内脂素Visfatin在结直肠癌细胞上皮间质化中的生物学功能研究

Accumulating evidence suggests that Visfatin expressions in tumor tissues and sera are closely associated with invasion and metastasis of colorectal cancer (CRC). However the mechanism is almost entirely unknown. Recent studies and our preliminary data revealed that Epithelial-Mesenchymal Transition (EMT) and invasive ability of CRC cells were significantly modified by alteration of Visfatin expression, indicating that Visfatin might regulate invasion and metastasis of CRC through promotion of EMT. These findings could be used as a novel clue for illustrating Visfatin-induced malignancy in CRC. In the meantime, Visfatin has been reported to regulate the activities of NAD-dependant molecules within the cell, and function as a pro-inflammatory cytokine outside the cell. These main biological functions of Visfatin could serve as the key point for understanding the mechanism of Visfatin-related EMT process. Thus, we plan to dissect the mechanism of Visfatin-induced EMT in CRC cells with multiple CRC models via multiple intervention methods, focusing on the main biological functions of Visfatin. Moreover, we would evaluate the feasibility and efficiency of these intervention methods to reduce the risk of metastasis.

日益增多的证据显示，内脂素（Visfatin）在肿瘤组织及血样中的含量与结直肠癌的侵袭与转移密切相关，但相关机制却鲜为人知。新近研究以及我们的前期工作发现，增加或抑制Visfatin的表达可明显改变肿瘤细胞的间质化特征和侵袭能力，提示Visfatin有可能通过促进上皮细胞间质化（EMT）参与结直肠癌的转移与侵袭过程。这为阐明Visfatin促进结直肠癌恶性生物学行为的作用机制提供了新的线索，而Visfatin具有细胞内烟酰胺腺嘌呤二核苷酸(NAD) 合成限速酶功能及细胞外促炎症细胞因子的两大生物学活性则是探明其参与EMT转化机制的切入点。据此，本课题拟在前期所建模型基础上，通过多种干预措施，重点从Visfatin的两大生物学活性着手，深入解析其诱发结直肠癌EMT过程的内在机制，并评价相关干预措施对降低肿瘤转移风险的有效性和可行性。

内脂素（Visfatin）在肿瘤组织及血样中的含量与结直肠癌的侵袭与转移密切相关，但相关机制却鲜为人知。本项目从Visfatin具有细胞内烟酰胺腺嘌呤二核苷酸(NAD) 合成限速酶功能及细胞外促炎症细胞因子的两大生物学活性着手，研究Visfatin通过促进上皮细胞间质化（EMT）参与结直肠癌的转移与侵袭的分子机制，并评价相关干预措施对降低肿瘤转移风险的有效性和可行性。 在临床样本分析中，我们明确了visfatin表达与结肠癌恶化转移及不良预后负相关，肿瘤组织内visfatin定位随着肿瘤恶化增加，由细胞质转向细胞核。患者血液中visatin随着肿瘤恶化含量增加，且与IL1-β、IL6等成正相关。在细胞水平上：（1）我们以人结肠癌细胞HCT-116和SW480为模型，证明Visfatin能通过调控Snail的表达量促进肠癌细胞EMT。进一步的实验证明visfatin重组蛋白能通过影响Snail和GSK-3β结合，抑制Snail的泛素化降解；（2）通过构建不同的visfatin表达质粒转染结肠癌细胞，我们发现提高细胞内visfatin表达量能有效促进肿瘤EMT，且这种恶化行为与证visfatin的核转运密切相关。具有核定位信号的表达质粒，能提高visfatin的入核效率，促进结肠癌细胞EMT及转移侵袭;（3）我们采用visfatin重组蛋白处理内皮细胞（HUVEC）、佛波豆蔻醚乙酸盐(phorbol myristate aceta，PMA)诱导THP-1分化的巨噬细胞，发现visfatin能显著上调这两种细胞IL1-β、IL6的表达分泌。将两种细胞分别与HCT-116和SW480细胞共培养后，均能促进肿瘤细胞EMT。在动物实验中，采用BALB/c小鼠和小鼠结肠癌细胞CT26为模型，尾静脉注射经过鼠visfatin重组蛋白、转染visfatin表达质粒、FK866、siRNA处理的CT26细胞，结果发现，visfatin蛋白处理和上调细胞内visfatin表达量均能促进肿瘤细胞肺转移，而抑制visfatin酶活性和细胞内表达，能降低肿瘤的肺转移。本项目初步阐明了visfatin通过EMT促进结肠癌转移的分子机制，抑制其酶活性和细胞内的表达能有效降低结肠癌的侵袭转移，为临床肿瘤转移治疗策略提供了新的思路。