ADAM17介导EGFR信号通路在中耳胆脂瘤角质形成细胞过度增殖中的机制研究

ADAM17 belongs to a disintegrin and metalloproteases (ADAMs) family of typeⅠ transmembrane Zn2+-dependent proteases that play a role in the proteolytic ectodomain shedding of multiple membrane-tethered ligands and cytokines implicated in diverse biological processes, including growth and inflammation. It has protease activity against multiple epidermal growth factor receptor (EGFR) ligands, thus activating EGFR-related proliferative signaling pathway. Cholesteatoma is a pathological condition characterized with keratinocyte hyper-proliferation, however, the molecular mechanism underlying the pathogenesis of cholesteatoma remains unknown. Our previous studies have demonstrated that EGFR signaling pathway is active in cholesteatoma and may play a crucial role in keratinocyte hyper-proliferation. However, the mechanism of the EGFR signaling pathway activation in cholesteatoma remains unknown, and whether ADAM17 participants in the activation is unreported. Accordingly, we hypothesize that ADAM17 may be activated in cholesteatoma tissues, and involved in the activation of EGFR signaling pathway through cleaving and releasing various EGFR ligands, leading to the keratinocyte hyper-proliferation responsible for cholesteatoma. In this study, we will investigate the role of ADAM17-mediated EGFR signaling pathway in the keratinocyte hyper-proliferation of cholesteatoma through in vivo and in vitro experiments. So that it could clarify the molecular mechanism of keratinocyte hyper-proliferation and explore a novel target for the prevent and treatment of keratinocyte hyper-proliferation of middle ear cholesteatoma.

ADAM17是一种跨膜金属蛋白酶，可通过蛋白水解作用剪切、脱落出多种EGFR配体，从而激活EGFR相关增殖性信号通路，是EGFR信号通路的“启动者”。中耳胆脂瘤是以角质形成细胞过度增殖为特征的疾病，其增殖的内在分子生物学机制不明。我们前期研究发现EGFR信号通路在中耳胆脂瘤上皮组织中处于激活状态，在胆脂瘤角质形成细胞过度增殖中起关键作用，但其激活的具体作用机制尚未明确，ADAM17是否参与其中目前尚无文献报道。由此，我们首次提出如下假说：ADAM17在中耳胆脂瘤中处于激活状态，并通过剪切、脱落出多种EGFR配体进而激活EGFR相关增殖性信号通路，最终导致胆脂瘤角质形成细胞过度增殖。本课题将通过体内、外实验揭示ADAM17介导EGFR信号通路在中耳胆脂瘤角质形成细胞过度增殖中的作用，为明确中耳胆脂瘤角质形成细胞过度增殖的内在分子机制提供实验依据，并为防治中耳胆脂瘤上皮过度增殖开辟新思路。

中耳胆脂瘤是以角质形成细胞过度增殖为特征的疾病，其增殖的内在分子生物学机制尚不明确。本研究运用免疫组织化学方法、Western blot法和RT-qPCR法等从体内和体外实验检测ADAM17和EGFR信号通路主要组成分子的表达情况，明确ADAM17是否通过EGFR信号通路调控胆脂瘤角质形成细胞增殖，深入探讨ADAM17介导EGFR信号通路调控胆脂瘤角质形成细胞增殖的信号转导机制。研究发现EGFR信号通路主要组成蛋白P-EGFR、P-ERK、P-Akt、NF-κB、PCNA等在中耳胆脂瘤上皮组织中处于高表达（激活）状态，同时对EGFR的主要配体进行了检测，发现一个新的配体HBEGF处于高表达状态，且HBEGF蛋白和胆脂瘤骨质破坏程度显著相关；表明HBEGF通过和中耳胆脂瘤角质形成细胞膜上的受体EGFR结合后激活EGFR，活化了的EGFR可以激活下游PI3K/Akt信号通路和ERK信号通路，发挥促进中耳胆脂瘤角质形成细胞增殖分化的作用。同时RT-qPCR实验发现ADAM17在中耳胆脂瘤上皮组织中表达较对照组正常皮肤标本显著增高，差异有统计学意义，表明ADAM17可能通过对EGFR配体HBEGF进行胞外段的切割加工，使HBEGF由无活性状态的前体转变为有活性状态的单体从细胞膜上释放出来，进而激活EGFR。与此同时，课题组还在实验过程中发现和EGFR通路有密切关系的破骨因子PTHrP在中耳胆脂瘤组织中处于高表达状态。课题组还运用microRNA芯片技术检测中耳胆脂瘤标本和正常皮肤标本的microRNA差异表达谱，同时应用生物信息学方法预测相关靶基因进而筛选出和EGFR/PI3K/Akt及EGFR/ERK信号通路靶向相关的2个关键性microRNA,分别为hsa-miR-16-1-3p和hsa-miR-10a-5p，进一步扩大样本量行PCR验证，结果发现：hsa-miR-16-1-3p在中耳胆脂瘤组织中表达上调，而hsa-miR-10a-5p在中耳胆脂瘤组织中表达下调，差异具有统计学意义。