基于IL-6介导的JAK2/STAT3/TWIST通路探讨化痰祛瘀方抑制肝癌上皮间质转化的作用机制

Epithelial-mesenchymal transition (EMT) is the key point of tumor invasion and metastasis, and IL-6-mediated JAK2/STAT3/TWIST signaling pathway is one of the most important pathways in EMT occurring. Our previous studies have shown that HuaTanQuYu herbs was related to down-regulation of bcl-2 and cyclin D1 gene expression, and inducing tumor cell apoptosis, and reducing the amount of VEGF protein expression in hepatoma cells; the downstream target genes of STAT3 have been identified, including cyclin D1, bcl-xl and VEGF. Therefore, we hypothesized that the anti-hepatoma mechanisms of HuaTanQuYu herbs may be achieved through regulating STAT3 signaling pathways. In this study, liver cancer spontaneous lung metastasis model in nude mice, and IL-6-induced epithelial-mesenchymal transition hepatoma cell model, will be studied; we will employ the methods including three-dimensional cell culture, FITC-phalloidin staining, confocal laser scanning microscopy and other techniques; the effects of HuaTanQuYu herbs on IL-6-mediated JAK2/STAT3/TWIST signaling pathway and EMT markers will be based on multi-angle observation from four levels covering overall-tissue-cells-molecular. This study will reveal the targets and mechanisms of HuaTanQuYu herbs inhibiting the recurrence and metastasis of liver cancer and provide new scientific evidence for the prevention and treatment of Traditional Chinese Medicine in liver cancer.

上皮间质转化（EMT）的形成是引起肿瘤侵袭和转移的关键环节，而IL-6介导的JAK2/STAT3/TWIST通路是EMT发生的重要信号通路。我们前期研究显示，化痰祛瘀方下调肝癌细胞bcl-2、cyclin D1基因的表达，有效诱导肿瘤细胞凋亡，并降低VEGF蛋白表达，而cyclin D1、bcl-xl、VEGF等为STAT3的下游靶基因。因此，我们推测化痰祛瘀方抗肝癌机制可能通过对STAT3相关信号通路调节而起效。本研究利用肝癌裸鼠自发性肺转移模型和IL-6诱导的肝癌上皮间质转化细胞模型，应用三维细胞培养、FITC-phalloidin染色、激光共聚焦等技术，从整体-组织-细胞-分子4个层次，多角度观察化痰祛瘀方对IL-6激活的JAK2/STAT3/TWIST通路及上皮间质转化特征性分子表达的影响，揭示化痰祛瘀方抑制肝癌复发和转移的作用靶点和机制，为肝癌的中医药防治提供新的科学依据。

目的：探讨化痰祛瘀方抑制肝癌上皮间质转化的分子机制。方法：1.MTT法及Annexin V-FITC/PI法检测化痰祛瘀方各浓度含药血清对HCCLM3增殖和凋亡的影响。2.检测化痰祛瘀方各浓度含药血清及JSI-124对EMT特征性蛋白(E-cadherin， N-cadherin 及Vimentin)，JAK2/STAT3/TWIST信号通路激活的关键蛋白(JAK2, p-JAK2, STAT3, p-STAT3, TWIST)的表达量的影响。3.建立高转移潜能肝癌皮下移植瘤自发性肺转移模型，应用免疫组化，Western Blot及RT-PCR分别检测各浓度化痰祛瘀方对肿瘤组织中EMT及JAK2/STAT3/TWIST信号通路特征性蛋白(JAK2, p-JAK2, STAT3, p-STAT3, TWIST)进行定位及定量分析。5.Transwell和肺组织HE染色，分别检测各浓度化痰祛瘀方及JSI-124对肝癌细胞侵袭能力及肺转移的影响。结果：1.高剂量化痰祛瘀方含药血清对HCCLM3增殖的抑制作用最大。 2.高剂量化痰祛瘀方含药血清干预后，HCCLM3的凋亡率最高。3.在体外实验中，高剂量化痰祛瘀方抑制IL-6诱导HCCLM3 EMT的形成，从而降低HCCLM3的侵袭能力。4.在体内试验中，高剂量化痰祛瘀方抑制HCCLM3皮下移植瘤 EMT的形成，进而降低HCCLM3皮下移植瘤自发性肺转移的能力。6.在体外实验中，高剂量化痰祛瘀方通过抑制JAK2/STAT3/TWIST信号通路激活，抑制IL-6诱导HCCLM3 EMT的形成。7.在体内实验中，高剂量化痰祛瘀方通过抑制IL-6介导的JAK2/STAT3/TWIST信号通路激活，进一步抑制HCCLM3皮下移植瘤 EMT的形成。结论：化痰祛瘀方通过干预炎症因子IL-6介导的JAK2/STAT3信号通路激活，调节肝癌上皮间质转化特征分子表达，抑制肝癌上皮间质转化，从而降低肝癌侵袭和转移能力，延缓复发转移。这一研究结果为临床研究中IL-6，STAT3以及TWIST的高表达是影响肝癌复发转移以及预后的因素，阐释了其可能的作用机制。同时为化痰祛瘀方改善肿瘤炎性微环境，防治肝癌复发转移提供了新的作用靶点和基础理论支持。