More modifiable sites on the nucleoside motif may need to be explored for developing novel(p)ppGpp molecular tools.Herein,we report for the first time that the C7-substituted deazapurine nucleoside triphosphates beari...More modifiable sites on the nucleoside motif may need to be explored for developing novel(p)ppGpp molecular tools.Herein,we report for the first time that the C7-substituted deazapurine nucleoside triphosphates bearing small modifications as substrates could be effectively accepted by RelSeqNTD protein to react with ATP to give pppGpp derivatives with 65%—89%yields.Further structural derivatization via metal-coupling reaction was performed to produce C7-substituted GTP derivatives with larger bulkiness,and those GTP derivatives were also proven to be good substrates of RelSeqNTD protein.Alkynyl modified pppGpp could be coupled with probes by click reactions as the potential molecular tools for fishing proteins in biological research.We further explored whether the C7-alkynyl-pppGpp(pppGEpp)could be recognized by pppGpp interaction proteins.A micromolar level binding affinity(with a KD value of less than 10μM)between pppGpp(pppGEpp)and its binding proteins was obtained from the Isothermal Titration Curve(ITC).All those illustrate that these easily accessible functionalized C7-substituted pppGpp derivatives were suitable tools for further exploring the molecular interaction between pppGpp and its binding proteins.展开更多
目的:探讨膀胱癌细胞中细胞色素c氧化酶亚基7A2(COX7A2)基因的表达,及其与顺铂联用对膀胱癌J82细胞增殖、凋亡及线粒体功能影响。方法:采用生物信息学方法分析COX7A2在膀胱癌患者中的表达,并在J82细胞中进行验证。功能实验分为对照组(...目的:探讨膀胱癌细胞中细胞色素c氧化酶亚基7A2(COX7A2)基因的表达,及其与顺铂联用对膀胱癌J82细胞增殖、凋亡及线粒体功能影响。方法:采用生物信息学方法分析COX7A2在膀胱癌患者中的表达,并在J82细胞中进行验证。功能实验分为对照组(仅转染阴性对照siNC)、siRNA组(仅转染COX7A2的siRNA)、对照组+顺铂组(先转染阴性对照后用顺铂处理)和siRNA+顺铂组(先敲低COX7A2后用顺铂处理)。CCK-8、Transwell迁移能力测试和克隆增殖实验检测对照组和siRNA组中J82细胞的增殖、迁移能力。采用相应试剂盒检测各组细胞的ATP水平、活性氧(ROS)水平及线粒体膜电位(ΔΨm),以评估线粒体功能。流式细胞术检测各组细胞凋亡,以反映细胞的线粒体状态与对顺铂治疗的响应性关系。进一步通过癌症治疗响应基因标识数据库(CTR-DB),分析COX7A2与接受顺铂联合治疗的膀胱癌患者预后的关系。结果:生物信息学分析与生存曲线显示,COX7A2在膀胱癌患者中高表达并且与患者预后不良有关联。COX7A2在J82细胞中呈高表达(P<0.05)。在未经顺铂处理时,与对照组相比,siRNA组J82细胞增殖、迁移和克隆形成能力均显著下降(均P<0.001),而线粒体的ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),凋亡水平增加(P<0.05);顺铂处理后,与对照组+顺铂相比,siRNA+顺铂组ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),线粒体功能受损,凋亡水平增加(P<0.001)。CTR-DB数据库生信分析显示,5例接受顺铂+多柔比星+甲氨蝶呤+长春碱联合治疗的膀胱癌患者中,有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:4501 vs 5009),12例铂类药物联合治疗的膀胱癌患者中有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:2947 vs 3035),由于样本量有限,虽观察到趋势但无统计学意义。结论:敲低COX7A2可通过损伤线粒体功能,抑制膀胱癌细胞增殖与迁移,并可能由此增强细胞对顺铂诱导凋亡的敏感性。展开更多
基金supported by the National Natural ScienceFoundation of China(U23A20106,22111530224,22304044 and 22473040)the Outstanding Youth Innovation Research Group in Henan Province(242300421005)Doctoral Initiation Fund(202103084).
文摘More modifiable sites on the nucleoside motif may need to be explored for developing novel(p)ppGpp molecular tools.Herein,we report for the first time that the C7-substituted deazapurine nucleoside triphosphates bearing small modifications as substrates could be effectively accepted by RelSeqNTD protein to react with ATP to give pppGpp derivatives with 65%—89%yields.Further structural derivatization via metal-coupling reaction was performed to produce C7-substituted GTP derivatives with larger bulkiness,and those GTP derivatives were also proven to be good substrates of RelSeqNTD protein.Alkynyl modified pppGpp could be coupled with probes by click reactions as the potential molecular tools for fishing proteins in biological research.We further explored whether the C7-alkynyl-pppGpp(pppGEpp)could be recognized by pppGpp interaction proteins.A micromolar level binding affinity(with a KD value of less than 10μM)between pppGpp(pppGEpp)and its binding proteins was obtained from the Isothermal Titration Curve(ITC).All those illustrate that these easily accessible functionalized C7-substituted pppGpp derivatives were suitable tools for further exploring the molecular interaction between pppGpp and its binding proteins.
文摘目的:探讨膀胱癌细胞中细胞色素c氧化酶亚基7A2(COX7A2)基因的表达,及其与顺铂联用对膀胱癌J82细胞增殖、凋亡及线粒体功能影响。方法:采用生物信息学方法分析COX7A2在膀胱癌患者中的表达,并在J82细胞中进行验证。功能实验分为对照组(仅转染阴性对照siNC)、siRNA组(仅转染COX7A2的siRNA)、对照组+顺铂组(先转染阴性对照后用顺铂处理)和siRNA+顺铂组(先敲低COX7A2后用顺铂处理)。CCK-8、Transwell迁移能力测试和克隆增殖实验检测对照组和siRNA组中J82细胞的增殖、迁移能力。采用相应试剂盒检测各组细胞的ATP水平、活性氧(ROS)水平及线粒体膜电位(ΔΨm),以评估线粒体功能。流式细胞术检测各组细胞凋亡,以反映细胞的线粒体状态与对顺铂治疗的响应性关系。进一步通过癌症治疗响应基因标识数据库(CTR-DB),分析COX7A2与接受顺铂联合治疗的膀胱癌患者预后的关系。结果:生物信息学分析与生存曲线显示,COX7A2在膀胱癌患者中高表达并且与患者预后不良有关联。COX7A2在J82细胞中呈高表达(P<0.05)。在未经顺铂处理时,与对照组相比,siRNA组J82细胞增殖、迁移和克隆形成能力均显著下降(均P<0.001),而线粒体的ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),凋亡水平增加(P<0.05);顺铂处理后,与对照组+顺铂相比,siRNA+顺铂组ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),线粒体功能受损,凋亡水平增加(P<0.001)。CTR-DB数据库生信分析显示,5例接受顺铂+多柔比星+甲氨蝶呤+长春碱联合治疗的膀胱癌患者中,有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:4501 vs 5009),12例铂类药物联合治疗的膀胱癌患者中有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:2947 vs 3035),由于样本量有限,虽观察到趋势但无统计学意义。结论:敲低COX7A2可通过损伤线粒体功能,抑制膀胱癌细胞增殖与迁移,并可能由此增强细胞对顺铂诱导凋亡的敏感性。
