CircFOXP1通过介导异常糖酵解过程促进胆囊癌化疗耐药机制研究

Gallbladder cancer (GBC) is most common malignancy of the biliary tract. Surgery is the only curative treatment for patients who are diagnosed at early stage. Chemoresistance is remarkable characteristic of GBC. Patients who are diagnosed at advanced stage present highly lethal with less than 5% overall 5-year survival rate. In present study, we performed RNA sequencing from gemcitabine-resistant GBC cells (NOZ/GEM) and corresponding controls (NOZ/Ctr) to analyze relative circRNA candidates. Our results show that circFOXP1 expression is significantly upregulated in NOZ/GEM cells compared with the NOZ/Ctr cells. By performing functional assays, we propose a hypothesis that circFOXP1 interacts with FOXM1 to activate HK2 and LDHA transcription, which promotes cell glycolysis capacity (termed Warburg effect) and subsequently lead to chemotherapy resistance of GBC cells. By performing fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down assays, Dual luciferase assay and orthotopic xenograft model, we try to prove that the glycolysis capacity in NOZ/GEM cells is significantly increased compared to NOZ/Ctr cells; upregulation of circFOXP1 significantly enhances the glycolysis capacity in NOZ/GEM cells; downregulation of circFOXP1 reverses the gemcitabine resistance of GBC cells via reducing the glycolysis capacity in NOZ/GEM cells. Taken together, we reveal the significant association among circFOXP1, cells glycolysis, and gemcitabine resistance in GBC, which provide some valuable strategies for GBC treatment.

胆囊癌手术根治率低，对化疗较易耐药，预后极差。本课题利用高通量测序技术发现环状RNAcircFOXP1在胆囊癌吉西他滨耐药株较对照株中表达显著升高，通过部分前期实验结果，我们提出假说：circFOXP1可通过与FOXM1蛋白结合上调己糖激酶2(HK2)及乳酸脱氢酶(LDHA)表达促进耐药细胞糖酵解水平进而诱导化疗耐药。本课题拟从分子、细胞及小动物等多层次并运用荧光原位杂交（FISH），RNA免疫共沉淀技术、RNA-pull down技术、双荧光素酶报告基因及小鼠原位胆囊癌模型等手段以期明确：1）胆囊癌吉西他滨耐药株较对照株细胞糖酵解能力显著增强；2）circFOXP1表达升高促进耐药细胞糖酵解能力；3）下调circFOXP1表达可抑制耐药细胞糖酵解水平进而逆转化疗耐药。由此，阐明circFOXP1、细胞异常糖酵解水平及化疗耐药三者的调控关系，为临床逆转胆囊癌化疗耐药提供新的研究线索。

胆囊癌手术根治率低，对化疗较易耐药，预后极差。本课题利用高通量测序技术发现环状RNAcircFOXP1在胆囊癌吉西他滨耐药株较对照株中表达显著升高，通过实验结果证实circFOXP1在胆囊癌耐药株较正常细胞株中表达升高，进一步通过体外细胞实验证实耐药株细胞升高的circFOXP1水平促进细胞增殖及糖酵解过程，机制研究发现circFOXP1可通过与FOXM1蛋白结合上调己糖激酶2(HK2)及乳酸脱氢酶(LDHA)表达促进耐药细胞糖酵解水平。此外，我们通过体内胆囊癌耐药株细胞小鼠原位胆囊癌模型证实敲除circFOXP1能够抑制肿瘤增殖及糖酵解过程。由此，阐明circFOXP1能够通过调控FOXM1促进糖酵解关键酶己糖激酶2(HK2)及乳酸脱氢酶(LDHA)表达，为临床逆转胆囊癌化疗耐药提供新的研究线索。