SIRT1/6对细胞老化过程中生物钟改变的调控作用

Circadian rhythms show profound changes with ageing. On the other hand, many pathways related to ageing, such as reactive oxygen species, DNA damage response and cell cycle, are under the control of the circadian clocks. Sirtuins are a family of proteins that are involved in the ageing and longevity regulation of various species.SIRT 1 as key regulator of peripheral circadian clocks, counterbalancing the histone acetyltransferase activity of CLOCK. But little is known as for the regulatory roles of SITR1/6 in age-related circadian clock changes.Most recent studies implicated SIRT1 as a key regulator of the mammalian circadian clock. The current proposal focuses on the hypothesis that Sirtuins are involved in the modulation of age-related changes of circadian clocks in cells. Using young and senescent cells as models of peripheral clocks, we will systematically investigate changes in clock gene expression/localization, circadian rhythmicity and expression/activity changes in SIRT1/6 during cellular ageing. We will also study the impacts of clock disruptions on the susceptibility to cellular senescence, as well as the effects of manipulating SIRT1/6 activities. Key techniques include luciferase reporter assay, real-time bioluminescence imaging, ChIP, Q-PCR, Western blotting and Immunofluorescence. This study aims to address the roles of Sirtuins as the potential molecular link between circadian clocks and cell senescence. Results could aid therapeutic drug design to ameliorate age-related diseases that involve circadian clock disruptions.

生物节律随个体老化发生改变。而多种与衰老相关的机制，如胞内活性氧分子、DNA损伤、细胞周期等均受生物钟调节。沉默信息调节因子（Sirtuins）家族蛋白参与多个种属的寿命及老化过程调节,其中SIRT1对平衡CLOCK基因活性及维持生物钟节律至关重要。但关于SIRT1/6是否调控细胞老化过程中生物钟的改变报道很少。综合国内外文献和我们已有的研究结果，我们提出SIRT1/6参与调控老化过程中生物钟节律、时钟基因表达和时钟蛋白亚细胞定位改变的假说。利用外周时钟细胞衰老模型，采用荧光素酶报告基因技术、实时生物发光计数系统、染色质免疫沉降、Q-PCR等技术，系统研究细胞老化过程中时钟基因及SIRT1/6表达和功能的改变，并探讨药物阻断生物钟节律对细胞老化过程的影响。旨在阐明生物钟与老化之间相互作用及SIRT1/6调控作用，从而辅助寻找针对生物钟调节来控制老龄化相关疾病的新药靶标。

沉默信息调节因子（Sirtuins）家族蛋白参与老化过程调节，其中SIRT1 对平衡生物钟基因活性及维持生物钟节律至关重要。最新国际研究表明 （Chang & Guarente, CELL 2013），老化过程中生物钟调节中心的SIRT1表达水平显著下降，导致老化过程的生物节律紊乱。过表达SIRT1的小鼠在其龄化过程中，其生物节律得以稳定。但关于SIRT6 是否调控细胞老化过程中生物钟的改变报道很少。综合国内外文献和我们已有的研究结果，我们提出SIRT1/6 参与调控老化过程中生物钟节律、时钟基因表达和时钟蛋白亚细胞定位改变的假说。本课题利用外周时钟细胞衰老模型，系统研究细胞老化过程中时钟基因及SIRT1/6 表达和功能的改变，并重点探讨SIRT6时钟蛋白稳定性如何参与细胞生物钟的调控。结果发现1）SIRT1/6和PER2，BMAL1时钟蛋白在细胞内共定位； 2）老化过程中，SIRT1/6和PER2, BMAL1蛋白表达显著下调；3） SIRT6基因过表达及抑制改变外周细胞水平生物节律；4）虽然药物阻断生物钟节律（CK1 抑制剂）对细胞老化过程并无显著影响，但BMAL1基因敲除细胞呈现明显细胞老化特征，提示该时钟基因与老化过程存在密切联系。5) 进一步研究揭示UBE3A泛素化调控BMAL1蛋白稳定性及细胞生物钟。 这些结果揭示（Sirtuins）家族蛋白参与老化过程生物钟的调节，并进一步锁定SIRT6及BMAL1作为将来进一步在体研究的重要靶标。部分研究结果已发表于Nucleic Acids Research 和Osteoarthritis and Cartilage。其他研究数据正在整理中，拟发表于[核酸研究]或相应水平的国际期刊。