MicroRNAs (miRNAs) are key regulators of gene expression and perform critical roles in various biological processes. To investigate the functional roles of miRNAs in the prolactin receptor (PRLR) signaling pathway in breast cancer, we constructed two small RNA libraries from human breast cancer T-47D cells treated with or without prolactin (PRL). The miRNA expression profiles were systematically screened using Solexa deep-sequencing technology. More than 40 miRNAs were significantly differentially expressed, from which 4 miRNAs were chosen for validation by stem-loop real-time PCR. In addition, 3 novel miRNAs were selected for verification by PCR. Furthermore, upstream miRNA target genes were predicted using different algorithms, GO and KEGG analyses revealed that these targets were highly related to the PRLR signaling pathway. This study provides a reference for elucidating the complex miRNA-mediated regulatory networks of PRL/PRLR signaling pathway that affect breast cancer tumorigenesis and progression. (C) 2013 Elsevier Inc. All rights reserved.