The highly conserved target of rapamycin(TOR)pathway plays an important role in aging across species.Previous studies have established that inhibition of the TOR complex 1(TORC1)significantly extends lifespan in Caeno...The highly conserved target of rapamycin(TOR)pathway plays an important role in aging across species.Previous studies have established that inhibition of the TOR complex 1(TORC1)significantly extends lifespan in Caenorhabditis elegans.However,it has not been clear whether TORC1 perturbation affects aging in a spatiotemporal manner.Here,we apply the auxin-inducible degradation tool to knock down endogenous DAF-15,the C.elegans ortholog of regulatory associated protein of TOR(Raptor),to characterize its roles in aging.Global or tissue-specific inhibition of DAF-15 during development results in various growth defects,whereas neuron-specific knockdown of DAF-15 during adulthood significantly extends lifespan and healthspan.The neuronal DAF-15 deficiency-induced longevity requires the intestinal activities of DAF-16/FOXO and PHA-4/FOXA transcription factors,as well as the AAK-2/AMP-activated protein kinaseαcatalytic subunit.Transcriptome profiling reveals that the neuronal DAF-15 knockdown promotes the expression of genes involved in protection.These findings define the tissue-specific roles of TORC1 in healthy aging and highlight the importance of neuronal modulation of aging.展开更多
目的:探讨沉默雷帕霉素不敏感的哺乳动物雷帕霉素靶蛋白伴侣(rapamycin insensitive companion of mammalian target of rapamycin,RICTOR)基因表达后食管鳞癌细胞对依维莫司敏感性的变化,及其可能的分子机制。方法:蛋白质印迹法检测食...目的:探讨沉默雷帕霉素不敏感的哺乳动物雷帕霉素靶蛋白伴侣(rapamycin insensitive companion of mammalian target of rapamycin,RICTOR)基因表达后食管鳞癌细胞对依维莫司敏感性的变化,及其可能的分子机制。方法:蛋白质印迹法检测食管鳞癌ECa109、EC9706、TE1、KYSE450和KYSE790细胞中RICTOR蛋白的表达情况。采用脂质体转染法将携带有RICTOR-shRNA和Control-shRNA(对照组)的重组质粒分别转入ECa109细胞中,并筛选稳定转染的细胞株(命名为ECa109-RICTORshRNA和ECa109-control-shRNA)。采用CCK-8法检测沉默RICTOR基因表达后依维莫司对ECa109细胞增殖的影响,再用蛋白质印迹法检测磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB,又称Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路中相关因子RICTOR、Akt、磷酸化Akt(phospho-Akt,p-Akt)(Ser473)、核糖体蛋白S6激酶70 kDa肽(ribosome protein subunit 6 kinase of 70 kDa,p70S6K)、p-p70S6K、40 kDa富含脯氨酸的Akt底物(proline-rich Akt substrate of 40 kDa,PRAS40)和p-PRAS40(r246)蛋白的表达水平。最后,用ECa109-RICTOR-shRNA和ECa109-control-shRNA细胞建立裸鼠移植瘤模型,并用依维莫司进行治疗,观察依维莫司对裸鼠体内移植瘤生长的影响。结果:RICTOR蛋白在5株食管鳞癌细胞中均有表达,且在ECa109细胞中表达水平相对较高。与ECa109-control-shRNA细胞相比,ECa109-RICTOR-shRNA细胞的增殖速度明显变慢(P<0.05);依维莫司对ECa109-RICTOR-shRNA和ECa109-control-shRNA细胞的增殖均有明显抑制作用,依维莫司对ECa109-RICTOR-shRNA和ECa109-controlshRNA细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)值分别为(17.68±1.25)μmol/L和(36.84±1.57)μmol/L。沉默RICTOR基因表达后,ECa109细胞中p-Akt(Ser473)和p-PRAS40(r246)蛋白的表达水平明显降低(P值均<0.001),而依维莫司使p-p70S6K蛋白的表达水平下调(P<0.001),但促进了p-Akt(Ser473)和p-PRAS40(r246)蛋白表达水平的上调(P值均<0.001);与ECa109-controlshRNA+依维莫司组相比,ECa109-RICTOR-shRNA+依维莫司组细胞中p-Akt(Ser473)和p-PRAS40(r246)蛋白的表达水平明显降低(P值均<0.001),p-p70S6K蛋白的表达水平无明显变化(P>0.05),提示RICTOR-shRNA能够抑制依维莫司引起的Akt和PRAS40蛋白的磷酸化激活。RICTOR-shRNA和依维莫司均能抑制裸鼠体内ECa109细胞移植瘤的生长(P值均<0.05),并以RICTOR-shRNA联合依维莫司对肿瘤生长的抑制作用最强(P<0.001)。结论:沉默RICTOR基因表达能够提高ECa109细胞对依维莫司的敏感性,其分子机制可能与下调RICTOR表达抑制了依维莫司引起的Akt和PRAS40的磷酸化有关。展开更多
基金the National Key R&D Program of China(2021YFA0805802 to D.C.)the National Natural Science Foundation of China(31971092 and 32171156 to D.C.)Some strains were provided by the Caenorhabditis Genetics Center(CGC),which is funded by NIHOffice of Research Infrastructure Programs(P40 OD010440).
文摘The highly conserved target of rapamycin(TOR)pathway plays an important role in aging across species.Previous studies have established that inhibition of the TOR complex 1(TORC1)significantly extends lifespan in Caenorhabditis elegans.However,it has not been clear whether TORC1 perturbation affects aging in a spatiotemporal manner.Here,we apply the auxin-inducible degradation tool to knock down endogenous DAF-15,the C.elegans ortholog of regulatory associated protein of TOR(Raptor),to characterize its roles in aging.Global or tissue-specific inhibition of DAF-15 during development results in various growth defects,whereas neuron-specific knockdown of DAF-15 during adulthood significantly extends lifespan and healthspan.The neuronal DAF-15 deficiency-induced longevity requires the intestinal activities of DAF-16/FOXO and PHA-4/FOXA transcription factors,as well as the AAK-2/AMP-activated protein kinaseαcatalytic subunit.Transcriptome profiling reveals that the neuronal DAF-15 knockdown promotes the expression of genes involved in protection.These findings define the tissue-specific roles of TORC1 in healthy aging and highlight the importance of neuronal modulation of aging.
