基于TGF-β/LncRNA-ATB/miR-200a通路研究片仔癀抑制大肠癌转移的作用机制

Tumor metastasis is one of the major reasons for the failure in the clinical treatment of colorectal cancer (CRC). TGF-β-mediated aberrant expression of LncRNA-ATB and miR200a is strongly associated with CRC metastasis. Pien Tze Huang (PZH), a well-known traditional Chinese formula prescribed 450 years ago in the Ming Dynasty, has been demonstrated to be clinically effective in the treatment of colorectal cancer. However, the underlying mechanism of its anti-cancer activity remains to be further elucidated. Our previous studies found that PZH treatment could significantly inhibit CRC metastasis and modulate the expression of TGF-β and miR-200a, suggesting that PZH may exert its anti-metastasis function via affecting the TGF-β/LncRNA-ATB/miR-200a regulatory network. To further investigate the precise mechanism of PZH’s anti-cancer activity, in present project we will evaluate the in vivo and in vitro inhibitory effect of PZH on CRC metastasis by IVIS Spectrum Imaging System and transwell assay. In addition, we will determine PZH’s effects on the activation of TGF-β/lncRNA-ATB/miR-200a signaling pathway as well as the expression of target genes. Our findings in this project will further elucidate the mechanism whereby PZH treats colorectal cancer.

肿瘤转移是大肠癌临床治疗失败的最主要原因；TGF-β介导的LncRNA-ATB和miR-200a表达异常与肿瘤转移密切相关。片仔癀治疗大肠癌疗效确切，但作用机制尚未完全阐明。我们前期研究发现片仔癀可抑制大肠癌转移并显著改变TGF-β和miR-200a的表达，提示“调控TGF-β/LncRNA-ATB/miR-200a通路”可能是其抑制大肠癌转移的重要机制。本课题拟通过接种荧光素酶标记稳转细胞株制备结肠原位移植瘤动物模型，采用TGF-β刺激、LncRNA-ATB高表达质粒转染构建高转移大肠癌细胞模型。运用小动物活体成像系统、Transwell、qPCR和Western-Blot等体内外实验研究片仔癀对大肠癌转移的抑制作用和对TGF-β/LncRNA-ATB/miR-200a通路的影响。研究结果将有助于从表观遗传学水平深入阐明片仔癀治疗大肠癌的分子机制和作用靶点，为其临床抗癌应用提供实验依据。

目的：探讨片仔癀干预对大肠癌细胞原位移植瘤转移和体外迁移和侵袭的影响，并探讨其对TGF-β/LncRNA-ATB/miR-200A通路的调控作用。.方法：体外培养大肠癌细胞，并在有、无TGF-β刺激条件下，采用划痕实验、Transwell实验检测片仔癀干预对大肠癌细胞迁移和侵袭的影响。构建荧光素酶标记的HCT116细胞，通过原位种植构建大肠癌移植瘤模型并给予片仔癀干预，通过小动物活体成像、HE染色等评价片仔癀对大肠癌体内转移的影响。通过QPCR、Western-blot等实验检测片仔癀干预对大肠癌细胞、原位移植瘤中TGF-β及其下游关键调节因子在RNA和蛋白水平的表达。.结果：片仔癀干预显著抑制HCT-8和HCT116细胞迁移、侵袭能力，同时下调HCT-8细胞TGF-β、ZEB1、N-cadherin和上调E-cadherin在蛋白水平的表达。体内实验发现片仔癀干预显著降低HCT116细胞体内转移，下调移植瘤TGF-β、ZEB1、N-cadherin和上调E-cadherin在mRNA和蛋白水平的表达,同时还具有下调LncRNA-ATB和上调miR-200A表达的作用。TGF-β刺激具有促进HCT116细胞迁移、侵袭和上调ZEB1、N-cadherin、LncRNA-ATB表达和下调miR-200A的作用；相反，片仔癀干预能够显著逆转上述改变。.结论：片仔癀干预通过抑制TGF-β/LncRNA-ATB/miR-200A通路及其下游转移相关调节因子表达抑制大肠癌细胞体内外转移。