肿瘤相关成纤维细胞来源的外泌体中miR-32-5p促进胆囊癌吉西他滨耐药的作用及机制研究

Cancer-associated fibroblasts (CAFs)-derived exosomes play important roles in tumor chemoresistance. However, the roles and underlying mechanisms of CAFs-derived exosomes with respect to gemcitabine resistance of gallbladder cancer (GBC) are elusive. Our previous study found that miR-32-5p was highly expressed in CAFs-derived exosomes and could promote gemcitabine resistance in GBC cells. In addition, miR-32-5p could targetedly regulate the expression of KLF2 that was reported to be related with glycolysis. Upregulation of glycolysis could enhance tumor chemoresistance. Thus, we hypothesized that CAFs-derived exosomes could transmit miR-32-5p into GBC cells, which could downregulate the expression of KLF2 and then enhance glycolysis levels of GBC cells, eventually leading to gemcitabine resistance. Therefore, the objective of this research is to clarify the mechanism of CAFs-secreted exosomal miR-32-5p in gemcitabine resistance of GBC cells based on experiments in vitro and in vivo, which will help to explore the possibility of miR-32-5p as a novel effective target of gemcitabine resistance in GBC cells, and provide a new theoretical basis for the diagnosis and treatment of GBC.

肿瘤相关成纤维细胞（CAFs）来源的外泌体在肿瘤耐药中发挥重要作用，但其在胆囊癌吉西他滨耐药中的作用和机制尚不清楚。我们前期研究发现miR-32-5p在CAFs源性外泌体中显著高表达，且miR-32-5p可促进胆囊癌细胞对吉西他滨的耐药性；另外，研究还发现miR-32-5p可靶向调控KLF2的表达，KLF2已被报道与细胞内的糖酵解过程有关，而糖酵解途径的增强可诱导或者促进肿瘤细胞耐药。因此，我们推测CAFs源性外泌体通过将miR-32-5p传递给胆囊癌细胞，靶向调控KLF2的表达，增强胆囊癌细胞的糖酵解水平，从而促进吉西他滨耐药。因此，本项目拟从体外和体内实验探究CAFs源性外泌体中miR-32-5p在胆囊癌吉西他滨耐药中的作用及机制，从而明确miR-32-5p作为胆囊癌吉西他滨耐药新靶点的临床意义，为胆囊癌的诊断和治疗提供新的理论依据。

胆囊癌是胆道系统最常见的恶性肿瘤，其恶性度极高，早期诊断困难，对放化疗不敏感，患者的预后很差。肿瘤微环境中的肿瘤相关成纤维细胞（CAFs）在肿瘤耐药中扮演着重要角色。近些年来，CAFs来源的外泌体在肿瘤耐药中作用受到越来越多的关注，其在胆囊癌吉西他滨耐药中的作用和机制尚不清楚。我们课题组从胆囊癌组织中的CAFs提取其中的外泌体，并证明CAFs源性的外泌体对吉西他滨耐药的促进作用。然后我们通过转录组芯片筛选出CAFs源性外泌体中差异性表达上调的分子miR-32-5p。通过敲减和过表达外泌体中的miR-32-5p，证实miR-32-5p对胆囊癌细胞吉西他滨耐药的促进作用。为了深入探究miR-32-5p参与吉西他滨耐药的机制，我们通过生物信息分析、荧光素酶报告实验、Western Blot和RT-PCR实验证明miR-32-5p对KLF2的表达的直接调控作用，并且通过回复实验验证miR-32-5p通过调控KLF2进而影响吉西他滨的耐药。鉴于KLF2与糖酵解过程的研究报道以及糖酵解与肿瘤细胞化疗耐药的关系，我们通过Seahorse实验验证miR-32-5p增强胆囊癌细胞的糖酵解水平，从而促进吉西他滨耐药。本项目证实CAFs源性的外泌体对胆囊癌吉西他滨耐药的促进作用，以及发现了一个胆囊癌吉西他滨耐药的新的靶点miR-32-5p，为胆囊癌的诊断和治疗提供新的方向。