PRAS40为富含脯氨酸、分子量为40kD的Akt底物蛋白,能够与雷怕霉素哺乳动物细胞靶点复合物1(mTORC1)结合,其苏氨酸183位点(Ser183)可被mTORC1磷酸化。为了制备PRAS40(Ser183)磷酸化多克隆抗体,本实验通过蛋白疏水性抗原性分析设计多肽抗...PRAS40为富含脯氨酸、分子量为40kD的Akt底物蛋白,能够与雷怕霉素哺乳动物细胞靶点复合物1(mTORC1)结合,其苏氨酸183位点(Ser183)可被mTORC1磷酸化。为了制备PRAS40(Ser183)磷酸化多克隆抗体,本实验通过蛋白疏水性抗原性分析设计多肽抗原,用其免疫家兔获得抗血清,ELISA检测其效价为1:10000;Western blotting法检测发现,通过rProtein A Sepharose亲和层析纯化并经非磷酸化的抗原条吸附处理后的抗体可以明显提高磷酸化抗体的特异性;用PRAS40抗体及PRAS40(Ser183)磷酸化抗体对正常细胞HL7702、HEK293及肿瘤细胞HepG2、A549、S180的检测显示:磷酸化的Ser183在不同细胞中表达差异不显著,而在经细胞饥饿处理的HEK293细胞中却明显观察到了S183磷酸化水平随氨基酸含量降低而减弱的现象。因此,本实验所制备的抗体可用于PRAS40(Ser183)磷酸化位点的功能研究。展开更多
Objective:Mesenchymal subtype of glioblastoma(mesGBM)is a refractory disease condition characterized by therapeutic failure and tumor recurrence.Hyperactive transforming growth factor-β(TGF-β)signaling could be a si...Objective:Mesenchymal subtype of glioblastoma(mesGBM)is a refractory disease condition characterized by therapeutic failure and tumor recurrence.Hyperactive transforming growth factor-β(TGF-β)signaling could be a signature event in mesGBM,which leads to dysregulation of downstream targets and contribute to malignant transformation.In this study we aimed to investigate the hyperactive TGFβsignaling-mediated pathogenesis and possible downstream targets for the development of novel therapeutic interventions for mesGBM.Methods:GBM-BioDP is an online resource for accessing and displaying interactive views of the TCGA GBM data set.Transcriptomic sequencing followed by bioinformatic analysis was performed to identify dysregulated microRNAs.Target prediction by MR-microT and dual luciferase reporter assay were utilized to confirm the predicted target of novel_miR56.CCK-8 assays was used to assesse cell viability.The miRNA manipulation was proceeded by cell transfection and lentivirus delivery.A plasmid expressing GFP-LC3 was introduced to visualize the formation of autophagosomes.Orthotopic GBM model was constructed forin vivo study.Results:TGFβ1 and TGFβreceptor type II(TβRII)were exclusively upregulated in mesGBM(P<0.01).Dysregulated miRNAs were identified after LY2109761(a TβRI/II inhibitor)treatment in a mesGBM-derived cell line,and novel_miR56 was selected as a promising candidate for further functional verification.Novel_miR56 was found to potentially bind to PRAS40 via seed region complementarity in the 3'untranslated region,and we also confirmed that PRAS40 is a direct target of novel_miR56 in glioma cells.In vitro,over expression of novel_miR56 in tumor cells significantly promoted proliferation and inhibited autophagy(P<0.05).The expression levels of P62/SQSTM was significantly increased accompanied by the decrease of BECN1 and LC3B-II/I,which indicated that autophagic activity was reduced after novel_miR56 treatment.In addition,over expression of novel_miR56 also promoted tumor growth and inhibited autophagyin vivo,which is associated with worse prognosis(P<0.05).Conclusions:In summary,we provide novel insight into TGFβsignaling-mediated pathogenesis in mesGBM and TGFβsignaling-induced novel_miR56 may be a novel target for mesGBM management.展开更多
文摘PRAS40为富含脯氨酸、分子量为40kD的Akt底物蛋白,能够与雷怕霉素哺乳动物细胞靶点复合物1(mTORC1)结合,其苏氨酸183位点(Ser183)可被mTORC1磷酸化。为了制备PRAS40(Ser183)磷酸化多克隆抗体,本实验通过蛋白疏水性抗原性分析设计多肽抗原,用其免疫家兔获得抗血清,ELISA检测其效价为1:10000;Western blotting法检测发现,通过rProtein A Sepharose亲和层析纯化并经非磷酸化的抗原条吸附处理后的抗体可以明显提高磷酸化抗体的特异性;用PRAS40抗体及PRAS40(Ser183)磷酸化抗体对正常细胞HL7702、HEK293及肿瘤细胞HepG2、A549、S180的检测显示:磷酸化的Ser183在不同细胞中表达差异不显著,而在经细胞饥饿处理的HEK293细胞中却明显观察到了S183磷酸化水平随氨基酸含量降低而减弱的现象。因此,本实验所制备的抗体可用于PRAS40(Ser183)磷酸化位点的功能研究。
基金This work was supported by the Beijing Tianjin Hebei Basic Research Cooperation Project(Grant 18JCZDJC45500,H2018201306)the Science and Technology Fund of Affiliated Hospital of Hebei University(Grant No.2019Z001)+1 种基金Training Program for the Young Talents of the Fujian Health System(Grant 2017-ZQN-90)the Natural Science Foundation of Fujian Province(Grant 2018J01399).
文摘Objective:Mesenchymal subtype of glioblastoma(mesGBM)is a refractory disease condition characterized by therapeutic failure and tumor recurrence.Hyperactive transforming growth factor-β(TGF-β)signaling could be a signature event in mesGBM,which leads to dysregulation of downstream targets and contribute to malignant transformation.In this study we aimed to investigate the hyperactive TGFβsignaling-mediated pathogenesis and possible downstream targets for the development of novel therapeutic interventions for mesGBM.Methods:GBM-BioDP is an online resource for accessing and displaying interactive views of the TCGA GBM data set.Transcriptomic sequencing followed by bioinformatic analysis was performed to identify dysregulated microRNAs.Target prediction by MR-microT and dual luciferase reporter assay were utilized to confirm the predicted target of novel_miR56.CCK-8 assays was used to assesse cell viability.The miRNA manipulation was proceeded by cell transfection and lentivirus delivery.A plasmid expressing GFP-LC3 was introduced to visualize the formation of autophagosomes.Orthotopic GBM model was constructed forin vivo study.Results:TGFβ1 and TGFβreceptor type II(TβRII)were exclusively upregulated in mesGBM(P<0.01).Dysregulated miRNAs were identified after LY2109761(a TβRI/II inhibitor)treatment in a mesGBM-derived cell line,and novel_miR56 was selected as a promising candidate for further functional verification.Novel_miR56 was found to potentially bind to PRAS40 via seed region complementarity in the 3'untranslated region,and we also confirmed that PRAS40 is a direct target of novel_miR56 in glioma cells.In vitro,over expression of novel_miR56 in tumor cells significantly promoted proliferation and inhibited autophagy(P<0.05).The expression levels of P62/SQSTM was significantly increased accompanied by the decrease of BECN1 and LC3B-II/I,which indicated that autophagic activity was reduced after novel_miR56 treatment.In addition,over expression of novel_miR56 also promoted tumor growth and inhibited autophagyin vivo,which is associated with worse prognosis(P<0.05).Conclusions:In summary,we provide novel insight into TGFβsignaling-mediated pathogenesis in mesGBM and TGFβsignaling-induced novel_miR56 may be a novel target for mesGBM management.
