人食道癌相关基因-4(Ecrg4)在房颤发生中的作用及机制研究

Accumulating epidemiological evidence has shown that atrial fibrillation (AF) and tumor are closely correlated; however the molecular link underlying the correlation remains to be explored. Esophageal cancer related gene-4 (Ecrg4), originally characterized as a tumor suppressor gene, functions as a ‘sentinel’ molecule monitoring tissue homeostasis: presence of Ecrg4 signals homeostasis, whereas loss of which activates inflammation and tissue proliferation. It has been shown that Ecrg4 is highly expressed in atrioventricular(A-V) node, and that HL1 cells express significantly lower level of Ecrg4 after a 24-hour of rapid electrical stimulation, suggesting a potential role of Ecrg4 in atrial electrical remodeling. Our preliminary data have shown that expression of Ecrg4 is higher in both atria and conduction systems than ventricles in rat; that atrial appendages in patients with AF showed significantly decreased Ecrg4 expression compared to their counterparts in Sinus rhythm; that expression of Ecrg4 in atria was significantly down-regulated in a canine AF model; and that knockdown Ecrg4 in neonatal rat atrial myocytes significantly shortened action potential duration. Therefore， we propose that Ecrg4 play a critical role in the pathogenesis of AF. Here we plan to create a heart-specific Ecrg4 knockout （KO） rat using the Cre-LoxP systems. The susceptibility to AF will be analyzed in the KO at resting, after forced exercise and rapid atria pacing; the expression of proinflammatory cytokines and molecules commonly involved in atrial remodeling will be analyzed by real-time PCR and ELISA; and the electrical and structural remodeling of atria will be analyzed by patch-clamping in isolated atrial myocytes and immunohistochemistry respectively. Then expression of Ecrg4 in the heart will be restored using inverse coronary artery reperfusion of a lentivirus expressing Ecrg4, and the therapeutic effect will be evaluated by analyzing the aforementioned variables. If confirmed, this proposal will further enhance our knowledge in the pathogenesis of AF and provide a therapeutic target for AF.

流行病学分析表明：肿瘤与房颤（AF）发生密切相关。人食道癌相关基因4（Ecrg4）具有抑制炎症和细胞增殖反应的功能。Ecrg4在房室结及心房高表达；HL1细胞经电刺激后，Ecrg4表达明显降低。我们前期研究发现（1）Ecrg4在心脏传导系统高表达；（2）AF患者心耳及犬AF模型心房，Ecrg4表达明显降低；（3）敲低乳鼠心房肌细胞Ecrg4后，动作电位时程明显缩短。因此我们假说：Ecrg4在AF发生和发展中起重要的作用。本研究拟运用Cre-LoxP技术构建心脏特异性Ecrg4敲除大鼠，观察大鼠自发AF、和对运动及快速心房起搏诱发AF的易感性；利用实时PCR和ELISA分析炎性因子及心房重构相关分子的表达；利用IHC分析心房炎性细胞浸润和纤维化。然后，经逆向冠脉灌流恢复Ecrg4的表达，观察其对AF的预防和治疗作用。从而阐明Ecrg4在AF发生中的作用及机理，为AF的治疗提供理论基础和靶点。

人食道癌相关基因-4（ECRG4）虽然最初以肿瘤抑制基因在食道上皮组织发现，但ECRG4远远不止是一个简单的肿瘤抑制基因。ECRG4肿瘤抑制功能的丧失主要源于其基因启动子的甲基化，而非基因突变；ECRG4在多种组织如脑垂体、白细胞、及心脏/传导组织等高表达；ECRG4编码分泌蛋白，可以被组织蛋白酶水解成多个功能各异的小肽。因此，ECRG4不仅具有肿瘤抑制功能，而且具有其它重要生物学功能:如ECRG4参与心脏稳态的维持。.利用基因敲除小鼠、RNAseq、细胞培养、免疫组织化学、启动子分析等一系列分子生物学技术，我们分析了ECRG4在心脏稳态维持中的作用。研究发现：（1）ECRG4在心肌细胞及其心脏传导组织高表达；（2）ECRG4在房颤病人及犬房颤模型的心肌组织表达下调，敲低心肌细胞的ECRG4显著改变了心肌细胞动作电位的时程，激活炎症信号通路及心脏重构基因的表达，因此ECRG4在房颤的发生及发展中起重要的作用；（3）阿霉素通过抑制ECRG4基因启动子而抑制其表达;心肌细胞特异性敲除ECRG4显著增加了阿霉素所致的心脏毒性。提示肿瘤病人心脏ECRG4的表达的下调可能是肿瘤病人阿霉素所致心脏毒性的分子基础；（4）心肌缺血及心肌细胞缺氧显著抑制ECRG4的表达，同时伴随心肌细胞凋亡的增加；而恢复ECRG4可以显著抑制缺血/缺氧所导致的心肌细胞凋亡。进一步研究发现ECRG4基因启动子包含缺氧反应元件。此外，在该基金的资助下我们进行了如下探索并取得了显著的成绩：（1）ECRG4通过抑制‘BC200长链非编码RNA-金属硫蛋白酶’信号通路而抑制口腔鳞癌转移；（2）健康人血清外泌体富含ECRG4 mRNA，提示其在肿瘤抑制及治疗中的作用；（3）利用噬菌体展示技术及配体挖掘潜技术发现了一个新的ECRG436-71配体，为ECRG4的成药提供了理论基础。