miR-23a调控细胞自噬及其在皮肤光老化进程中的作用与机制研究

Recent studies find that abnormally reduced autophagy is one of the important factors that cause aging of the skin and the whole body. More than 30 proteins are required to participate in the procedure of autophagy. Our preliminary studies found that 1) the autophagy activity was significantly reduced in photoaged skin cells; 2) autophagy activator can reverse many performance of skin photoaging; 3) miR-23a expression was significantly increased in skin photoaging models which was confirmed by miRNAs chip and RT-PCR, and miR -23a predicted downstream target sites have some relationship with autophagy. In this condition, we propose the hypothesis: miR-23a may participate in the development of skin photoaging through regulation of autophagy. The present project intends to observed the impact of miR-23a and autophagy on established skin photoaging cells, tissue and animal models. By selective regulation of miR-23a expression in photoaged cells and dual-luciferase assay to verify the downstream target genes of miR-23a. On the basis of verify the roles of miR-23a in autophagy, clarify the exact mechnisms of miR-23a regulation of photoaging. Our expeirments will provide an experimental basis of specific mechanisms of new targets for the prevention and treatment of skin photodamage.

新近发现细胞自噬异常减少是导致机体老化的重要因素之一；发生细胞自噬需要30余种蛋白参与。前期研究发现：皮肤光老化细胞的自噬活性显著降低，自噬激活剂可逆转皮肤光老化诸多表现；miRNAs芯片及RT-PCR检测证实皮肤光老化模型中miR-23a表达显著升高，且miR-23a预测的下游靶位点为调控自噬相关基因表达有关及自噬相关特异蛋白编码基因。为此提出假说：miR-23a可能通过调控细胞自噬而参与皮肤光老化的发生发展。本项目拟通过已建立的皮肤老化细胞、组织及动物模型，多层次观察miR-23a与细胞自噬对皮肤光老化的影响，通过老化细胞选择性差异调控miR-23a表达技术及荧光素酶报告载体等研究方法验证miR-23a调控的下游靶基因。在确证"紫外线-miR-23a-细胞自噬-皮肤光老化"调控环节的基础上，明确miR-23a调控光老化的具体机制，为防治皮肤光老化明确新靶点提供理论基础和实验参考。

研究背景和主要研究内容：.自噬是与生物体的老化进程密切相关。微小RNA是一种非编码单链小RNA分子，已有研究发现微小RNA也参与了自噬的过程。MiR-23a可能是老化发生过程中的一个潜在靶位点。本篇研究的目的旨在找出在提前衰老的模型中miR-23a与自噬之间的关系，并找到miR-23a的下游靶位点，阐明miR-23a在自噬和老化调控中的机制。.结果：.1. UV-SIPS模型中自噬水平、老化水平以及miR-23a ~27a ~24-2的表达量.UV组GFP-LC3阳性率明显下降，LC3II/LC3I明显下降，p62/SQSTM1明显升高。SA-β-半乳糖苷酶阳性率、p16、 p53和p21的蛋白表达量、G1期阻滞率明显上升，EdU阳性率明显下降。miR-23a的表达量在UVB和PUVA模型均明显下降，miR-24-2的表达量无明显改变，miR-27a的表达量在UVB模型明显下降。.2. 沉默miR-23a对UV-SIPS模型自噬水平、老化水平的影响.与UV组相比，miR-23a+UV组GFP-LC3阳性率明显上升，LC3II/LC3I明显增加，p62/SQSTM1明显下降。SA-β-半乳糖苷酶阳性率、p16、 p53和p21的蛋白表达量、G1期阻滞率明显下降，EdU阳性率明显升高。.3. AMBRA1可能是miR-23a的下游靶位点，沉默miR-23a可抑制AMBRA1表达。.4. 过表达miR-23a对雷帕霉素引起的自噬和老化水平变化的影响.Rapa+UV组SA-β-半乳糖苷酶阳性率、p16、 p53和p21的蛋白表达量、G1期阻滞率明显下降，EdU阳性率明显升高。Ago-23a+Rapa+UV组SA-β-半乳糖苷酶阳性率、p16、 p53和p21表达量、G1期阻滞率明显下降。Rapa+UV组GFP-LC3阳性率明显上升，LC3II/LC3I明显增加，p62/SQSTM1明显下降。.5. 过表达miR-23a对上调AMBRA1引起的自噬和老化水平变化的影响.Ad-AMBRA1+Agot-23a+UV组SA-β-半乳糖苷酶阳性率、p16、 p53和p21表达量、G1期阻滞率明显下降。.结论：. MiR-23a可能与通过调控其下游靶位点AMBRA1来自噬水平进而进一步改善老化水平，可能成为治疗皮肤光源性损伤及光源性皮肤病的新靶点。