外泌体circ-FOXK2介导细胞异常自噬促进胆囊癌化疗耐药机制研究

Gallbladder cancer（GBC） is an aggressive neoplasm and presents a poor prognosis in patients, due to the limited options of surgery and chemotherapy resistance for advanced GBC. Thus, how to enhance chemosensitivity in GBC patients is urgently needed. In the present study, by RNA sequencing data from the exosomes of Gemcitabine(GEM) resistant cell line and control cell line, we characterized circular RNA-FOXK2 (circ-FOXK2) was significantly upregulated in the resistant cell line, compared with the control cell line. Furthermore, we found that circ-FOXK2/FOXO1/ATG7 and miR-130a/ATG12 signaling pathways may lead to upregulated cell autophagy and then enhanced cell chemoresistance in GBC. Therefore, the aim of this study is to investigate associations between exosome circ-FOXK2, cell autophagy and chemotherapy resistance for GEM by multiple aspects of molecules, cells, animals and combined with the Northern-blot assay, Fluorescence in situ hybridization (FISH), dual-luciferase report assay, RNA Binding Protein Immunoprecipitation (RIP), RNA-pull down and CRISPR/Cas9 assay for constructing nude mice tumor formation model. Finally, we attempt to demonstrated that (1) Exocrine secretion was association with cell chemotherapy resistance; (2) Activation of circ-FOXK2/FOXO1/ATG7 and miR-130a/ATG12 signaling pathways lead to up-regulated cell autophagy and enhanced cell chemoresistance; (3) Inhibition of circ-FOXK2 could reverse chemotherapy resistance in GBC by suppressing the cell autophagy. Thus,these results provide a novel molecular target for reversing chemotherapy resistance of GBC patients.

胆囊癌手术根治率低，化疗极易耐药，预后差，逆转耐药亟待解决。我们前期利用高通量测序发现circRNA-FOXK2在吉西他滨耐药株较对照株外泌体中高表达;通过部分实验结果提出假说：circRNA-FOXK2可能通过FOXO1/ATG7及miR-130a/ATG12两条信号轴促进细胞自噬升高进而诱导化疗耐药。本课题拟从分子、细胞、动物等多层次并运用Northern-blot实验、荧光原位杂交技术、双荧光霉素报告基因、RNA免疫共沉淀,RNA-pull down及CRISPR/Cas9基因敲除技术建立动物模型等手段以期明确：（1）外泌体分泌参与胆囊癌化疗耐药过程；（2）外泌体中circRNA-FOXK2调控细胞异常自噬的分子机制；（3）抑制circRNA-FOXK2可下调细胞自噬水平进而逆转化疗耐药。阐明外泌体环状RNA、细胞自噬、化疗耐药三者之间的调控关系，为逆转胆囊癌化疗耐药提供分子靶点。

胆囊癌(GBC)占全世界胆道系统恶性肿瘤的85-90%，众多的证据表明lncRNAs的失调参与了肿瘤的发生发展。LncRNA PVT1已有报道在各种肿瘤中发挥重要作用，但其在胆囊癌中的作用机制尚不明确。在该研究中，我们发现PVT1在GBC组织和细胞中表达均明显上调，且其上调与GBC患者不良预后相关。PVT1促进GBC细胞在体内、外的增殖。在机制上，PVT1通过EZH2将DNMT1招募到miR-18b-5p DNA启动子，并通过DNA甲基化抑制miR-18b-5p的转录 。此外，HIF1A被证实是miR-18b-5p和PVT1调控GBC细胞增殖的下游靶基因。总之，我们的研究阐明了PVT1/miR-18b-5p/HIF1A调节轴参与胆囊癌的调控，并表明PVT1可能是GBC潜在的治疗靶点。