片仔癀调控miRNA逆转大肠癌多药耐药的作用机制研究

Deregulation of miRNA in cancer cells is strongly associated with the multi-drug resistance (MDR) of colorectal cancer, which therefore is one of the major reasons for the failure in the clinical treatment of colorectal cancer. 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 mechamism of its anti-cancer activity remains largely unclear. Our previous studies suggest that, as a multi-component and multi-target agent, PZH may exert its anti-cancer function via affecting the miRNA in cancer cells leading to the reversal of drug resistance. To further investigate the precise mechanism of PZH’s anti-tumor activity, in present project we will generate colorectal cancer MDR cells, and analyze miRNA expression profile in MDR cancer cells. In addition, we will study the in vitro effects of PZH on the expression of those specifically expressed miRNAs. Our findings in this project will provide new insight into the correlation between miRNA regulation and (MDR) of colorectal cancer, as well as fully elucidate the mechanism of action of how PZH treats colorectal cancer.

肿瘤多药耐药是目前临床大肠癌化疗失败的主要原因之一；大肠癌多药耐药的产生与肿瘤细胞的miRNA调控异常密切相关。临床及基础研究均表明片仔癀治疗大肠癌疗效确切，但作用机制尚未完全阐明。我们的前期研究提示“调控miRNA及其相关信号转导通路而逆转多药耐药” 可能是片仔癀治疗大肠癌的重要机制。本课题在此基础上拟构建人大肠癌多药耐药细胞模型，采用组学技术检测大肠癌耐药细胞中miRNA的差异性表达；进一步研究片仔癀对大肠癌多药耐药的逆转作用，并运用荧光定量PCR技术探讨片仔癀对耐药相关miRNA表达的影响。研究结果将有助于进一步明确miRNA调控与大肠癌耐药的关系；深入而系统地阐明片仔癀治疗大肠癌的作用机制，以期为片仔癀临床抗癌应用提供理论根据；并为中药复方逆转肿瘤多药耐药研究提供新的思路。

为进一步探讨片仔癀对大肠癌多药耐药的逆转作用及其机制，本研究通过MTT法检测5-FU（5氟尿嘧啶）、ADM（阿霉素）和片仔癀等抑制HCT-8/5-FU和HCT-8细胞活力的IC50值，计算HCT-8/5-FU细胞的耐药指数(RI)验证其耐药性。结果显示，HCT-8/5-FU细胞对5-FU和ADM的耐药指数（RI）分别为＞34.59和4.22（RI＞1.5即认为对该药耐药），表明HCT-8/5-FU细胞具有多药耐药性。而片仔癀干预后对HCT-8/5-FU和HCT-8细胞的IC50值未见显著性差异，即HCT-8/5-FU细胞对片仔癀无耐药性（RI<1.5）；片仔癀分别联合5-FU和ADM干预HCT-8/5-FU细胞，探讨片仔癀的逆转作用，结果显示片仔癀具有明显的逆转HCT-8/5-FU耐药性的作用，其逆转指数（RE）分别为＞1.96和1.51（RE＞1.5即认为具有逆转作用）。miRNA表达谱芯片检测结果表明，与HCT-8细胞相比，在HCT-8/5-FU细胞中有miR-483等7个miRNA显著上调，miR-494-3p、miR-5787等34个miRNA发生下调（大于2倍）；Q-PCR检测结果表明miR-494-3p、miR-5787在HCT-8/5-FU细胞中显著下调，且片仔癀干预能够显著上调二者的表达。研究结果证实，通过调控miR-494-3p、miR-5787等多个miRNA的表达可能是片仔癀逆转大肠癌多药耐药的内在机制。在本基金项目的资助下，课题组已发表SCI源论文3篇。