细胞核分布基因NudCL2在细胞迁移及小鼠胚胎发育过程中的作用及机制研究

It is reported that cell migration contributes to amount of biological processes, including various events of early embryonic development. However, the underlying molecular mechanism that controls cell migration remains unclear. We focus on the biological function of nuclear distribution gene C (Nuclear distribution gene C, NudC) family for many years. We established NudCL2 (NudC-like gene 2) gene knockout mice and found that homozygous state is lethal in mid-gestation with severe embryonic malformations related to the defects of migration such as neural tube defects. Furthermore, Our data showed that knockout of NudCL2 significantly inhibits cell migration process in cells. Moreover, quantitative mass spectrometry was applied to identify differentially expressed proteins in NudCL2 knockout and wildtype mouse embryo. The data indicated that knockout of NudCL2 results in the decrease of TSC2 (Tuberous Sclerosis Complex 2) protein level. The similar role of TSC2 in cell migration and embryonic development is reported and prompted us to investigate the biochemical relationship between it and NudCL2. Our immunoprecipitation data revealed that NudCL2 can interact with TSC2 protein in vivo. Thus, we propose that NudCL2 may play important role in cell migration during mouse embryonic development by stabilizing TSC2. In this proposal, we will systematically characterize the biological function of NudCL2 in cell migration and mouse embryonic development, and elucidate its possible molecular mechanism at molecular, cellular and animal levels. This study will provide novel potential therapeutic targets for the human diseases related to the dysfunction of cell migration.

细胞迁移在早期胚胎发育等生物学过程中发挥重要作用，但其分子调控机制尚不清楚。本课题组一直致力于细胞核分布基因C（Nuclear distribution gene C，NudC）家族的功能研究。近期结果显示，细胞核分布基因NudCL2（NudC-like gene 2）敲除将导致小鼠胚胎致死且伴随脑部发育缺陷等细胞迁移异常相关表型；在细胞水平敲除NudCL2则可抑制细胞迁移；定量质谱分析NudCL2敲除后下调的分子，发现细胞迁移调控相关基因TSC2（Tuberous Sclerosis Complex 2）敲除胚胎表型与NudCL2敲除最相似，并可与NudCL2互作。上述结果提示，NudCL2可能通过调节TSC2蛋白稳定性来参与细胞迁移调控，进而影响小鼠胚胎发育。本项目拟在分子、细胞及动物水平研究NudCL2在细胞迁移及小鼠胚胎发育过程中的作用及机制，为细胞迁移异常相关疾病的诊治提供线索。

细胞运动在许多生物学过程中起着关键作用，如胚胎发育、组织修复、免疫应答等，其异常涉及多种人类疾病，例如自身免疫综合征、智力低下、癌症等。但是，其潜在机制仍不清楚。我们的研究发现，NudC样蛋白2（NudC-like protein 2，NudCL2）热休克蛋白90（Heat shock protein 90，Hsp90）的分子共伴侣蛋白，全身性敲除将导致小鼠胚胎致死且伴随脑部发育缺陷等细胞迁移异常相关表型。通过蛋白质定量质谱分析NudCL2敲除后显著性下调的蛋白分子（如Annexin1、myosin-9、Nedd4l及TSC2等）。蛋白质印迹分析表明，NudCL2的下调不仅会降低myosin-9和TSC2的蛋白水平，而且还会导致肌动蛋白actin的结构及功能紊乱。外源过表达myosin-9可有效地逆转NudCL2敲低所引起的上述表型，提示NudCL2可能主要通过myosin-9调控细胞迁移过程。此外，与在MEF细胞中敲除NudCL2的表型不同，在人肿瘤细胞中敲除NudCL2可显著促进单细胞运动，但对集体细胞运动无明显影响。我们的研究显示，敲除myosin-9会使单细胞和集体细胞运动均增快，这与下调NudCL2只促进单细胞运动的表型有所不同。我们之前的研究显示，下调NudCL2可降低LIS1蛋白水平。已有的报道及我们的研究结果显示，敲低LIS1不仅抑制单细胞运动，同时也抑制了集体细胞的运动，这与myosin-9下调的表型相反。有趣的是，当我们共同敲低myosin-9和LIS1后发现其对细胞运动的影响与NudCL2敲低引起的表型相似。进一步的机制研究显示，myosin-9不仅与Hsp90相互作用，且抑制Hsp90的ATPase活性可降低myosin-9的蛋白稳定性并促进单细胞运动。外源过表达Hsp90可有效逆转NudCL2下调所致的myosin-9蛋白的不稳定性和细胞运动异常相关表型，但反之则不然。综上所述，这些数据表明，Hsp90可作为NudCL2的上游调控因子，并通过稳定myosin-9和LIS1蛋白，进而参与细胞运动的精确调控。总之，我们的研究揭示了NudCL2在细胞运动调控中的新功能，为细胞运动相关疾病（如肿瘤）的诊治提供了潜在的新思路和新靶点。