肿瘤转移是90%以上实体瘤病人死亡的原因，由于缺少对机制的深入了解，依然没有特异性治疗药物。Regnase-1是最近发现的在免疫细胞中发挥重要作用的核糖核酸内切酶。而我们的研究发现，Regnase-1是一个重要的未报道的转移抑制基因。在高转移能力的乳腺癌细胞中，Regnase-1蛋白被转录后失活。转移的乳腺癌组织中Regnase-1蛋白显著降低。将Regnase-1稳定表达到高转移能力肿瘤细胞后，其通过酶活性能基本完全抑制转移。据此，本课题将深入研究：(1) Regnase-1是如何在转移细胞中被转录后失活的; (2) Regnase-1是如何识别和降解mRNA的; (3) Regnase-1是通过哪些下游靶基因抑制转移的; (4) 通过转基因和基因敲除小鼠研究其在自发性肿瘤转移中的作用和机制。本研究将为理解肿瘤转移提供新的切入点，即肿瘤细胞如何通过控制一部分mRNA稳定性来实现远处转移。

Cancer metastasis is a leading cause of cancer-related death but without efficient treatment due to poorly defined molecular mechanisms. Regnase-1 is a recently identified ribonuclease, which play critical role in the regulation of T cell and macrophage activation. Here, we found that Regnase-1 is a novel metastatic suppressor, which is inactivated through post-transcriptional mechanism in metastatic cancer cells. The mRNA level of Regnase-1 in high metastatic cancer cells is very high but the Regnase-1 protein is undetectable in these cells. Stable expression of Regnase-1 in cancer cells could dramatically inhibit metastasis through its ribonuclease activity. The protein level of Regnase-1 in breast cancer tissues with metastasis is significantly lower than it in breast cancer tissues without metastasis. Taken together, we identified a novel metastatic suppressor but the mechanism is less clear. In this proposal, we will study this novel metastatic suppressor systematically. First, we will study the pathways that trigger the post-transcriptional inactivation of Regnase-1 in metastatic cancer cells. Second, we will study how regnase-1 recognizes and degrades mRNAs in cancer cells. Third, we will study the Regnase-1 downstream targets that play important role in cancer metastasis. Finally, we will use conditional transgenic and conditional knockout mouse models to study the role of Regnase-1 in spontaneous cancer metastasis. Whereas extensive efforts have focused on genetic mechanism for the understanding of cancer metastasis, our results provide new evidence to show post- transcriptional inactivation of key metastatic checkpoints in metastatic cancer cells to enable the occurrence of cancer metastasis. This proposal will not only provide us a better understanding on the role of Regnase-1 in cancer metastasis, but also provide several novel therapeutic targets to fight cancer metastasis.

肿瘤转移是导致肿瘤病人死亡的主要原因，目前依然没有特异性的治疗药物。原因是我们对于肿瘤转移的机制理解较少，需要开发新的具有治疗价值的靶点。我们的前期结果表明Regnase-1是一个新的肿瘤转移抑制基因，强烈抑制肿瘤转移。更重要的是，在转移的肿瘤细胞内，Regnase-1通过蛋白降解失活。因此，Regnase-1有望成为肿瘤转移特异性治疗的重要靶点。为了进一步研究Regnase-1的调控机制，本研究建立了高效灵敏的GFP报告细胞，此细胞中的GFP荧光强度能反应Regnase-1的表达量及功能。此GFP报告细胞的建立，为我们后续高通量研究Regnase-1的调控机制及功能提供了强有力的工具。同时我们购买和建立了人和小鼠的全基因组SgRNA文库。我们发现TRAF6通过其泛素连接酶活性稳定Regnase-1. 在TRAF6缺陷细胞内，Regnase-1虽然有mRNA表达，但无蛋白产物。我们还发现，A20可以促进Regnase-1的功能。A20对Regnase-1功能的促进作用依赖其去泛素化酶活性。在分子机制上，A20可以和Regnase-1竞争结合到MALT1的C末端，从而增强Regnase-1的功能。综上所述，TRAF6和A20是调控Regnase-1稳定及功能的关键蛋白。理解Regnase-1的精细调控可以为肿瘤转移特异性治疗提供重要的靶点。除了以上本课题研究内容外，我们还对Regnase-1的调控机制进行了广泛的研究：我们发现在稳态条件下，Regnase-1的表达由位于其启动子区的超增强子维持，Regnase-1基因的第二内含子在此超增强子的功能中发挥了关键作用；Regnase-1 可以通过下调5.8S RNA而抑制总蛋白合成，5.8S RNA有Regnase-1识别的发夹结构；根据已报道的结果结合我们自己的发现，我们推测Regnase-1是一个重要的快速反应应急基因；我们还设计了一段小肽，包含Regnase-1的C端的一段氨基酸序列，可以特异地杀死HBL-1细胞。以上这些发现推动了我们对于Regnase-1的理解，并为可能的临床应用找到了多个切入点。