盐酸小檗碱通过抑制wnt信号通路调控ALDH1A1表达在结直肠癌肺转移中的作用及机制研究

The 5-year overall survival rate of lung metastasis from colorectal cancer is less than 7%, and the prognosis is very poor. Traditional and Modern medicine have little research on it. In previous studies, we found that berberine hydrochloride can inhibit the growth of colorectal cancer and the occurrence of lung metastasis, but the specific mechanism is unclear. Subsequent studies have found that berberine hydrochloride can significantly reduce the activity of Wnt pathway and down-regulate the levels of β-catenin and ALDH1A1 mRNA in colorectal cancer. The classical Wnt pathway has been reported to regulate the expression of ALDH1A1. Therefore, we postulated that berberine hydrochloride affects the transcriptional activity of β-catenin/TCF-4 complex by interfering with β-catenin, thereby regulating the expression of ALDH1A1 and inhibiting the occurrence of lung metastasis of colorectal cancer. To verify this hypothesis, molecular biological technologies such as bioinformatics, gene chip, plasmid construction and transfection, chromatin immunoprecipitation and immunohistochemistry will be used to identify the molecular targets of berberine hydrochloride in the prevention and treatment of lung metastasis of colorectal cancer, enrich the scientific connotation of “colorectal exterior-interior” in TCM holistic view, and provide theoretical support and experimental evidence for the research and development of Berberine hydrochloride “New Functions of Old Drugs” as a novel antitumor durg.

结直肠癌肺转移5年总生存率不足7%，预后极差，传统及现代医学对其均鲜有研究。我们前期研究发现盐酸小檗碱能够抑制结直肠癌原发灶的生长以及肺部转移的发生，但其具体机制不清。后续研究发现，盐酸小檗碱能够显著降低Wnt通路的活性，并下调β-catenin和ALDH1A1 mRNA水平。文献报道，Wnt经典通路可调控ALDH1A1表达。据此提出“在结直肠癌肺转移中，盐酸小檗碱通过干预β-catenin，影响β-catenin/TCF-4复合体转录活性，进而调控ALDH1A1的表达，抑制结直肠癌肺部转移的发生。”的假说。为验证该假说，拟通过生物信息学、基因芯片、质粒构建与转染、染色质免疫共沉淀、免疫组化等分子生物学技术，明确盐酸小檗碱抑制结直肠癌肺转移的分子作用靶点，丰富中医整体观“肺与大肠相表里”科学内涵，为临床现有抗炎药物盐酸小檗碱“老药新用”治疗结直肠癌肺转移提供理论基础和实验依据。

结直肠癌（CRC）肺转移5年总生存率不足7%，预后极差，传统及现代医学对其均鲜有研究。结直肠癌转移性疾病的主要治疗方法是手术，但其益处有限。大量研究揭示了盐酸小檗碱对肠道疾病的改善包括对CRC的调控具有至关重要的作用，成为研究肠道疾病重要的天然药物，但盐酸小檗碱在结直肠癌肺转移中的作用仍不清楚。本研究旨在揭示盐酸小檗碱对CRC细胞二次归巢形成肺部转移灶的调节机制。本项目前期实验发现盐酸小檗碱能够有效地抑制结直肠癌各细胞株的增殖、迁移和转移，且盐酸小檗碱能够干预人CRC肺转移灶标本中跟转移相关蛋白的表达。基于此，本项目围绕盐酸小檗碱抑制CRC肺转移发生的转移相关通路及转移相关基因的作用靶点开展研究，研究发现：①在“肺与大肠相表里”传统医学理论的指导下，盐酸小檗碱不仅能够有效干预CRC细胞增殖侵袭，而且对CRC原发灶的生长和肺部转移癌的发生均有显著地抑制作用；②微阵列数据分析发现盐酸小檗碱干预后，CRC细胞株中差异表达基因（DEGs）与癌症进展和转移相关通路Wnt、Rap 1等通路有关；③盐酸小檗碱对CRC转移进展中Wnt通路的活性以及ALDH1A1的表达皆具有一定的调控作用；④盐酸小檗碱对癌症转移和进展相关通路（如Wnt、Hippo和Rap 1信号通路）具有负性调节作用，而HEY2是与癌症进展和转移中生存相关的唯一中枢基因，受上述Wnt、Hippo通路调控；⑤HEY2在CRC肺转移阶段显示高表达以及低生存率；⑥盐酸小檗碱降低CRC肺转移部位间充质上皮转化（MET）过程中HEY2、转移相关蛋白E-cadherin、β-catenin和Cyclin D1的表达；⑦盐酸小檗碱和转移相关蛋白HEY2表现出显著的相互作用，盐酸小檗碱在残基HIS-99界面以1.9A的氢键距离与HEY2蛋白结合。以上研究成果丰富了中医整体观“肺与大肠相表里”的科学内涵，为临床现有抗炎药物盐酸小檗碱“老药新用”治疗结直肠癌肺转移提供理论基础和实验依据。

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