γ-分泌酶对角质形成细胞生物学行为及Wnt通路调控的研究

Previous researches have found that gamma secretase was associated with Alzheimer's disease. In 2010, we firstly reported gamma secretase gene mutation was related with familial acne inverse (AI), but its mechanism was unknown, Currently, the pathogenic similarity and difference of these two diseases are focus of international academic community. Abnormal proliferation of keratinocytes was related to AI, and Wnt signaling not only regulated growth of epidermal cells but also was influenced by gamma secretase. So, in this study stable transfection HaCaT cell modes were established by short hair-pin RNA interfereing to silencing presenilin-1 and nicastrin subunits of gamma secretase respectively. Through fluorescent quantitative PCR, Western Blot, ELISA and laser confocal microscopy combined with microRNA and long noncoding RNA expression profile, changes of adhesion and other biological behaviors as well as Wnt signal pathway in different gamma secretase subunits interference cells were compared. The above results were further verified in gamma secretase subunit gene knockout mice model and lesions of AI patients. This study may reveal the role of gamma secretase gene mutation in AI pathogenesis, and contribute to understanding the different molecular pathway of AI and Alzheimer's disease which both related to gamma secretase.

研究发现，γ-分泌酶与阿尔茨海默病有关，2010年我们在国际上最早报道γ-分泌酶基因突变与家族性反常性痤疮相关，但机制不详，两种不同疾病发病机制的异同是目前学术界关注的热点。反常性痤疮的发生与角质形成细胞异常增殖相关，Wnt信号通路调控表皮细胞的生长，并受γ-分泌酶的影响。本课题拟通过shRNA分别干扰γ-分泌酶的PS-1和NCT亚基，建立稳定转染的HaCaT细胞模型，采用荧光定量PCR、Western Blot、ELISA、激光共聚焦显微镜等技术结合MicroRNA芯片和长链非编码RNA芯片，对比研究γ-分泌酶不同亚基干扰后细胞生长分化等生物学行为的变化以及Wnt信号通路的改变，并使用γ-分泌酶PS-1和（或）NCT亚基基因敲除小鼠模型和患者皮损，验证细胞实验结果。本研究可能揭示γ-分泌酶基因突变引起反常性痤疮的发病机制，有助于了解γ-分泌酶引起反常性痤疮与阿尔茨海默病分子通路的差异。

γ-分泌酶中NCT亚基的突变已被证实与家族性反常性痤疮密切相关，但其在家族性反常性痤疮发病过程中的功能未完全明确。我们构建了家族性反常性痤疮患者mRNA数据库以及miRNA表达谱数据库，筛选了一系列差异表达的基因和miRNA，通过qPCR技术我们发现在NCT干扰后的角质形成细胞中Wnt4和Wnt11的mRNA水平显著下调，而Wnt5a的mRNA水平显著上调。另外我们也重点探讨了家族性反常性痤疮发病过程中NCT突变导致miR-30a-3p和miR-100-5p水平下降的潜在机制以及在角质形成细胞增殖分化中的功能。通过基因敲除小鼠，qPCR, Western blot和免疫荧光等实验我们揭示了NCT突变可通过miR-30a-3p/RAB31轴促使EGFR降解，导致角质形成细胞分化异常，同时我们发现NCT可以靶向AKT，进而激活AKT的磷酸化水平，促进角质形成细胞的过度增殖。上述研究全方位阐释了家族性反常性痤疮发病过程中重要的分子包括miR-30a-3p、miR-100-5p及EGFR作为治疗靶点的可能性，为家族性反常性痤疮治疗策略提供新的线索。