核仁素亚细胞定位的改变在UVA所致人皮肤成纤维细胞衰老中的作用及机制研究

Oxidative stress is an important mechanism of skin photoaging. Nuceolus is the center of cell stress receptor, among which nucleoclin (NCL) is the most abundant protein expressed. Under stress, NCL moves out of the nucleolus, participating in the regulation of DNA replication and repair. As shown by the pre-experiment, UVA irradiation increased the expression of NCL in the cytoplasm and on the cell membrane of human skin fibroblasts (hSFB). Blocking cell membrane NCL or silencing NCL significantly aggravates cell senescence induced by UVA. In this study, Co-IP-MS and some other techniques will be used to identify the senescence-associated proteins that combine with extranuclear NCL, the binding sites, and to explore the binding effects on the pathway and UVA-induced hSFB senescence. Then, the senescence-associated RNA/miRNAs that combine with extranuclear NCL will be detected by RIP and RNA/Small RNA Sequencing, to reveal the effects of NCL binding on the stability and maturation of RNA/miRNAs and UVA-induced hSFB senescence. Ultimately, the post-translational modification of NCL and the expression of MyH9 will be detected to determine the mechanism of the increased extranuclear NCL induced by UVA irradiation. Overall, this study aims at exploring the mechanism and the effect of NCL on UVA induced cell senescence, provides a new target for prevention and treatment against skin photoaging.

氧化应激是皮肤光老化发生的重要机制。核仁是细胞应激感受的中心，其间表达最丰富的蛋白是核仁素（NCL）。应激下，NCL能移出核仁，参与DNA复制和修复的调控。预实验显示：人皮肤成纤维细胞（hSFB）中，UVA照射能使NCL在胞浆和胞膜上表达增多，封闭胞膜NCL或沉默NCL能明显加重UVA所致的细胞衰老。本研究我们先利用Co-IP-MS等技术鉴定能与核外NCL结合的衰老相关蛋白、结合位点、NCL与之结合对相关通路及UVA所致hSFB衰老的影响；再利用RIP和RNA/小RNA测序等鉴定能与核外NCL结合的衰老相关RNA/miRNAs，明确NCL结合对RNA/miRNAs稳定、成熟及对UVA所致hSFB衰老的影响；最后通过检测NCL蛋白翻译后修饰和MyH9的表达明确UVA照射后NCL核外表达增多的机制。本研究旨在明确NCL在皮肤细胞光老化中的作用机制及作用方式，为防治皮肤光老化提供新的靶点。

氧化应激是皮肤光老化发生的重要机制。核仁是细胞应激感受的中心，其间表达最丰富的蛋白是核仁素（NCL）。应激下，作为穿梭蛋白，NCL能移出核仁，通过结合RNA或蛋白质，调控细胞内信号通路，参与DNA复制和修复的调控。我们的实验显示人皮肤成纤维细胞（hSFB）中，UVA照射能使NCL在胞浆和胞膜上表达增多，封闭胞膜NCL或沉默NCL能明显加重UVA所致的细胞衰老，首次证明了核仁素在光老化中的重要作用。进一步，我们发现核仁素这种抑制衰老的作用是通过抑制活性氧（ROS）生成及激活丝裂原活化蛋白激酶（MAPK）通路来完成的。我们通过免疫共沉淀和质谱分析和RNA-seq筛选与NCL结合的蛋白和miRNA，发现核仁素可以和细胞膜上的表皮生长因子受体（EGFR）相结合，同时抑制其磷酸化，进而抑制其下游的MAPK通路。另外，利用RNA-IP和RNA-seq检测人皮肤成纤维细胞中与核仁素结合的microRNA，我们首次发现核仁素可以和microRNA138结合，通过促进其成熟，抑制p38通路，起到抑制光老化的作用。综上，我们发现了一条以核仁素为中心，通过其蛋白结合能力和RNA结合能力多途径抑制UVA诱导的人皮肤成纤维细胞光老化的机制，并为为临床治疗⽪肤衰⽼及相关疾病提供新的思路和理论依据。