SIRT6调控线粒体-内质网结构偶联损伤在银屑病中的作用机制研究

Psoriasis is a chronic inflammatory skin disease which characterized by hyperplasia and abnormal differentiation of keratinocytes, however, the mechanism is not clarified. Recent studies have found that endoplasmic reticulum stress plays a role in the pathogenesis and development of psoriasis, and mTORC1 can regulate endoplasmic reticulum stress. Endoplasmic reticulum stress mediates mitochondria-associated endoplasmic reticulum membrane (MAM) injury and further affects mitochondrial function. Preliminary experiments showed that Sirt6 was highly expressed in the skin of patients and mice with psoriasis. Sirt6 silencing inhibited the expression of mTORC1. Results from preliminary experiments indicated that the expression of MAM marker protein GRP75 was increased in the skin tissue of mice with psoriasis. Therefore, we speculated that Sirt6 mediates MAM injury by regulating mTORC1/ endoplasmic reticulum stress, thereby affecting mitochondrial function, keratinocytes proliferate, differentiate, and inflammation. This project intends to study the effect of Sirt6 on keratinocytes proliferation, differentiation and inflammation by using cell and animal experiments. The effects of Sirt6 on endoplasmic reticulum stress were studied through exploring the regulation of Sirt6 on mTORC1. Further, the influence and related mechanisms of Sirt6 on MAM and mitochondrial function were studied. This project will clarify the role and mechanism of MAM in the pathogenesis of psoriasis and lay the foundation for exploring the new therapy for this disease.

银屑病是慢性炎症性皮肤病，角质形成细胞过度增生和分化异常是主要病理特征，但具体机制未阐明。最近研究发现，内质网应激在银屑病发病机制中发挥作用，而mTORC1能够调控内质网应激；内质网应激介导线粒体-内质网结构偶联（MAM）损伤，进而影响线粒体功能。我们预实验显示，Sirt6在银屑病患者及银屑病小鼠模型皮肤中高表达，Sirt6沉默可抑制mTORC1表达；小鼠模型皮肤组织中MAM标志蛋白GRP75表达升高。因此推测，Sirt6通过调控mTORC1/内质网应激介导MAM损伤，进而影响线粒体的功能，影响角质形成细胞增殖、分化和炎症。本项目拟从细胞与动物实验入手，研究Sirt6对角质形成细胞增殖、分化以及炎症的影响；通过探索Sirt6对mTORC1的调控作用，介导内质网应激，进而研究Sirt6对MAM以及线粒体功能的影响和相关机制。该课题将阐明MAM在银屑病发病机制中的作用，并为探索新疗法奠定基础。

银屑病是慢性炎症性皮肤病，角质形成细胞过度增生和分化异常是其主要表现，但机制未阐明。研究内容主要包括以下三部分：.（1）研究Sirt6是否通过mTOR信号通路促进银屑病的进程：采用M5诱导人角质形成细胞HaCaT制备银屑病细胞模型。结果显示，M5处理后sirt6及mTORC1-Raptor、mTORC1-PRAS40、mTORC2-Rictor、mTORC2-SIN1基因表达水平均显著上调。sirt6 shRNA慢病毒感染显著上调Rictor及SIN1的表达水平，但对Raptor和PRAS40以及mTORC2下游基因FOXO1A和FOXO3A表达水平无显著影响。银屑病皮损部位sirt6、Rictor及SIN1的表达水平显著升高。以上结果未能证实sirt6是否能调控mTORC2。.（2）miR-142-3p在M5诱导的角质形成细胞中的作用及其机制：M5诱导人角质形成细胞HaCaT中miR-142-3p表达水平显著升高。M5刺激促进了细胞增殖、抑制了细胞凋亡、促进了炎性细胞因子的分泌，而miR-142-3p inhibitor转染抑制了M5的以上作用。荧光素酶报告基因实验及功能实验证实Sema3A是miR-142-3p的靶分子，Sema3A敲低部分逆转了miR-142-3p对HacaT细胞增殖、凋亡和细胞因子分泌的影响。综上，miR-142-3p敲减可以通过靶向Sema3A改善M5诱导的银屑病细胞模型的增殖、凋亡和炎症反应。.（3）CircVAPA在M5诱导的角质形成细胞中的作用及其机制：CircVAPA在银屑病患者皮损部位和M5诱导银屑病细胞模型中高表达。在M5诱导的HacaT细胞中，敲低CircVAPA，能抑制炎性因子分泌，降低细胞活性，抑制细胞增殖，促进细胞凋亡。miR-125b-5p在HacaT银屑病细胞模型中以及银屑病患者皮肤组织中低表达；银屑病患者皮肤组织中CircVAPA与miR-125b-5p表达水平呈负相关。荧光素酶报告基因实验、RIP实验及功能实验证实miR-125b-5p是CircVAPA靶分子。Sirt6在HacaT银屑病细胞模型中以及银屑病患者皮肤组织中高表达；银屑病患者皮肤组织中sirt6与miR-125b-5p表达水平呈负相关，CircVAPA与sirt6表达水平呈正相关。荧光素酶报告基因实验、RIP实验及功能实验证实sirt6是miR-1