Department of Obstetrics and Gynaecology, Third Affiliated Hospital of Guangzhou Medical College, Guangzhou 510150, China.
Gynecol Oncol. 2011 Jul;122(1):149-54. doi: 10.1016/j.ygyno.2011.03.026. Epub 2011 May 6.
MicroRNA-200a (miR-200a) has been reported to be a prognostic marker and to play an important role in ovarian cancer progression. The aim of the study was to elucidate the mechanism of miR-200a involved in migration and invasion in CD133/1+ ovarian cancer stem cells (OCSCs).
The expression of miR-200a between CD133/1+ and CD133/1- cells was performed using real-time PCR, and wound healing assay and matrigel invasion assay were used to detect migration and invasion of CD133/1+ cells, respectively, target gene regulated by miR-200a was detected using Dual Luciferase Reporter system, The expression levels of target gene were confirmed using real-time PCR and western blot.
miR-200a was downregulated in CD133/1+ cells compared with CD133/1- cells, and overexpression of miR-200a significantly reduced CD133/1+ cells migration and invasion compare with negative control (NC) (p<0.05). The 3'-UTR of ZEB2 mRNA, a transcriptional repressor of E-cadherin, was found to be regulated directly by miR-200a. In addition, when miR-200a was overexpressed in CD133/1+ cells, the mRNA and protein levels of ZEB2 were both suppressed, which resulted in an increase in the E-cadherin expression level, suggesting that ZEB2 was a functionally important target of miR-200a in CD133/1+ cells.
Our results suggest that loss of expression of miR-200a may play a critical role in the repression of E-cadherin by ZEB2, thereby enhancing migration and invasion in CD133/1+ cells.
microRNA-200a（miR-200a）已被报道为一种预后标志物，并在卵巢癌进展中发挥重要作用。本研究旨在阐明 miR-200a 参与 CD133/1+ 卵巢癌干细胞（OCSCs）迁移和侵袭的机制。
采用实时 PCR 检测 CD133/1+和 CD133/1-细胞中 miR-200a 的表达，采用划痕愈合试验和基质胶侵袭试验分别检测 CD133/1+细胞的迁移和侵袭，采用双荧光素酶报告系统检测 miR-200a 调控的靶基因，采用实时 PCR 和 Western blot 验证靶基因的表达水平。
与 CD133/1-细胞相比，CD133/1+细胞中 miR-200a 表达下调，过表达 miR-200a 可显著降低 CD133/1+细胞的迁移和侵袭能力，与阴性对照（NC）相比差异有统计学意义（p<0.05）。ZEB2 mRNA 的 3'-UTR 被发现可被 miR-200a 直接调控，ZEB2 是 E-钙黏蛋白的转录抑制因子。此外，当 miR-200a 在 CD133/1+细胞中过表达时，ZEB2 的 mRNA 和蛋白水平均受到抑制，导致 E-钙黏蛋白表达水平升高，表明 ZEB2 是 miR-200a 在 CD133/1+细胞中功能重要的靶基因。
本研究结果表明，miR-200a 的表达缺失可能在 ZEB2 抑制 E-钙黏蛋白中发挥关键作用，从而增强 CD133/1+细胞的迁移和侵袭能力。
