ZNF-148-miR-335-EphA4联动信号干预乳腺癌细胞转移的机制研究

Accumulating evidences have shown miR-335 act as a suppressor in breast cancer development, however, the regulation and signaling pathway of miR-335 in breast cancer metastasis still in discussing. Using detection software, our previous work shows EphA4 as a target gene of miR-335 and found that the down-regulate miR-335 but up-regulate EphA4 in breast cancer tissue, we also found that miR-335 over-expression may inhibit cancer cell migration. Depend on the findings which show EphA4 may regulating cell migration by activating Rho pathway result in cytoskeleton reorganization and zinc-finger protein 148 (ZNF-148) contain sequences may bind to miR-335, we hypothesized that the expression of miR-335 has been regulated by ZNF-148, meanwhile, miR-335 repress EphA4 and then control Rho signaling, which may down cell adhesion through cytoskeleton reorganization directly, and finally inhibit cell migration according to non-classical epithelial- mesenchymal transition pathway. Therefore, we want to figure out the relations between miR-335 and ZNF-148, and confirm miR-335 target at EphA4, to clarify the mechanism involved in breast cancer metastasis mediated by miR-335 in this study, in aim to demonstrate our hypothesis “ZNF-148-miR-335-EphA4 signaling mediate breast cancer cell migration and invasion”.

研究发现miR-335能显著抑制乳腺癌细胞的生长，但其作用机制尚未完全清楚，应用microRNA靶基因分析软件Targetscan对可能的miR-335目标靶基因分析，我们证实了EphA4为miR-335的下游靶基因，并观察到miR-335在癌组织中表达下调而EphA4上调。结合EphA4可能联动Rho信号影响细胞骨架重构直接干预细胞移动及锌指蛋白148（ZNF-148）存在miR-335结合序列的新近研究，我们推测miR-335表达受ZNF-148等上游信号因子的调控并通过下调EphA4的表达，进而干预Rho信号传导，影响细胞骨架重构直接改变细胞粘附力，从而介导非上皮-间质转化机制的细胞转移。因此，本研究拟阐释乳腺癌中miR-335与ZNF-148级联及其与EphA4的靶向定位，分析miR-335细胞浸润机制，验证“ZNF-148-miR-335-EphA4信号干预乳腺癌转移”假说。

研究发现miR-335能显著抑制乳腺癌细胞的生长，但其作用机制尚未完全清楚，前期的研究我们证实了EphA4为miR-335的下游靶基因，并观察到miR-335在癌组织中表达下调而EphA4上调。通过实验的开展，我们发现ZNF-148在乳腺癌组织和乳腺癌细胞中高表达，miR-335在乳腺癌组织和乳腺癌细胞中低表达，过表达ZNF-148抑制miR-335促进乳腺癌细胞增殖、侵袭和迁移，抑制细胞凋亡。 ZNF-148 敲除促进ROS 产生，ROS(NAC)清除剂消除ZN F-148 敲除 miR-335/SOD2 调控ROS 的产生， 触发凋亡和焦细胞凋亡，并促进乳腺癌细胞的氧化应激，ZNF-148的沉默通过触发氧化应激促进细胞凋亡。我们还发现miR-335是通过靶向SOD2调控ROS的产生对乳腺癌细胞增殖/侵袭/迁移和凋亡的影响。EphA4沉默，至Rho、Cdc42的表达量明显下降，miR-335过表达时，细胞运动迁侵袭力明显减弱，上皮标志物（CDH1和ZO1两个指标）表达增强和间质细胞标志物（FN、SNAI2和ZEB1三个指标）表达减弱，鬼笔环肽标记和β-Actin免疫荧光染色实验，观察到miR-335过表达时，细胞骨架没有支撑细胞延伸变形，而是细胞团缩，伸展变形能力减弱。. 实验结果证实了.①ZNF-148 在乳腺癌起到促乳腺癌及细胞的侵袭、迁移的作用，ZNF-148通过抑制miR-335的表达发挥作用，ZNF-148- miR-335通过靶向SOD2调控ROS的产生，触发凋亡和焦细胞凋亡，并促进乳腺癌细胞的氧化应激，对乳腺癌细胞增殖/侵袭/迁移和凋亡的影响，.②miR-335-EphA-RhoGTP 激酶下游通路信号是通过改变细胞骨架，细胞骨架没有支撑细胞延伸变形，而是细胞团缩，伸展变形能力减弱而导致乳腺癌细胞侵袭、迁移能力减弱。.③证实了“ZNF-148- miR-335-EphA4-RhoGTP 联动信号干预乳腺癌侵润转移。. 研究结果为未来的乳腺精分子诊断及分子靶向治疗提供思路及潜在的治疗靶点，具备应用潜力。