半枝莲靶向调控miR-17逆转大肠癌干细胞EMT的机制研究

Cancer stem cells (CSCs) is strongly associated with the tumor recurrence and metastasis. Epithelial-to-mesenchymal transition(EMT) is based to tumorigenicity, invasion and metastasis of CSCs. miR-17, one of specific miRNAs in colorectal cancer stem cells (CRC-SCs), regulates the EMT via regulation of Wnt/β-catenin pathway. Our previous studies suggest that Scutellaria barbata D. Don (SB) inhibited the growth，increasd the expression of EMT marker protein E-cadherin and inhibited the expression of N-cadherin and β-catenin in CRC-SCs. So, SB may exert its function of inhibiting tumorigenicity, invasion and metastasis via reversing EMT by targeting miR-17 in colorectal cancer stem cells. In present project, we will study SB on the invasion, metastasis, tumorigenicity, miR-17 and its target genes in CRC-SCs by using transwell, in vivo imaging system, Q-PCR and Western-blotting. And finding the target of SB by cell transfection with miR-17 inhibitor. Therefore, we will further explore the mechanism and the target of SB treats colorectal cancer by reversing EMT, providing theoretic foundation for clinical application of SB.

肿瘤干细胞(CSCs)是导致肿瘤转移和复发的重要因素。上皮间质转化（EMT）是CSCs获得起始致瘤性、高侵袭及转移性的前提基础。miR-17可通过Wnt/β-catenin通路调控大肠癌干细胞（CR-CSCs）的EMT。预实验发现半枝莲可抑制CR-CSCs生长，促进EMT标记蛋白E-cadherin，抑制N-cadherin及β-catenin的表达。提示：靶向调控miR-17逆转EMT可能是半枝莲抑制CR-CSCs致瘤性、高侵袭、转移性的重要途径。故本项目在此基础上，采用迁移，侵袭、小动物活体成像系统、Q-PCR、Western blot等实验探讨半枝莲对CR-CSCs迁移、侵袭、致瘤、miR-17及靶基因的影响，并通过转染miR-17 inhibitor验证半枝莲的作用靶点。将从表观遗传学水平阐明半枝莲可作为EMT逆转剂治疗大肠癌的作用机制和靶点，为半枝莲临床抗癌应用提供实验依据。

肿瘤干细胞是导致肿瘤转移和复发的重要因素。上皮间质转化（EMT）是肿瘤干细胞获得起始致瘤性、高侵袭及转移性的前提基础。miR-17-5p及Wnt/β-catenin通路具有调控大肠癌干细胞生长、EMT及转移的作用。半枝莲是对肿瘤治疗有明确疗效的中药之一，为了进一步阐明其抑制肿瘤生长的作用机制，我们以肿瘤干细胞为切入点，采用CCK-8、克隆球形成实验、Transwell、划痕实验、小动物活体成像系统、Q-PCR及Western-blotting等实验技术观察半枝莲对大肠癌干细胞增殖、克隆球形成、迁移、EMT、瘤体生长及转移的影响，及对miR-17-5p和Wnt/β-catenin通路活化的调控作用及作用机制。体外实验结果表明，半枝莲可显著抑制大肠癌干细胞增殖、克隆球形成数量、迁移能力及miR-17-5p的表达，并具有调控E-cadherin，N-cadherin、Vimentin、β-catenin及Twist等基因表达的作用，且显著下调β-catenin的核内转移；体内实验结果表明，半枝莲明显抑制大肠癌干细胞的瘤体生长及转移，并具有调控E-cadherin、Vimentin、β-catenin及Twist等基因表达的作用。另外，转染miR-17-5p mimic后，大肠癌干细胞的增殖能力、克隆球形成及迁移能力明显增加，N-cadherin、β-catenin及Twist蛋白表达显著增高；且半枝莲可以逆转这些功能并下调N-cadherin、β-catenin及Twist蛋白的表达。综上所述，半枝莲具有抑制大肠癌干细胞增殖、克隆球形成、迁移及EMT的作用，通过靶向调控miR-17-5p的表达和Wnt/β-catenin信号通路的活化是其发挥作用的重要机制。研究结果为半枝莲在临床治疗肿瘤提供了实验依据，并为大肠癌的临床治疗提供新的方向和思路。