miR-210在乳腺癌干细胞自我更新和侵袭转移中的作用及机制研究

Breast cancer stem cells（BCSCs）are a small cell population with unique characteristics such as self-renewal, invasion and migration, all of which are believed to contribute to the development and overall aggressiveness of the recurrent or metastatic lesions．But the mechanism under this is incompletely understood. In this study, we detected miR-210 was up-regulated in BCSCs, base on this discovery, first we research the effect of miR-210 on BCSCs self-renew and metastasis by overexpressing or knocking down miR-210 in breast cancer cells in vitro and in vivo, in combination with 3D semi-solid system culture technology, secondary spheroids-forming assay, MTT assay, wound healing assay, transwell invasion assay and orthotopic injection of BCSCs into the mammary fat pad of nude mice. Secondly, we define the target gene and concrete mechanism of miR-210 in regulating self-renew and metastasis of BCSCs by using bioinformatics,luciferase reporter gene system, RNA interference and function restoration assay. Finally, we try to clarify the blocking effect of knocking down miR-210 in BCSCs by applying hyaluronic acid(HA)-conjugated liposome nanoparticles as vector to deliver miR-210 sponge to the nude mice bearing orthotopically implanted breast tumors, rely on HA specific binding to CD44, the surface marker of BCSCs, and liposome fusing with the cellular membrane. The successful completion of this study will reveal a novel role of miR-210 in BCSCs self-renew and metastasis, suggest new ideas of mechanisms of breast tumor recurrence and metastasis, and provide a potential new target for developing gene drugs aim at BCSCs in breast cancer therapy in the future.

乳腺癌干细胞（BCSCs）的自我更新及侵袭转移可造成乳腺癌的复发转移，但具体的调控机制尚不清楚。本研究在发现miR-210高表达于BCSCs的基础上，利用慢病毒介导的miR-210敲减或过表达策略，结合三维半固体培养技术、二次克隆、transwell及小鼠植瘤等实验确定miR-210对BCSCs在体内外的自我更新、成瘤性及转移能力的影响；利用荧光素酶报告系统确定miR-210调节BCSCs自我更新及侵袭转移的靶基因，通过RNA干扰及功能回复实验阐明其调控机制；以透明质酸(HA)修饰的脂质体为载体，利用HA与BCSCs表面标记CD44特异性结合，在原位乳腺癌荷瘤鼠体内导入miR-210 海绵体慢病毒，阐明靶向BCSCs沉默miR-210对乳腺癌的阻断作用。本研究将有助于阐明miR-210在BCSCs自我更新及侵袭转移中的作用与调控机制，为研制针对BCSCs的RNA干扰型基因药物提供新靶点。

乳腺癌作为全球女性中最常见的恶性疾病其发病率仍在持续上升。目前认为乳腺癌的复发和远端转移与乳腺癌中存在的乳腺癌干细胞（BCSCs）密切相关。BCSCs具有自我更新能力、高增殖迁移能力、多分化潜能、和放化疗抵抗等多种肿瘤干细胞特性，这些都有助于乳腺癌的转移和复发。因此，以BCSCs为研究对象，探寻乳腺癌发生发展的具体机制，寻求新的治疗靶点成为现阶段乳腺癌研究的热点和重点。随着高通量测序技术的发展，越来越多的BCSCs相关基因被挖掘出来，尤其是非编码RNA被发现在BCSCs促进乳腺癌的发展过程中发挥一定的作用。在本研究中，我们通过芯片测序技术，筛选出在BCSCs中异常表达的MicroRNA（miRNAs），探究它的生物学功能和作用机制，旨在为探索乳腺癌的发病机理和BCSCs的靶向治疗提供新的靶点。. 我们首先建立了新型三维半固体培养体系，并成功富集得到MCF-7微球体细胞，体内外实验验证该微球体细胞具有BCSCs的干样特性。接着通过miRNA芯片测序发现miR-210在BCSCs中表达显著上调且受到缺氧微环境诱导。功能上，miR-210能够促进MCF-7细胞在体内外的迁移，增殖和自我更新能力。机制研究发现，miR-210既能够通过与E-cadherin的ORF区直接结合，还能够通过调节E-cadherin转录抑制因子，SNAIL，来共同降低E-cadherin的表达，促进乳腺癌发展。. 综上所述，我们建立了三维半固体培养体系并成功富集到了BCSCs，通过对BCSCs进行高通量测序，筛选得到在BCSCs中表达显著上调的miR-210，其能够通过调节BCSCs功能促进乳腺癌发展，上述研究结果为探索乳腺癌的发病机理和挖掘新的BCSCs治疗靶点提供理论指导。