEX-KG.The recombinant expression vector achieved high expression in E.coli BL21 as a GST fusion protein.NBEAL1 recombinant protein was purified by affinity chromatography.Monoclonal antibody was prepared against the recombinant NBEAL1 protein.Its bioactivity was identified by Western Blotting analysis.Anti-NBEAL1 antibody was conjugated with CdTe quantum dots.Resultant anti-NBEAL1 antibody-conjugated nanoprobes were injected into mice via tail vessel.After 12h,it is clearly observed that prepared nanoprobes located in brain tissues of mice model with glioma by IVIS Imaging system.In conclusion,NBEAL1 protein was successfully expressed and its monoclonal antibody was successfully prepared.Anti-NBEAL1 antibody-conjugated quantum dots may be used to image glioma.These prepared nanoprobes have great potential in early detection of glioma.
文摘Although temozolomide （TMZ） is the first-line chemotherapeutic agent for glioblastoma, it is often non-curative due to drug resistance. To overcome the resistance of glioblastoma cells to TMZ, it is imperative to identity prognostic markers for outcome prediction and to develop chemo-sensitizing agents. Here, the gene expression profiles of TMZ-resistant and TMZ-sensitive samples were compared by microarray analysis, and mitogen-activated protein kinase kinase 2 （MEK2） was upregulated specifically in resistant glioma cells but not in sensitive tumor cells or non-tumor tissues. Moreover, a comprehensive analysis of patient data revealed that the increased level of MEK2 expression correlated well with the advancement of glioma grade and worse prognosis in response to TMZ treatment. Furthermore, reducing the level of MEK2 in U251 glioma cell lines or xenografted glioma models through shRNA-mediated gene knockdown inhibited cell proliferation and enhanced the sensitivity of cells toward TMZ treatment. Further analysis of tumor samples from glioma patients by real-time PCR indicated that an increased MEK2 expression level was closely associated with the activation of many drug resistance genes. Finally, these resistance genes were downregulated after MEK2 was silenced in vitro, suggesting that the mechanism of MEK2-induced chemo-resistance could be mediated by the transcriptional activation of these resistance genes. Collectively, our data indicated that the expression level of MEK2 could serve as a prognostic marker for glioma chemotherapy and that MEK2 antagonists can be used as chemo-sensitizers to enhance the treatment efficacy of TMZ.
