从miR-200c介导的Notch信号通路研究增免抑瘤方及有效组分抑制卵巢癌干细胞侵袭转移的作用机制

ZMYL is used for the treatment of ovarian cancer in clinical practice and has obtained the satisfactory curative effect, but the mechanism and the specific pathway are still unclear. The existence of ovarian cancer stem cells (OCSCs) is the root cause of recurrence、metastasis and chemotherapy drug resistance of ovarian cancer, in the process of invasion and metastasis of ovarian cancer stem cells occurred epithelial-mesenchymal transition (EMT). MiRNA-200c can mediate the Notch signaling pathway which plays an important role in cell proliferation, invasion, metastasis and other biological behavior. Our previous study found ZMYL can reverse EMT and inhibit the proliferation and metastasis of OCSCs. So we peculated that ZMYL may regulate the invasion and metastasis of OCSCs through Notch signaling pathways to inhibit the progress of ovarian cancer. This project is proposed on the basis of previous study, culturing ovarian cancer cell lines SKOV3 and cancer stem cells in vitro, building a high expression of miR-200c ovarian cancer stem cells, to explore the molecular mechanisms of ZMYL and the effective components inhibiting the invasion and metastasis of OCSCs through Notch pathway mediated by miR-200c, and finally verified in vivo, to provide new scientific and theoretical basis for ovarian cancer clinical treatment by ZMYL.

增免抑瘤方（ZMYL）为治疗卵巢癌的经验方，用于临床治疗取得了满意疗效，但该方治疗卵巢癌具体作用途径与机理尚不清楚。卵巢癌干细胞（OCSCs）的存在是卵巢癌复发转移及化疗耐药的根源，卵巢癌干细胞侵袭转移的过程中发生了上皮间转化（EMT）。miR-200c介导的Notch信号通路在细胞的增殖、侵袭、转移等多种生物学行为中发挥重要作用。我们前期研究发现ZMYL可以逆转OCSCs发生EMT并抑制卵巢癌干细胞的增殖和转移。推测ZMYL可能是通过Notch信号通路调控OCSCs侵袭和转移达到抑制卵巢癌的进展。本课题拟在既往研究的基础上，体外培养卵巢癌细胞株SKOV3和肿瘤干细胞，构建高表达miR-200c的卵巢癌干细胞，探讨ZMYL及有效组分通过miR-200c介导的Notch通路抑制OCSCs侵袭和转移的分子机制，最后进行动物体内验证，为ZMYL临床治疗卵巢癌提供新的科学依据和理论基础。

卵巢癌的死亡率高居妇科癌症之首，严重威胁女性的健康和生命。卵巢癌干细胞（OCSCs）的存在是卵巢癌复发转移及化疗耐药的根源，OCSCs侵袭转移的过程中发生了上皮间质转化。增免抑瘤方为治疗卵巢癌的经验方，用于临床治疗取得了满意疗效。本课题在原有研究基础上，通过无血清悬浮培养法富集OCSCs，建立裸鼠OCSCs移植瘤模型，观察增免抑瘤方及有效组分（PAWU）抑制卵巢癌细胞和肿瘤干细胞的侵袭转移作用并探讨可能的机制。研究结果显示：卵巢癌SKOV3细胞悬浮培养7d可形成三维球样OCSCs，同时高表达0ct-4、Nanog、SOX2等干性标志基因。增免抑瘤方四个有效组分复方可以显著抑制OCSCs的增殖、侵袭和转移能力，下调Snail、Slug、Vimentin、N-cadherin和Fibronectin mRNA的表达，提高E-cadherin mRNA的表达。利用细胞转染技术转染Hsa-miR-200c-inhibitor，进一步证实了PAWU可以提高miR-200c、E-cadherin的表达，下调Notch信号通路Notch1、Jagged1、Hes1基因和蛋白的表达水平。并在体内OCSCs移植瘤模型实验中进行了验证，增免抑瘤方及有效组分可以抑制卵巢癌干细胞裸鼠移植瘤的生长，下调Ki67、PCNA和Notch通路关键蛋白的表达，PAWU与顺铂联合应用有显著的协同增效作用。提示增免抑瘤方抗卵巢癌作用机制可能是通过上调miR-200c的表达，抑制Notch/Jagged1信号通路的激活，从而抑制卵巢癌细胞和OCSCs的侵袭和转移，为临床治疗提供科学依据。