NCSTN基因通过IGF-1R介导的PI3K/AKT信号通路在反常性痤疮发病中的作用及机制研究

Loss-of-function mutations of NCSTN gene, known as the γ- secretase subunit, were related to acne inversa (AI). Hyperproliferation and abnormal differentiation of keratinocytes is the initial cause of AI. PI3K/AKT signaling pathway is related with proliferation and differentiation of keratinocytes and its activation is regulated by NCSTN gene, but the specific mechanism is unknown. Insulin-like growth factor 1 receptor (IGF-1R) is a substrate of γ- secretase and it activates PI3K/AKT signaling pathway by combining its ligand. We found that after disturbing NCSTN expression of HaCaT cells, the expression of IGF-1R was increased significantly, indicating that IGF-1R may involve the regulation of NCSTN on PI3K/AKT signaling pathway. This topic plans to establish a primary human keratinocytes model, of which the expression of NCSTN is silenced by RNA interference, to study the influence of NCSTN knockdown on biological behaviors like cell proliferation and differentiation, and on IGF-1R-medicated PI3K/AKT signaling pathway and its downstream genes; to detect the difference in the listed biological behaviors of primary human keratinocytes before and after adding IGF-1R or PI3K/AKT specific inhibitors; to detect the expression of IGF-1R/PI3K/AKT molecules in skin lesions of patients with NCSTN gene mutations and make clear its clinical relevance with AI. The study may contribute to further clarify the pathogenesis of AI.

γ-分泌酶亚基NCSTN基因功能缺失性突变与反常性痤疮(AI)相关。角质形成细胞(KC)过度增殖及异常分化是AI的初始致病因素。PI3K/AKT信号通路与KC的增殖和分化相关，其活化受NCSTN基因调控，但机制不详。IGF-1R是γ-分泌酶底物，通过结合其配体激活PI3K/AKT通路。我们发现干扰HaCaT细胞NCSTN表达后IGF-1R表达明显增加，提示IGF-1R可能参与NCSTN对PI3K/AKT通路的调控。本课题拟采用RNA干扰技术沉默人原代KC的NCSTN基因表达，研究其对细胞增殖、分化等生物学行为及对IGF-1R介导的PI3K/AKT通路及下游基因表达的影响；检测原代KC分别加入IGF-1R或PI3K/AKT特异性抑制剂前后生物学行为的变化；在NCSTN基因突变患者皮损中检测IGF-1R/PI3K/AKT分子的表达，明确其与AI临床相关性。本研究有助于进一步明确AI的发病机制。

背景：.反常性痤疮（Acne Inversa，AI）是毛囊-皮脂腺-顶泌汗腺单元的慢性炎症性皮肤病，主要累及皱褶部位，皮损可表现为脓肿、窦道、瘢痕形成，严重影响患者的生活质量。γ分泌酶是家族性AI研究的热点，其包含四个组分，分别为：早老素(presenilin,PS）、纳卡斯楚因蛋白（nicastrin,NCT）、前咽缺陷蛋白(anterior pharynx defective-1,APH1)、早老素增强子（presenilin enhancer-2,PEN2）。PSENEN基因突变是AI的突变基因之一，但PSENEN基因在皮肤病的作用至今仍不清楚。.主要研究内容：.本课题旨在研究PSENEN敲减对角质形成细胞γ-分泌酶的影响和相关信号传导通路的改变，以及对角质形成细胞生物学如增殖、分化、凋亡的影响。同时，探讨反常性痤疮PSENEN基因突变的发病机制和治疗靶点，试图寻找有别与阿尔兹海默的独特分子机制。.重要结果及关键数据：.1.沉默HaCaT细胞PSENEN 72h PSENEN及PEN2蛋白水平表达均明显下调（p < 0.05），成功构建了PSENEN敲减的细胞模型。实验组γ-分泌酶组分：imNCT升高、mNCT降低、PS1-CTF下降、PS1增加（均p < 0.05），而APH1a不变。.2.转录组测序结果表明组间差异mRNA生物学功能绝大部分与代谢有关；分子功能绝大部分与分子间的结合有关；KEGG分析（差异2倍，p < 0.05）：EGFR、VEGFR、m-TOR信号通路表达升高，胰岛素、m-TOR、代谢信号通路表达下降。.3.转染72h内，组间Ki67mRNA及蛋白水平不变，CCK-8和细胞凋亡组间差异无统计学意义。EGFR mRNA及蛋白水平表达均升高（p < 0.05），但PI3K-AKT-mTOR信号通路相关蛋白差异无统计学意义。.科学意义：.1. HaCaT细胞PSENEN敲减，PEN2蛋白表达下降，引起γ-分泌酶NCT成熟障碍和PS1磷酸化障碍。PSENEN敲减引起HaCaT细胞EGFR信号通路升高。