miR-655 Is an EMT-Suppressive MicroRNA Targeting ZEB1 and TGFBR2

Recently, the epithelial-to-mesenchymal transition (EMT) has been demonstrated to contribute to normal and disease processes including cancer progression. To explore EMT-suppressive microRNAs (miRNAs), we established a cell-based reporter system using a stable clone derived from a pancreatic cancer cell line, Panc1, transfected with a reporter construct containing a promoter sequence of CDH1/E-cadherin in the 5′ upstream region of the ZsGreen1 reporter gene. Then, we performed function-based screening with 470 synthetic double-stranded RNAs (dsRNAs) mimicking human mature miRNAs using the system and identified miR-655 as a novel EMT-suppressive miRNA. Overexpression of miR-655 not only induced the upregulation of E-cadherin and downregulation of typical EMT-inducers but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift toward the epithelial phenotype. In addition, we found a significant correlation between miR-655 expression and a better prognosis in esophageal squamous cell carcinoma (ESCC). Moreover, ZEB1 and TGFBR2, which are essential components of the TGF-b signaling pathway, were identified as direct targets of miR-655, suggesting that the activation of the TGF-b-ZEB1-E-cadherin axis by aberrant downregulation of miR-655 may accelerate cancer progression.

近年来，上皮间质转化（epithelial-to-mesenchymal transition, EMT）已被证实参与包括癌症进展在内的正常生理与疾病进程。为筛选具有上皮间质转化抑制活性的微小RNA（microRNAs, miRNAs），本研究构建了基于细胞的报告基因系统：以胰腺癌细胞系Panc1的稳定克隆为宿主，转染携带ZsGreen1报告基因5'上游区域包含CDH1/E-钙粘蛋白（CDH1/E-cadherin）启动子序列的报告载体。随后，利用该系统对470条模拟人成熟miRNA的合成双链RNA（double-stranded RNAs, dsRNAs）进行功能筛选，鉴定出miR-655为新型EMT抑制性miRNA。过表达miR-655不仅可诱导E-钙粘蛋白的上调与典型EMT诱导因子的下调，还能抑制间质样癌细胞的迁移与侵袭能力，并伴随细胞形态向上皮表型的转变。此外，本研究发现miR-655的表达水平与食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）患者的良好预后显著相关。进一步研究证实，转化生长因子β信号通路的核心组分ZEB1与TGFBR2为miR-655的直接靶标，提示miR-655的异常下调可激活TGF-β-ZEB1-E-钙粘蛋白轴，进而加速癌症进展。