盐霉素介导PAF/Wnt信号通路调控胃癌干细胞自噬的研究

Due to the tumor relapse after surgery resection and resistance to Chemotherapy drugs, prognosis of gastric cancer is very poor. Recently, more and more reports have demonstrated that cancer stem cell is closely associated with postoperative recurrence and drug resistance, and it would be a promising target for cancer treatment. It’s showed that salinomycin could specifically inhibit the growth and renewal of cancer stem cell, and PNCA-associated Factor(PAF)/Wnt signaling pathway is required for maintenance of Breast Cancer Cell stemness. In our previous studies, it’s found that salinomycin suppressed the proliferation of CD44 + MKN45 gastric cancer cell significantly with the trait of cancer stem cells. In our preliminary experiment, we also observed the negatively correlation between β-catenin and autophagy related gene beclin-1 in the specimen of patients with gastric cancer. In addition, salinomycin could trigger the enhancement of autophagy in CD44+ MKN45 cells, and the levels of PAF and β-catenin were decreased simultaneously. Consequently, we speculate that salinomycin would up-regulate the autophagy of gastric cancer stem-like cells, and PAF/Wnt signaling pathway may play a pivotal role in it. In this study, we will explore the correlation between β-catenin and beclin-1 in the gastric caner tissues, the effects of salinomycin on CD44+ MKN45 gastric cancer cell proliferation and autophagy in vitro and vivo experiments, and investigate the potential molecular mechanism by many techniques such as CRISPR/Cas9, Luciferase reporter gene and gene expression profile analysis. Ultimately, we would provide scientific and theoretical basis to the use of salinomycin for treatment of gastric cancer patients, and expand our knowledge about the function of PAF/Wnt signaling pathway and the mechanism of salinomycin.

术后复发和药物抗拒影响胃癌患者预后，肿瘤干细胞在其中起重要作用。新近报道盐霉素能特异性抑制肿瘤干细胞生长，PAF/Wnt是维持乳腺癌干细胞特性的必需信号通路。我们前期研究证实盐霉素对具备干细胞特性的CD44+ MKN45胃癌细胞有显著杀灭作用；预实验发现胃癌患者组织β-catenin和自噬基因Belcin-1呈负相关，盐霉素作用于CD44+ MKN45细胞后，细胞自噬增强，PAF和β-catenin表达下降。因此我们推测：盐霉素可能介导PAF/Wnt信号通路调控胃癌干细胞自噬活性。本项目拟在胃癌组织标本揭示PAF与自噬的关联，及其对患者生存的影响；体内外实验明确盐霉素对CD44+ MKN45细胞凋亡和自噬的作用，采用CRISPR/Cas9、荧光素报告基因和基因表达谱分析等技术初步探讨其调控自噬的分子机制，旨在拓宽我们对PAF/Wnt通路和盐霉素的认识，为盐霉素的临床应用提供理论基础和依据。

术后复发和药物抵抗影响胃癌患者的预后，肿瘤干细胞在其中起着重要的作用。既往研究报道盐霉素能特异性抑制肿瘤干细胞生长，PAF/Wnt是维持乳腺癌干细胞特性的重要信号通路。然而盐霉素对胃癌干细胞特性及自噬功能的影响及机制尚未阐明。本项目的研究结果发现，PAF及β-catenin在胃癌组织中的表达明显升高，且其高表达与肿瘤患者出现转移及预后差密切相关，而Beclin-1在胃癌组织中的表达则显著降低，其表达降低与患者肿瘤出现淋巴结转移、远处转移及预后差密切相关。经过流式细胞学成功筛选并培养CD44+MKN45胃癌干细胞，经顺铂组和盐霉素处理后的胃癌干细胞的增殖能力显著下降，细胞凋亡明显增多，PAF、β-catenin及Wnt下游通路靶基因cyclinD1、c-Myc的蛋白表达均显著降低，但Beclin-1的表达增多。此外，我们还发现高表达PAF后，Wnt通路下游靶基因cyclinD1、c-Myc的mRNA表达均显著上升，但β-catenin的mRNA未见明显改变，提示PAF对Wnt通路的调控是核心因子β-catenin的在转录后水平。以上结果提示盐霉素能通过调控PAF/Wnt信号通路活性，从而抑制细胞增殖、促进凋亡并增强CD44+MKN45胃癌干细胞的自噬活性。此外，构建裸鼠动物模型，发现干扰PAF能显著抑制胃癌细胞的体内生长能力，且干扰PAF后裸鼠瘤体内的Wnt信号通路的主要成分表达受到明显抑制，从体内角度进一步佐证了本项目的体外细胞实验的结论。综上，本相关的研究结果证实盐霉素可通过PAF/Wnt通路调控胃癌干细胞的自噬活性，揭示了PAF/Wnt信号通路新的调控机制，也为盐霉素的临床应用提供理论基础和依据。