Bmi-1通过miR-27a调控RKIP促进胃癌EMT和化疗耐药的机制研究

Epithelial-mesenchymal transition (EMT) and chemotherapy resistance are crucial for metastasis and prognosis of gastric cancer. Both B cell-specific MLV integration site-1 (Bmi-1) and Raf kinase inhibitor protein (RKIP) are involved in these processes. In our preliminary study, gastric cancer tissue chip and cellular express/silence experiments demonstrated that Bmi-1 and RKIP were negatively related. Furthermore, microRNA chips and miRNA reply analyses suggested that RKIP was repressed probably via miR-27a up-regulated by Bmi-1. However, the specific regulation mechanism between Bmi-1 and RKIP remains unknown. Therefore, this study intends to define the Bmi-1-miR-27a-RKIP mechanism and its role in EMT and chemotherapy resistance of gastric cancer, and find out effective targets to reverse EMT and chemotherapy resistance. Moreover, based on the nude mouse gastric cancer metastatic tumor model, miR-27a inhibitors are delivered by a MRI-visible vector targeting at gastric cancer constructed in our previous study, and that realizes body tracing of gastric cancer metastasis and evaluation the inhibition effect of miR-27a inhibitors on gastric cancer metastasis. Finally, combined with clinical and pathological data, we further analyze whether Bmi-1, miR-27a and RKIP can be used as diagnostic and prognostic markers for gastric cancer and chemotherapy resistance.

上皮间质转化（EMT）和化疗耐药是胃癌转移及影响预后关键环节，原癌基因Bmi-1和Raf激酶抑制蛋白（RKIP）均参与其中。我们已通过胃癌病理组织芯片和细胞过表达/沉默实验证实Bmi-1与RKIP存在负向调控关系，由microRNA芯片和miRNA回复实验发现Bmi-1可能通过miR-27a调控RKIP表达，但具体调控机制尚未明确。本课题将在此基础上进一步确定Bmi-1-miR-27a-RKIP通路及其促进胃癌EMT和化疗耐药作用机制，寻找有效逆转EMT和化疗耐药靶点。进一步利用前期已建立具有活体示踪能力胃癌靶向载药纳米技术，在鼠胃癌转移瘤模型中包载miR-27a抑制剂靶向输送，实现体内胃癌转移全过程的活体示踪及明确该通路靶点。并进行前瞻性临床研究，结合胃癌患者临床和病理资料验证Bmi-1、miR-27a和RKIP可作为靶向逆转化疗耐药重要靶点和判断预后的临床指标，具有重要的转化医学意义。

近年来尽管胃癌发病率和死亡率有所下降，但目前为止，仍然是人类最常见恶性肿瘤之一，全球预计每年新发胃癌93万例，中国占42%，且我国胃癌患者大多数为进展期。胃癌早期即发生淋巴结转移，进展期以腹膜转移为主，虽然以外科手术为主、化疗为辅的综合治疗可以提高患者无复发生存率，但由于进展期胃癌化疗敏感性低、耐药发生率高，总的复发率仍达到50%-70%。胃癌复发和转移与化疗耐药是最终预后不良的原因之一。然而，胃癌转移及化疗耐药机制尚未明确。Bmi-1与RKIP均参与调控肿瘤EMT和化疗耐药。在课题前期研究，我们通过胃癌病理组织芯片和细胞过表达/沉默实验证实Bmi-1与RKIP存在负向调控关系，但具体调控机制尚未明确。本课题在前期基础上分四部分完成。第一部分研究Bmi-1、RKIP在胃癌的表达及临床意义：①胃癌组织芯片Bmi-1、RKIP表达呈负性相关；②胃癌组织Bmi-1、RKIP表达与患者临床特征、预后、化疗耐药密切相关；③细胞实验证实Bmi-1负向调控RKIP表达。第二部分研究Bmi-1通过miR-27a、miR-155调控RKIP的具体机制：①胃黏膜上皮细胞GES-1过表达Bmi-1，miRNA芯片筛选差异表达miRNAs；②胃癌细胞中miR-27a、miR-155调控RKIP表达；③实验验证胃癌细胞Bmi-1-miR-27a-RKIP及Bmi-1-miR-155-RKIP轴。第三部分研究Bmi-1-miR-27a-RKIP、Bmi-1-miR-155-RKIP轴在胃癌EMT和化疗耐药的作用：①体外实验验证Bmi-1-miR-27a-RKIP、Bmi-1-miR-155-RKIP轴调控胃癌细胞增殖、侵袭、转移、凋亡、周期的作用；②体外实验验证Bmi-1-miR-27a-RKIP、Bmi-1-miR-155-RKIP轴调控胃癌细胞化疗耐药的作用；③动物实验验证Bmi-1-miR-27a-RKIP、Bmi-1-miR-155-RKIP轴调控胃癌细胞增殖、转移及化疗耐药的作用。第四部分靶向纳米研究方面：①载药免疫纳米微粒的合成与表征：②载药免疫纳米微粒体内肿瘤示踪及抑制肿瘤生长和转移。本研究阐明了Bmi-1调控RKIP机制以及Bmi-1-miR-27a -RKIP、Bmi-1-miR-155-RKIP在胃癌EMT和化疗耐药的作用，有望寻找有效的可逆转胃癌转移和耐药的靶点。