肿瘤相关成纤维细胞通过外泌体传递circRNAs调控三阴性乳腺癌进展转移及化疗耐药的机制研究

Breast cancer is a heterogeneous disease and triple negative breast cancer (TNBC) is characterized by unfavorable prognosis. Chemoresistance and metastasis are the main factors influencing prognosis in TNBC. Recent studies have revealed that tumor microenvironment plays important roles in the biological behaviors of tumor. Carcinoma-associated fibroblast (CAF) is an important component of tumor microenvironment. However, whether CAF could influence chemoresistance and metastasis in TNBC through exosome by delivering circular RNA (circRNA) remains unknown. To screen exosomal circRNAs modulating chemoresistance and metastasis in TNBC, we firstly established the CAF cell lines derived from TNBC primary tissue with or without metastasis (namely, CAF-M and CAF-N). With RNA-seq high-throughput sequencing analysis, three candidate circRNAs were identified for further study. We explored their roles in chemoresistance and metastasis both in vivo and in vitro and also in clinical breast cancer samples, and we further explored the underlying molecular mechanisms. This is the first study to investigate the circRNA expression in the exosome of CAF, illustrating their function and underlying mechanisms in chemoresistance and metastasis in TNBC. Our study may provide important markers for the prognostic evaluation of TNBC, and may also lay the foundation for the therapeutic strategy of targeting CAF in TNBC.

乳腺癌是一个异质性的疾病，三阴性乳腺癌是预后较差的分子亚型，化疗耐药和转移是影响预后的主要因素。近来研究表明肿瘤微环境影响着肿瘤的生物学行为，CAF作为肿瘤微环境的主要组成成分，能否通过外泌体传递circRNA调控三阴性乳腺癌转移及化疗耐药有待深入探讨。为了筛选三阴性乳腺癌CAF外泌体中有潜在功能的circRNA，我们构建了有无转移的三阴性乳腺癌CAF细胞系CAF-M和CAF-N，利用RNA-seq高通量测序筛选出3个circRNAs进行深入研究。从体外、体内实验和临床标本分析三个层面探讨候选circRNAs对三阴性乳腺癌化疗耐药和转移的影响，并明确其分子机制。本课题首次分析了CAF外泌体中circRNA的表达，从肿瘤微环境角度揭示CAF外泌体circRNA影响三阴性乳腺癌化疗耐药和转移的分子机制，为三阴性乳腺癌的预后提供重要的分子标志物，也将为靶向三阴性乳腺癌CAF的新型治疗奠定基础。

乳腺癌是全球女性最常见的恶性肿瘤之一，化疗耐药和转移是影响预后的主要因素。有报道肿瘤微环境中CAFs可通过外泌体介导的细胞通讯在肿瘤进展转移中发挥重要作用。然而，来自CAFs的外泌体环状RNAs（circRNAs）在乳腺癌进展转移中的作用和机制仍不明确。为筛选具有潜在功能的CAFs外泌体circRNAs，我们培养了有无转移的三阴性乳腺癌CAFs细胞系CAF-M和CAF-N，利用RNA-seq高通量测序筛选出4个circRNAs作为研究目标。通过体内外功能实验、临床样本分析及分子机制研究，揭示了候选circRNAs对三阴性乳腺癌进展转移的影响。首先功能实验和动物实验结果显示，候选circRNAs能够促进三阴性乳腺癌细胞的增殖和转移。CAFs外泌体通过递送circTBPL1促进三阴性乳腺癌的增殖和转移。接下来在机制方面，通过蛋白质谱分析及co-IP、RNA pull-down实验的分析验证，我们确定circEIF3H作为支架促进IGF2BP2和HuR的结合，并影响下游靶基因HSPD1/RBM8A/G3BP1 mRNA的稳定性；通过生物信息学分析结合双荧光素酶实验、qRT-PCR等实验的分析验证，我们确定了circHIF1A 和circTBPL1作为miRNA海绵分别与miR-149-5p和miR-653-5p结合并影响下游靶基因的表达；通过蛋白质谱分析及co-IP 、RNA pull-down实验的分析验证我们确定circ5081与IGF2BP2结合，circ5081过表达可以增加IGF2BP2下游靶分子c-myc和FSCN1的表达。最后我们通过临床样本分析发现circEIF3H、circHIF1A和circ5081在乳腺癌组织的表达水平明显高于正常组织，并且在三阴性乳腺癌组织中的表达水平高于其他分子类型并影响患者的预后。综上所述，我们的结果充分表明，候选circRNAs在乳腺癌中发挥促癌作用，能够增强乳腺癌的进展转移，并且与肿瘤预后密切相关，circRNAs可能成为乳腺癌新的预后预测标志物及治疗的潜在靶点。