青蒿琥酯诱导自噬下调Bmi1增强索拉菲尼抗肝癌效应的机制研究

Sorafenib remains the only approved systemic drug for patients with advanced hepatocellular carcinoma (HCC). However, sorafenib resistance has been implicated as a major challenge to anti-cancer therapy. The resistance was associated with complex changes in molecular pathways, cellular characteristics and microenvironment. Previously, we investigated artesunate could boost the anti-cancer effect of sorafenib by inducing autophagy and downregulating Bmi1. Here, based on our previous studies, we put forward the hypothesis that artesunate enhances sorafenib sensitivity in HCC by inhibiting Bmi1 via autophagy. In this proposed project, we aim to thoroughly investigate the mechanism underlying key targets in Bmi1-downstream that regulate artesunate sensitizaton to sorafenib by using Bmi1-overexpression HCC line, gene expression profile microarray, Chip-seq, Co-IP and promoter-luciferase reporter assays. Based on literatures, Human Transcriptome Array 2.0 and Agilent Human miRNA Array, we will screen potential molecules that are associated with autophagy and Bmi1 regulation, and use gene-knockdown technique or kinase inhibitors to reveal the mechanism for regulating Bmi1 by artesunate-induced autopahgy. Furthermore, the results in vitro will be verified by animal assay. The project will provide scientific evidence for designing and developing new strategy to sensitize sorafenib in HCC.

索拉非尼是治疗肝细胞癌（HCC）的有效靶向药物，但耐药问题亟待解决。耐药机制主要包括分子信号通路、细胞特性及肿瘤微环境的改变。我们前期研究发现青蒿琥酯可诱导自噬增强索拉非尼的抗肝癌效应，并下调Bmi1的水平。本研究根据前期积累，以Bmi1为靶点提出如下假说：青蒿琥酯通过诱导自噬抑制了Bmi1的表达增强了肝癌对索拉非尼的敏感性。本课题拟构建Bmi1稳定高表达的HCC细胞系，应用基因表达谱芯片、ChIP-seq、免疫共沉淀、启动子报告基因等方法明确青蒿琥酯增敏索拉非尼过程中Bmi1下游的关键分子作用机制。根据文献报道结合转录组芯片和非编码芯片技术，筛选出与自噬及调节Bmi1相关的分子，通过分子生物学技术明确青蒿琥酯诱导自噬调节Bmi1的机制。并通过动物实验系统验证体外实验结果，为寻找增敏索拉非尼的途径提供科学依据。

索拉非尼是治疗肝细胞癌的有效靶向药物，但是耐药问题亟待解决。青蒿琥酯可增强索拉非尼抗肝癌的效应。青蒿琥酯联合索拉非尼可以显著抑制肝癌细胞在裸鼠体内的成瘤能力。青蒿琥酯联合索拉非尼除了自噬还可引起凋亡途径的主要蛋白表达出现显著的变化, 提示青蒿琥酯联合索拉非尼主要通过诱导凋亡以抑制肝癌细胞的增殖。进一步进行分子机制研究发现,联合用药主要通过mTOR和ERK通路抑制肝癌细胞的增殖。lncRNA芯片结果显示青蒿琥酯引起了565个lncRNA表达上调，1105个lncRNA表达下调。生信分析显示EMT相关的MAPK,TGFβ信号通路发生显著变化。通过CNC分析lncRNA与肝癌细胞EMT的相关性，发现RP11与肝癌细胞EMT相关。进一步研究发现青蒿琥酯可通过RP11抑制肝癌细胞EMT。此项研究为后续青蒿琥酯抑制肿瘤及增敏化疗效果的研究提供大量研究基础，有望在将来应用于临床治疗之中。