从lncRNA-ANRIL/VEGF-C的调控探讨片仔癀抑制大肠癌淋巴管新生的作用机制

Lymphangiogenesis is an important factor in metastasis, recurrence and death of colorectal cancer (CRC). Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in the treatment of colorectal cancer. In previous study, we found that PZH could significantly inhibit the lymphangiogenesis and metastasis of CRC, down-regulate the expression of VEGF-C in CRC cells, and inhibit the migration and tube formation of human lymphatic endothelial cells (HLECs). However, the underlying mechanism of its anti-cancer activity remains to be further elucidated. Recent studies have shown that the abnormal regulation of long non-coding RNA (lncRNA)-ANRIL/VEGF-C is closely associated with lymphangiogenesis in colorectal cancer. Therefore, to further elucidate the mechanism of PZH treatment of CRC, in this project, an orthotopic xenograft model will be set up by CRC cells stably transfected luciferase reporter gene, and the HLECs or CRC cells will be uesd as the objects in vivo and in vitro, to study the mechanism of the inhibition of PZH on the lymphangiogenesis of CRC via regulating the lncRNA ANRIL/VEGF-C pathway by using IVIS Spectrum Imaging System, IHC, Transwell, tube formation, RT-qPCR, Northern Blot and Western Blot. Our findings in present project will further elucidate the mechanism whereby PZH treats CRC, and to provide reference for similar research of traditional Chinese medicine.

淋巴管新生所致的淋巴转移是大肠癌转移、复发及患者死亡的重要因素。片仔癀治疗大肠癌临床疗效确切，我们研究发现片仔癀可显著抑制大肠癌的淋巴管新生及转移，抑制大肠癌细胞VEGF-C的表达及淋巴管内皮细胞的迁移和淋巴管腔形成等，但其作用机制有待进一步阐明。最新研究表明，长链非编码RNA（lncRNA）-ANRIL/VEGF-C的调控异常与大肠癌淋巴管新生密切相关。故本课题拟在此基础上，以稳转荧光素酶标记的大肠癌细胞原位移植瘤动物模型及淋巴管内皮细胞、大肠癌细胞为研究对象，运用小动物活体成像、IHC、Transwell、管腔形成、RT-qPCR、Northern Blot、Western Blot等研究技术，从lncRNA-ANRIL/VEGF-C的调控通路探讨片仔癀抑制大肠癌淋巴管新生的作用机制，以期进一步阐明片仔癀治疗大肠癌的作用机制，为片仔癀临床抗癌应用提供实验依据，为中医药同类研究提供借鉴。

淋巴管新生所致的淋巴转移是大肠癌转移、复发及患者死亡的重要因素。片仔癀治疗大肠癌临床疗效确切，可显著抑制大肠癌的淋巴管新生及转移，抑制大肠癌细胞VEGF-C的表达及淋巴管内皮细胞的迁移和淋巴管腔形成等，但具体药效机制尚未阐明。本课题基于lncRNA-ANRIL/VEGF-C调控轴，稳转荧光素酶标记的大肠癌细胞原位移植瘤动物模型及淋巴管内皮细胞、大肠癌细胞为研究对象，运用小动物活体成像、IHC、Transwell、管腔形成、RT-qPCR、Northern Blot、Western Blot等研究技术，以明确片仔癀抑制大肠癌的关键药效作用机制。. 研究我们发现片仔癀可抑制瘤体体积和重量，显著抑制 LYVE-1、VEGF-C、VEGFR-3及 LncRNA ANRIL的基因表达，抑制原位移植瘤体积增大以及肿瘤细胞肝组织浸润。此外，我们还发现在体外，片仔癀可以抑制VEGF-C诱导的淋巴管内皮细胞HELC和大肠癌细胞HCT116的细胞活力和迁移能力，抑制HELC细胞的管腔形成和集落形成的能力，并且抑制了相关蛋白VEGF-C、VEGFR3、Bcl-2、Bax、Cyclin D1、CDK4、P21、MMP-2、MMP-9的表达和PI3K、AKT、ERK1/2、JNK1/2的活化，抑制了ANRIL的表达。. 综上，通过本课题基于中医药理论指导，以符合大肠癌的中医治则的传统名贵中药片仔癀作为药物研究对象，以研究前沿的长链非编码RNA（lncRNA）和肿瘤淋巴管新生为切入点，分析lncRNA-ANRIL/VEGF-C的表达调控及其介导促淋巴管新生相关信号通路的活化与相关因子的表达，从lncRNA表观遗传学调控、VEGF-C表达及其介导的多条促淋巴管新生通路调控等多个环节和多个层面系统地阐明了片仔癀抑制大肠癌淋巴管新生的关键作用机制。