Downregulation of MicroRNA-644a Promotes Esophageal Squamous Cell Carcinoma Aggressiveness and Stem-cell-like Phenotype via Dysregulation of PITX2.

Purpose: We previously reported the oncogenic role of paired-like homeodomain 2 (PITX2) in esophageal squamous cell carcinoma (ESCC). In this study, we aimed to identify the microRNA (miRNA) regulators of PITX2 and the mechanism underlying the pathogensis of ESCC. Experimental Design: Using miRNA profiling and bioinformatics analyses, we identified miR-644a as a negative mediator of PITX2 in ESCC. A series of in vivo and in vitro assays were performed to confirm the effect of miR-644a on PITX2-mediated ESCC malignancy. Results: ESCC cells and tissues expressed less miR-644a than normal epithelial controls. In patient samples, lower expression of miR-644a in ESCC tissues was significantly correlated with tumor recurrence and/or metastasis, such that MiR-644a, PITX2 and the combination of the two were independent prognostic indicators for ESCC patient's survival (P<0.05). Gain- and loss-of-function studies demonstrated that miR-644a inhibited ESCC cell growth, migration and invasion in vitro and suppressed tumor growth and metastasis in vivo. In addition, miR-644a dramatically suppress self-renewal and stem cell-like traits in ESCC cells. Further, the effect of up-regulation of miR-644a was similar to that of PITX2 knockdown in ESCC cells. Mechanistic studies revealed that miR-664a attenuates ESCC cells' malignancy and stem-cell-associated phenotype, at least partially, by inactivation of the Akt/GSK-3β/β-catenin signaling pathway through PITX2. Furthermore, promoter hypermethylation caused down-regulation of miR-644a in ESCC. Conclusions: Down-regulation of miR-644a plays an important role in promoting both aggressiveness and stem-like traits of ESCC cells, suggesting that miR-644a may be useful as a novel prognostic biomarker or therapeutic target for the disease.