甘氨鹅脱氧胆酸盐诱导自噬促进肝癌侵袭转移的机制研究

The overall outcomes of patients with hepatocellular carcinoma (HCC) are poor due to high metastasis and recurrence. Previous studies show that the HCC patients with cholestasis are easy to recurrence and metastasis. Glycochenodeoxcholate (GCDC) is an important bile salts in cholestasis. In our previous experiment, we found that GCDC could induce invasion in HCC cells, which was related with up-regulated of autophagy, but the mechanism was unclear. In this study, we aim to explore the invasion and metastatic effect of GCDC on HCC cells; Then we observed the role of autophagy in hepatocellular induced by GCDC and the effect of GCDC on hepatoma invasion and metastasis by using siRNA or inhibitor of autophagy. On the other hand, autophagy related gene will be detected by PCR microarray, and confirmed by western blot, which intend to explore the molecular mechanism of autophagy related-gene regulated by GCDC; Finally, the relationship between autophagy genes expression level in the clinical specimens and clinical prognosis of enrolled patients will be evaluated. The results of study can not only directly explain the effect and mechanism of high concentration of bile salts on HCC cells invasion and metastasis, but also deepen our understanding of the prognosis of patients with hepatocellular carcinoma with cholestasis. This investigation could elucidate the mechanism how cholestasis promotes metastasis and provide the experimental basis for exploring the new strategy of anti-tumor metastasis aiming at autophagy.

肝癌转移与复发严重影响其治疗效果。研究发现肝癌伴有黄疸的患者更易发生转移复发，预后更差。甘氨鹅脱氧胆酸盐（GCDC）是胆汁的主要成分，我们前期实验发现GCDC可增强肝癌细胞侵袭转移能力，而肝癌侵袭转移增加与其自噬水平升高有关，但具体机制尚不明确。本课题将通过体内外实验观察GCDC对肝癌细胞侵袭转移的影响；检测GCDC对肝癌细胞自噬发生的作用，并应用自噬抑制剂或siRNA抑制自噬后，明确自噬在GCDC促进肝癌细胞侵袭转移中的作用；采用PCR芯片和Western Blot筛选鉴定GCDC促进自噬的关键信号通路，明确GCDC促进自噬相关基因表达的分子机制；利用肝癌患者临床标本，分析胆盐水平、自噬相关分子表达与肝癌转移复发的相关性。本研究结果不仅可以明确胆盐对肝癌细胞侵袭转移能力的影响及内在机制，也有望阐明肝癌伴发胆汁淤积患者预后差的原因，为肝癌的临床治疗提供新的预后判断标准和治疗靶点。

肝癌伴胆汁淤积的患者预后差，肿瘤容易转移。我们利用胆盐刺激模拟体内胆汁淤积微环境，探讨胆盐对肝癌细胞侵袭转移的直接影响。我们首先利用GCDC刺激模拟胆汁淤积的微环境，经体内外实验观察GCDC对肝癌细胞侵袭转移能力的直接影响。结果发现，胆盐能够促进体内外肝癌细胞侵袭转移；进一步我们检测GCDC作用下肝癌细胞自噬激活情况，以明确自噬在胆盐促进肝癌细胞侵袭转移中的调控作用。结果发现，胆盐诱导自噬发生，从而促进了肝癌细胞的侵袭转移，抑制自噬能够减轻胆盐在体外对肝癌细胞侵袭转移的作用；进一步探讨胆盐影响肝癌细胞侵袭转移相关自噬基因的分子机制发现，GCDC 刺激下，肝癌细胞自噬激活的发生是依赖AMPK/mTOR途径作用的；最后我们利用胆盐表达差异的肝癌患者肿瘤标本，检测并分析自噬相关分子与肿瘤侵袭及预后的相关性。结果发现，胆汁酸的升高和自噬相关分子的表达及预后呈正相关，进一步使用统计学分析肝癌患者TBA的表达与临床病理特征的相关性分析发现，HCC肝癌组织中胆汁酸升高与肝纤维化的发生、更高的TMN分级、微血管侵袭和大血管侵袭发生有显著相关性。此外，除了上述微环境中的胆汁酸因素，课题组还围绕肿瘤微环境中的肝星状细胞和间质细胞与肿瘤侵袭转移的关系开展了相关研究。我们发现肝星状细胞可以通过分泌细胞因子HGF促进Hep3B肝癌细胞系的侵袭转移，并诱导其发生上皮间质转换；而这种促进作用依赖于p53的缺失及其下游分子c-Met的活化。进一步延伸研究发现癌旁中的成纤维细胞可以分泌趋化因子募集肝癌干细胞，同时分泌细胞因子HGF等维持其干性来促进肝细胞癌的发展。