神经降压素/白介素8（NTS/IL-8）通路调控炎性微环境诱导肝细胞肝癌上皮间质转化(EMT)的分子机制探讨

The epithelial mesenchymal transition(EMT) induced by chronic inflammation promotes invasion and metastasis of malignancy. However, the concrete molecular mechanisms involved in inflammatory microenvironment-mediated EMT are still unclear. Our previous research on global gene expressing profiling and IHC of primary HCC samples distinguished a special subtype of HCC with worse prognosis, in which activation of neurotensin/IL-8 (NTS/IL-8) pathway accompanied by significantly enhanced EMT, upregulated expression of inflammatory cytokines and increased infiltration of immunocytes were observed. The preliminary results implied that activation of NTS/IL-8 pathway promoted EMT in HCC and favored formation of inflammatory microenvironment. Therefore, in this study 100 cases of primary HCC samples will be collected to validate the new pattern of "NTS-inflammation-EMT" in HCC. Different HCC models with varied sensitivity to NTS stimulation will be established to elucidate the regulatory signal network of NTS/IL-8 pathway and pivotal molecular mechanisms modulating EMT and formation of inflammatory microenvironment, as well as to demonstrate the therapeutic effects of small specific inhibitors targeting to NTS/IL-8 pathway in inhibiting EMT and decreasing metastasis of HCC. Our research will provide solid experiment data to screen novel target molecules for HCC treatment.

慢性炎症可通过诱导上皮间质转化(EMT)促进肿瘤侵袭转移，但肝细胞肝癌(HCC) 中炎性微环境介导EMT的分子机制尚不明确。我们前期通过比较原发性HCC 标本的全基因组表达谱和免疫组化结果鉴别出一个预后不良的HCC亚型，以神经降压素/白介素8（NTS/IL-8）通路异常活化为分子特征，伴有显著增强的EMT过程,多种炎性因子的过表达和明显的炎细胞浸润，提示NTS/IL-8通路活化促进HCC中炎性微环境形成和EMT发生。在本研究中，拟收集100 例原发性HCC 标本验证"NTS-炎症-EMT"模式的存在，通过构建不同NTS易感性HCC模型明确NTS/IL-8通路在肝癌中的分子信号调控网络,阐明NTS/IL-8通路调控炎性微环境诱导肝癌细胞EMT的主要分子机制，观察阻断NTS/IL-8通路的小分子靶向药物抑制EMT和降低HCC转移的治疗作用，以期为筛选HCC 治疗新靶点提供有利的实验证据。

神经降压素（Neurotensin，NTS）是一种广泛分布于整个中枢神经系统和部分消化系统中的内源性神经内分泌肽。自从首次发现它在乳腺癌中异常表达以来，已经发现NTS及其高亲和力受体神经降压素受体1（Neurotensin receptor 1，NTR1）可以影响乳腺癌，结肠癌，前列腺癌，胰腺癌和肺癌的生长、凋亡、迁移和侵袭等多个过程。然而，目前在肝细胞肝癌（hepatocellular carcinoma，HCC）中关于NTS和NTR1的研究报道还十分鲜见。前期研究通过基因芯片发现一类以NTS高表达为分子特征的HCC亚型，该亚型存在IL-8的高表达和上皮间质转化（epithelial-mesenchymal transition ，EMT）的特征。本研究在100例HCC临床标本中观察到NTS与IL-8表达水平存在显著相关，且NTS、NTS/NTR1、NTS/IL-8的表达与HCC包膜完整性负相关，与门静脉癌栓侵润正相关，且NTS、NTS/NTR1、NTS/IL-8阳性患者生存时间明显缩短。NTS/IL-8通路异常活化的HCC组织存在明显的EMT的分子特征，并伴有显著增强的局部炎症反应。为了阐明具体分子机制，我们以Hep3B和HepG2细胞为基础，通过基因转染和siRNA干扰构建了不同NTR1表达水平的HCC细胞模型。体外实验证实给予外源性NTS刺激和提高NTR1的表达水平可以通过激活NTS/IL-8通路诱导HCC细胞EMT发生促进其迁移和侵袭，而对增殖和凋亡没有明显作用。NTS/IL-8通路的活化主要依赖于MAPK和NF-κB信号通路的激活。我们还发现NTS 刺激可以通过激活经典Wnt/β-catenin通路促进HCC细胞EMT而增强其侵袭性。而利用特异性抑制剂阻断NTR1和IL-8受体及NTS/IL-8通路中关键蛋白均可逆转HCC细胞EMT，降低其侵袭性，提示NTS/IL-8通路激活是HCC细胞发生EMT的另一个关键分子机制。而且NTS/IL-8通路活化可以诱导单核细胞向M2型TAM细胞分化，促进TAM的迁移和向肿瘤的趋化作用，促进肿瘤局部炎性微环境的形成。体内实验表明NTR1阻断剂SR48692可以抑制荷瘤鼠中HCC肺转移，减少肺部转移灶数量，提示NTR1可能是HCC靶向治疗的新靶点，而针对NTR1的靶向治疗可有效降低HCC体内进展和转移。