肥胖通过visfatin-Sirt1/AMPK-YAP信号通路促进食管癌进展研究

Obesity affects the progression of esophageal cancer and the expression of YAP in the normal cells such as cardiac myocytes, and it could be related to visfatin regulating Sirt1 and AMPK which are closely associated with energy metabolism and glucose and lipid metabolism. Visfatin increases obviously in sera of the obese and acts as nicotinamide phosphoribosyltransferase to remedy the synthesis of NAD+. Sirt1 depending on NAD+ deacetylates YAP protein and promotes the growth of hepatic cancer cells. Under obese condition, AMPK is suppressed, and Sirt1 may play a suppressive role on AMPK, then the phosphorylation of YAP reduces to promote tumor. Sirt1 and YAP expression is elevated in esophageal squamous cell carcinomas. Therefore, obesity may contribute to esophageal cancer progression by visfatin regulating the Sirt1/AMPK-YAP signaling pathways which was found in recent studies and was closely related with tumor growth. The project intends to analyze the clinical and pathological features of patients with esophageal cancer; use high fat diet inducing C57BL/6 nude mice to establish obese mice model and their subcutaneous xenograft model; utilize exogenous visfatin for culturing human esophageal cancer cells; construct the recombinant plasmid pshRNA-Sirt1 and pshRNA-AMPK to transfect esophageal cancer cells, then the cells were stimulated by visfatin to explore the role of visfatin in regulating Sirt1/AMPK-YAP signaling pathway. Results of the study would demonstrate that obesity affects the progression of esophageal cancer and clarify its molecular mechanism for searching the target of esophageal cancer to improve the therapeutic effect.

肥胖影响食管癌进展及机体细胞YAP表达，这可能与肥胖时明显升高的内脂素调控与能量、糖脂代谢密切相关的Sirt1、AMPK有关。内脂素具有类似Nampt酶活性而补救合成NAD+，依赖NAD+作为辅酶的Sirt1去乙酰化YAP蛋白而促进肝癌生长；肥胖状态下，AMPK处于抑制状态且Sirt1可抑制AMPK表达，减少YAP磷酸化而促肿瘤进展；Sirt1、YAP在食管鳞癌中表达升高。因此，肥胖可能通过内脂素调控最近研究发现与肿瘤生长相关的Sirt1/AMPK-YAP通路促进食管癌进展。为此，本项目利用临床病理资料；高脂饮食诱导裸鼠建立肥胖模型进而建立皮下移植瘤模型；内脂素培养食管癌细胞；构建重组干扰质粒pshRNA-Sirt1、pshRNA-AMPK转染食管癌细胞，内脂素再对其进行刺激，以阐述肥胖及内脂素对食管癌进展的影响及对Sirt1/AMPK-YAP信号通路的调控以筛选提高食管癌治疗效果的靶点。

食管癌在我国发病率高且具高致死性。现阶段超重或肥胖人群明显增加，已有研究表明肥胖能促进多种恶性肿瘤发生与进展，而食管癌与肥胖之间关系复杂。本研究通过临床病理资料分析及建立肥胖裸鼠皮下人食管癌移植瘤模型，研究肥胖和内脂素对食管癌进展以及化疗敏感性的影响；通过内脂素体外刺激食管癌细胞及使用目的基因干扰质粒转染食管癌细胞，观察内脂素是否通过Sirt1/AMPK-YAP信号通路调控食管癌生长与进展。本研究结果显示：①通过临床病理资料分析发现，内脂素即eNampt在肥胖患者血清中明显升高，并与肥胖体征明显相关，但与食管癌细胞iNampt表达无相关性，食管癌细胞iNampt表达阳性或升高可降低食管癌病灶对新辅助化疗的敏感性，虽然iNampt不是新辅助化疗患者预后的独立影响因素，但ypN分期、肿瘤组织化疗后病理改变TRG能独立影响这群食管癌患者的预后。②通过裸鼠皮下移植瘤模型发现，肥胖组移植瘤生长明显较快，肥胖导致机体相关脂肪因子和代谢性因素发生显著改变，而这些肥胖相关脂肪因子和代谢性因素的改变可显著促进食管癌在体内的生长。利用含裸鼠血清的条件培养基处理食管癌细胞发现，肥胖组食管癌细胞有更快的生长、迁移和侵袭能力。肥胖组食管癌细胞AMPK表达在蛋白和mRNA水平均降低，MMP9、总YAP、p-YAP和非磷酸化YAP蛋白表达显著升高，YAP和MMP9 mRNA表达显著升高，这表明肥胖能通过包括内分泌在内的复杂综合效应调控食管癌细胞MMP9、AMPK-YAP信号通路而影响食管鳞癌细胞在体外的增殖、迁移和侵袭。③通过内脂素体外实验发现，内脂素可促进食管癌细胞生长、迁移和侵袭；用质粒敲除目的基因进行重复实验，结果提示内脂素能调控Sirt1-YAP通路并促进YAP入核，进而调控食管癌细胞生长、迁移和侵袭；移植瘤组织经免疫组化染色发现血清内脂素与Sirt1、YAP表达呈正相关。综上结果，肥胖可通过包括内脂素在内的多种因子促进食管癌进展，靶向iNampt的治疗可增加食管癌对化疗敏感性；针对内脂素-Sirt1/AMPK-YAP信号通路可筛选食管癌治疗新靶点。