核仁素对热变性成纤维细胞自噬的影响及其机制研究

Nucleolin is an abundant, multifunctional protein that exhibits an unusual range of activities, including DNA binding and RNA binding. Our previous studies found nucleolin enhanced the recovery of heat-denatured human dermal fibroblasts via increasing cell proliferation and migration. However, whether nucleolin enhances the autophagy during the recovery of heat-denatured human dermal fibroblasts or not is still unclear. Recently,our studies showed that autophagy increases during the recovery of heat-denatured dermis and fibroblasts and nucleolin ablation decreased the autophagy significantly. These results suggest nucleolin probably plays a vital role in autophagy. However, little is known about the molecular mechanism by which nucleolin regulates autophagy. Since nucleolin contains four RNA binding domains and can selectively bind to (T/G)CCCG(A/G),as nucleolin recognition element (NRE), we hypothesized that nucleolin might regulate the expression of autophagy-related genes through post-transcriptional mechanisms, i.e., stabilization of mRNA. We analyzed the mRNA sequence of autophagy-related genes by bioinformatics and found the mRNA molecules of Bnip3、ATG5、ATG3、Beclin 1、FOXO1、STMN1、RAB5A genes containing a NRE and some of genes expression were regulated by nucleolin. In this project, we will study the change and role of autophagy during the recovery of heat-denatured dermis and fibroblasts;the role of nucleolin in autophagy by using full-length nucleolin cDNA and nucleolin RNA interfere and approach the molecular mechanism by which nucleolin regulates cellular autophagy by using Bioinformatics, RNA Transcription, RNA Gel Mobility Shift Assays, UV Cross-linking, Coimmunoprecipitation of protein and RNA, Measurement of mRNA Stability, and luciferase reporter assay system. Carrying out this project can help us to explain the effect of nucleolin on autophagy and its molecular mechanism during the recovery of heat-denatured fibroblasts and to provide new ideas and experimental clues for the recovery of heat-denatured dermis.

变性真皮的转归机制仍是烧伤创面修复研究的热点。核仁素具有多生物学功能，申请者在前一个课题中发现核仁素在皮肤成纤维细胞热损伤转归中起着显著促修复作用，但其促修复机制尚不得而知。最近申请者发现热变性皮肤成纤维细胞转归中伴有自噬增多，与核仁素的表达存在一定相关性，而核仁素低表达可抑制自噬的发生，提示核仁素可能是重要的自噬调控分子，其可能通过调控自噬发生从而发挥促修复作用。本项目拟在上述研究的基础上，从整体及细胞水平探讨热变性皮肤成纤维细胞修复中自噬发生情况及其生物学意义；采用基因转染和RNA干扰技术探讨核仁素对皮肤成纤维细胞自噬的影响；采用RNA凝胶滞留实验、紫外交联、蛋白质-RNA免疫共沉淀、mRNA稳定性、报告基因分析及基因突变等方法深入探讨核仁素对自噬相关基因的调控机制。开展本项目有助于阐释核仁素对皮肤成纤维细胞热损伤转归中自噬的影响及其机制，为深度烧伤创面的治疗提供新的思路和实验线索。

变性真皮是指深度烧伤创面上虽然发生了组织细胞新陈代谢障碍、细胞功能降低，待创面局部环境改善后能恢复正常形态和功能的那部分真皮。变性真皮中包含成纤维细胞、血管内皮细胞两种主要的创面修复细胞，深入了解创面修复细胞在烧伤创面修复过程中的作用，对于进一步认识创面修复过程具有重要意义。细胞自噬是一种广泛存在于真核细胞中的生命现象。然而自噬是否在细胞热变性恢复过程中发挥作用，尚不清楚。.本项目的研究内容包括：（1）自噬在热变性成纤维细胞恢复过程中的作用；（2）自噬在热变性内皮维细胞恢复过程中的作用；（3）变性真皮与正常皮肤中LncRNA、mRNA的差异表达；（4）XIST/miR-29a/LIN28A信号在变性成纤维细胞恢复过程中的作用。.研究发现：（1）热变性皮肤成纤维细胞恢复过程中，核仁素的表达增加、自噬增强，核仁素可以调控热变性成纤维细胞自噬的发生，进一步研究发现核仁素直接调控自噬基因Bnip3、ATG5的表达，进而增强热变性成纤维细胞的恢复能力。（2）热变性内皮维细胞恢复过程中，自噬以时间依赖的方式增加，自噬进而促进内皮细胞增殖、迁移及血管生成，进一步研究发现：热变性内皮细胞通过激活AMPK/Akt/mTOR信号通路激活细胞自噬的发生，进而促进热变性内皮细胞形成血管。（3）与正常皮肤比较，在变性真皮中获得差异表达的lncRNAs4604个，其中表达上调的有2587个，表达下调的有2017个；获得了差异表达的mRNAs 2799个，其中表达上调的有857个，表达下调的有1942个。生物信息学进一步分析示：与miRNA-199a-5p存在反应元件的lncRNAs245个和mRNAs175个。（4）XIST通过竞争性结合miR-29a影响LIN28A的表达，进而调控热变性成纤维细胞的增殖、迁移及细胞外基质的合成，并促进变性真皮的恢复。.上述研究为烧伤创面修复以及保留变性真皮与整张自体薄中厚皮片移植技术，提供了理论依据。