14-3-3σ在皮肤角质形成细胞UVB辐射损伤应激中的作用及其机制的研究

Overexposure to ultraviolet B (UVB) has resulted in the increasing incidence of skin disorders,which enhances the research of radiation damage and its protection. The previous work showed that ①14-3-3σ expression was lower in cutaneous squamous cell carcinoma than that in normal tissues and act as a key molecule in UVB-induced G2/M phase block ; ② expression levels of miR-486-3p and 14-3-3σshowed opposite changes after UVB radiation and possible binding sites for miR-486-3p were located in the 3'UTR of 14-3-3σ mRNA;③ the peak time of UVB induced increasing expression of D53 was delaying compared with that of 14-3-3σ.Base on above，this research includes: exploring the functions of 14-3-3σ,miR-486-3p and D53 in response to UVB radiation respectively by up or down regulating their expression; confirming that miR-486-3p targets 14-3-3σ by luciferase report system; confirming interaction between 14-3-3σ and D53 directly or indirectly by confocal and pull-down assay to clarify miR-486-3p/14-3-3σ/D53 axis in UVB radiation response; investigating the probability of 14-3-3σ as a therapeutic target for cutaneous squamous cell carcinoma by nude mouse tumorigenicity. It could provide more knowledge of the mechanism of UVB radiation damage and a theories foundation for its protection research. It could also provide a new target for preventing and curing the tumors.

紫外线B（UVB）过量照射使皮肤病发病率增加的现状使人类加强了对其损伤和防护的研究。我们已证明：①14-3-3σ在皮肤鳞癌中低表达，且是皮肤角质形成细胞中被UVB诱导并引起G2/M期阻滞的关键分子；②miR-486-3p表达水平在UVB照后和14-3-3σ相反，且有14-3-3σ结合靶点；③D53在UVB照后升高峰值时间点略晚于14-3-3σ。本研究通过影响细胞中三个分子的表达水平等来研究它们在UVB应激中的作用；通过双荧光酶素实验明确miR-486-3p靶向14-3-3σ；通过激光共聚焦、pull down等明确14-3-3σ和D53直接调控，以最终明确UVB损伤应激中是否存在miR-486-3p-14-3-3σ-D53调控轴；最后用成瘤实验等分析14-3-3σ作为皮肤鳞癌防治靶点的可能性。该研究将有助于更全面认识UVB辐射损伤机理，并为其防护提供理论基础，也能为皮肤鳞癌防治提供新靶点。

臭氧空洞的不断扩大以及工业生产、航天航空活动的增多导致机体接触UVB机会大大增加，而UVB过量暴露是多种皮肤疾病的主要环境致病因素。因此，深入研究UVB的辐射损伤效应及相关的分子机制是该领域迫切解决的问题。.本项目以HaCaT细胞为主要研究对象，以波长为310nm的UVB为干预因子，探索14-3-3σ在UVB诱导的辐射损伤应激中的作用及机制。我们在前期的基础上发现：①在UVB介导的辐射损伤反应中，14-3-3σ表达增加，调控HaCaT细胞G2/M期阻滞。调控14-3-3σ的表达可以影响DNA合成代谢，并在UVB应激条件下与DAXX等DNA损伤相关蛋白结合，促进DNA损伤的修复和细胞内过剩ROS的清除，从而维持HaCaT细胞的存活；②UVB辐射诱导D53蛋白表达，14-3-3σ通过与D53结合促进UVB介导的细胞损伤修复，加速CPD和γH2AX的修复；③miR-486-3p可促进UVB照射条件下表达增加，因此不是14-3-3σ的上游调控因，但miR-486-3pHaCaT细胞的存活，但；④在皮肤鳞癌中，miR-486-3p通过靶向14-3-3σ负调控其表达，增强肿瘤细胞增殖和迁移能力；14-3-3σ过表达则会抑制皮肤鳞癌细胞的增殖和迁移能力，提示14-3-3σ可能在皮肤鳞癌的发生发展中发挥抑癌基因作用。. 综上所述，14-3-3σ是UVB辐射损伤应激信号通路中的关键分子，也具有应用于皮肤鳞癌临床治疗的可能性。本项目究为UVB辐射的防护提供理论基础，同时也为皮肤鳞癌的防治提供新的思路和靶点。