虫草素介导miR-21/PI3K/Akt信号通路抑制胆管癌增殖和侵袭转移的分子机制研究

Cholangiocarcinoma (CCA) is a malignant neoplasm derives from bile duct epithelium cells, which characterized by highly malignancy, poor prognosis, early metastasis and insensitive to chemoradiotherapy. Thus, it is important to explore a traditional Chinese medicine (TCM) as anti-tumor drug with low toxicity, high efficiency and multiple targets for the treatment of CCA. As a oncogenic factor, miR-21 plays an important role in cell proliferation and metastasis of CCA. In addition, miR-21 can up-regulate PI3K/Akt signaling pathway by targeting PTEN, and may be a novel therapeutic target for CCA. Cordycepin has shown anti-proliferation, pro-apoptosis and anti-tumor metastasis effects in several tumors. Our previous studies indicated that cordycepin can down regurate the miR-21 expression, induce apoptosis and autophagy, and inhibit cell migration in CCA. These results suggest that miR-21 may be the main mechanism of cordycepin on anti-cholangiocarcinoma effect. In this study, we use miR-21 high-expression cell line of CCA to clarify cordycepin anti-tumor effect and molecular mechanism through various methods of molecular biology and animal experiments in vivo and in vitro. This will provide a new theoretical basis and experimental evidence for the treatment of CCA and the development and utilization of cordycepin.

胆管癌是来源于胆管上皮细胞的恶性肿瘤，具有恶性度高、预后差、早期发生转移及对放化疗不敏感等特点。寻找低毒、高效、多靶点的抗肿瘤中药对胆管癌治疗具有重要意义。研究表明，MiR-21作为促癌因子在胆管癌的发生发展过程中起促增殖、转移的作用，并通过PTEN调控PI3K/Akt信号通路的激活，可能是治疗胆管癌的一个新靶点。虫草素对多种肿瘤具有抗增殖、促凋亡及抑制转移的作用。前期研究发现，虫草素可下调胆管癌细胞miR-21表达水平，并诱导胆管癌细胞凋亡和自噬，抑制迁移。提示miR-21可能为虫草素抑制胆管癌的主要靶点。因此，本项目用miR-21高表达胆管癌细胞株，通过多种分子生物学方法及动物实验，探讨虫草素对人胆管癌细胞及动物模型的抗肿瘤作用及分子机制，为胆管癌的临床治疗及虫草素的开发利用提供新的理论基础和实验依据。

胆管癌是一种来源于胆管上皮细胞的恶性肿瘤，预后较差，因此迫切需要更有效的治疗方案。虫草素是一种中药提取物，在抗肿瘤方面有多种药理作用，但其作用机制尚未完全阐明。本研究采用MTT和集落形成试验，观察了虫草素对胆管癌细胞活性和增殖能力的抑制作用。流式细胞术和hoechst 染色显示，虫草素通过细胞外信号调节激酶ERK1/2失活诱导肿瘤细胞凋亡。此外，管腔形成实验显示，虫草素显着降低血管生成能力。DEK是一种致癌蛋白，在各种胃肠道肿瘤中过度表达。蛋白印迹实验结果显示，虫草素可明显抑制胆管癌细胞的DEK表达。Western blot和流式细胞术检测DEK沉默对胆管癌细胞活性和血管生成有抑制作用，但对凋亡诱导无明显影响。此外，虫草素通过下调 DEK、ERK1/2 磷酸化水平和cd31和vWF的表达，显著抑制原位瘤的肿瘤生长和血管生成能力。总之，我们证明了虫草素通过下调 ERK1/2信号通路抑制 胆管癌细胞增殖和血管生成，并与 DEK相互作用。以上结果表明，虫草素可能作为一种新的药物用于临床治疗和预后的改善。