基于外泌体的iRGD-Exos-rMETase生物纳米囊泡在化疗诱导的胃癌干细胞富集及耐药中的干预作用及机制研究

It is an important reason that tumor cells acquired multidrug resistance by obtaining the characteristics of the cancer stem cells through epithelial mesenchymal transition in the resistance to chemotherapeutic drugs. Our previous studies have found that gastric cancer cells are sensitive to chemotherapy when methionine is starved. Meanwhile, the expression and activity of NF- kappa B and miR-21 are abnormal. They are closely related to epithelial mesenchymal transition. Studies have shown that miRNA can be regulated by nuclear transcription factors. Therefore, we assume that methionine starvation may prevent gastric cancer cells from being differentiated into cancer stem cells by NF- kappa B/miR-21 pathway, and overcome the drug resistance. Based on this new idea, First ,the project construct targeting iRGD-Exos-rMETase bio nano vesicles to induced methionine starvation by using exosomes. Secondly, we investigate the effect of iRGD-Exos-rMETase reversing the multidrug resistance of gastric cancer by gastric cancer xenograft model, in vivo imaging, flow cytometry and immunofluorescence technique from the whole and the cellular and molecular level of the system . We clarify that the role of NF- kappa B/miR-21 in the acquired resistance of gastric cancer cells. This study will provide a theoretical basis for the treatment of gastric cancer stem cells by methionine starvation therapy combined with chemotherapeutic drugs.

肿瘤细胞在抵抗化疗药物时通过上皮间质转化获得肿瘤干细胞特性是获得性多药耐药的重要原因。我们前期研究发现：胃癌细胞处于甲硫氨酸饥饿时对化疗药物的敏感性增加；同时，与上皮间质转化密切相关的NF-κB、miR-21表达及活性异常。研究表明miRNA可以被核转录因子调控，因此我们设想：甲硫氨酸饥饿可能通过NF-κB/miR-21途径阻止普通胃癌细胞逆分化成肿瘤干细胞，克服肿瘤耐药。基于此新设想，本项目利用外泌体这种新型的天然药物纳米载体，构建具有肿瘤靶向性的iRGD-Exos-rMETase生物纳米囊泡，诱导甲硫氨酸饥饿后综合运用胃癌移植瘤模型、活体成像、流式细胞术、免疫荧光等技术，从整体与细胞、分子等水平系统探讨iRGD-Exos-rMETase逆转胃癌多药耐药性的效应，明确NF-κB/miR-21在胃癌细胞获得性耐药中的作用。本研究将为甲硫氨酸饥饿疗法联合化疗药物清除胃癌干细胞提供理论依据。

肿瘤细胞在抵抗化疗药物时通过上皮间质转化获得肿瘤干细胞特性是获得性多药耐药的重要原因。我们前期研究发现：甲硫氨酸饥饿可以增加胃癌细胞对化疗药物的敏感性及抑制胃癌干细胞的自我更新等特性，但是其作用机制不明。本项目首先利用外泌体这种新型的天然药物纳米载体，构建具有肿瘤靶向性的iRGD-Exos-rMETase生物纳米囊泡，诱导胃癌细胞甲硫氨酸饥饿；其次研究了甲硫氨酸饥饿逆转胃癌细胞化疗耐药性的作用及分子机制；同时研究了肿瘤相关巨噬细胞及胃癌细胞来源的外泌体与胃癌细胞化疗耐药及增殖的关系。研究结果如下:（1）成功构建了基于外泌体的生物纳米囊泡iRGD-Exos-rMETase 并进行了体内外生物学评价：体外细胞实验证实iRGD-Exos-rMETase生物纳米囊泡能够有效将rMETase传递进胃癌细胞中并杀伤肿瘤细胞；荷胃癌动物模型体内实验表明，和非靶向性的药物相比，iRGD-Exos-rMETase生物纳米囊泡抑瘤效果是较强的。(2）甲硫氨酸饥饿通过调控长链非编码RNA HULC下调FoxM1蛋白抑制自噬，进而抑制胃癌细胞对顺铂的耐药性；（3）胃癌组织微环境中肿瘤相关巨噬细胞来源的外泌体可以转运入胃癌细胞内，该外泌体内高表达的长链非编码RNA CRNDE可以通过增强NEDD4-1介导的PTEN泛素化来增加胃癌细胞对顺铂（CDDP）的耐药性。（4）胃癌细胞来源的外泌体可以进入肿瘤相关巨噬细胞，通过高表达的长链非编码RNA HCG18负性调节巨噬细胞中miR-875-3p的表达来增加KLF4的表达，调节M2巨噬细胞的极化来促进胃癌细胞的生长，抑制胃癌细胞来源的外泌体中HCG18表达可以抑制胃癌的生长。（5）甲硫氨酸饥饿可以通过长链非编码RNA PVT1 调控BNIP3 基因启动子去甲基化来促进胃癌细胞的有丝分裂，进而抑制胃癌细胞的增殖和转移。本研究有助于拓展重组甲硫氨酸酶的临床应用，可为筛选胃癌治疗的分子靶点、设计更加高效的抗肿瘤药物提供理论基础，具有重要的临床意义。