miR-205调控转录因子RunX2表达在乳腺癌干细胞侵袭转移中的作用及其机制研究

Breast cancer metastasis is the main reason that caurses therapy failure and death. Recent investigation indicates breast cancer stem cell plays a critical role in it. The mechanism is complicated and not very clear. Our previous study found miR-205 was down-regulated in breast cancer stem cell and targeted transcriptional factor RunX2, a promoter of breast cancer metastasis. Therefore we hypothesize that miR-205 is involved in breast cancer stem cell metastasis by regulating RunX2 expression. To confirm it, we will identify the direct regulation of RunX2 by miR-205 in breast cancer stem cell using report gene assay, immunohistochemisty,etc. Then we will set up breast cancer stem cell lines with enforced-expression of miR-205 or deficient expression of RunX2 by miR-205 lentiviral vector or RunX2 antisense. Compare the cell ability of metastasis,stem cell population and self-renewal properties using the methods of mobility and invasion assay, colony formation assay, flow cytometry, mammosphere formation analysis,etc.Observe tumor formation ability,bone metastatic rate and the expression of breast cancer stem cell markers in the null mice transplanted tumor models using above cell lines respectively. This study will clarify the regulatory role of miR-205 and RunX2 in breast cancer stem cells. It will provide the theoretical foundation for using RunX2 and miR-205 as the targets to intervene breast cancer metastasis in the future.

乳腺癌侵袭转移是导致治疗失败和患者死亡的最主要原因，近年来的研究表明乳腺癌干细胞（BCSC）在此过程中起决定性作用，机制复杂不明。我们前期研究发现miR-205在BCSC中表达下调，并靶向一种乳腺癌骨转移密切相关的转录因子RunX2，提示miR-205可能通过调节RunX2参与BCSC侵袭转移。因此我们将通过报告基因、免疫组化等方法证明miRNA-205对RunX2表达的直接调控作用；然后通过构建miR-205慢病毒载体、RunX2反义核酸载体等在BCSC中过表达miRNA-205、敲低RunX2或同时过表达二者，体外检测对BCSC侵袭性、BCSC比例和自我更新等特性的影响；体内裸鼠移植瘤模型观察不同表达水平的miR-205和RunX2对成瘤率、骨转移率和BCSC标记物表达水平的影响。本研究将阐明miR-205和RunX2对BCSC调节作用及机制，为今后以其为靶点对乳腺癌进行干预奠定基础。

乳腺癌侵袭转移是导致治疗失败和患者死亡的最主要原因，近年来的研究表明乳腺癌干细胞（BCSC）在此过程中起决定性作用，机制复杂不明。我们前期研究发现miR-205在BCSC中表达下调，并靶向一种乳腺癌骨转移密切相关的转录因子RunX2，提示miR-205可能通过调节RunX2参与BCSC侵袭转移。因此本研究的主要研究目的是明确在乳腺细胞中miR-205 对RunX2 的直接调控作用；观察miR-205 和RunX2 如何调节乳腺癌细胞干细胞的侵袭性和“干性”；证明外源性过表达miR-205 或敲低内源性RunX2 表达对乳腺癌细胞的成瘤性、侵袭性和“干性”等恶性表型具有抑制作用。研究结果显示，在乳腺癌细胞中RunX2 是直接受miR-205调节的功能靶基。体外抑制miR-205或过表达RunX2增加乳腺癌干细胞比例，增强细胞增殖、迁移、侵袭、克隆集落形成能力。相反，过表达miR-205或敲低RunX2抑制乳腺癌干细胞比例，抑制细胞增殖、迁移、侵袭、克隆集落形成能力。体内稳定转染RunX2的乳腺癌细胞裸鼠成瘤能力增强。而稳定转染miR-205的乳腺癌细胞裸鼠成瘤能力减弱。因此我们得出结论：在乳腺癌的发展过程中，由于miR-205下调导致RunX2表达上调，引起乳腺癌干细胞群体增加，最终导致肿瘤的生长和转移能力增强。本研究将阐明miR-205和RunX2对BCSC调节作用及机制，为今后以其为靶点对乳腺癌进行干预奠定基础。