In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the invasiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in invasive cell lines when compared to normal and less invasive cell lines. Transfection of miR-200c, miR-205, and miR-375 mimics into MDA-MB-231 cells led to the inhibition of in vitro cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that miR-200c plays a pivotal role in regulating the invasiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer invasiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression by translational repression or mRNA degradation. Although the functions of specific miRNAs in breast cancer development and progression have been extensively investigated, few studies have focused on the systematic identification of miRNAs and their target genes related to breast cancer aggressiveness. In this study, we sought to discover the signatures of miRNA-mRNA target pairs in aggressive breast cancer cell lines through a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction and functional assays. MiRNA and mRNA expression profiles of 12 breast cancer cell lines including 8 less aggressive lines (MCF10A, MCF12A, BT474, MCF7, MDA-MB-468, SK-BR3, T-47D and ZR-75-1) and 4 aggressive lines (BT549, HS578T, MDA-MB-231 and SUM159) were generated using Affymertrix miRNA Array and U133 Plus 2.0 Array, respectively. The miRNA expression array analysis has shown that 44 out of 847 miRNAs were differentially expressed (p<0.01) between less aggressive and aggressive cell lines. Among the 44 differentially expressed miRNAs, 30 miRNAs (i.e. mir-141, mir-200c, mir-203, mir-205 and mir-34a) were down-regulated in aggressive lines as compared to less aggressive lines, while 14 miRNAs (i.e. mir-100, mir-125b, mir-138 and mir-146a) were up-regulated. The integrated analysis of miRNA and mRNA expression was performed in Partek Genomic Suites and the predicted miRNA-mRNA target pairs by TargetScan5.1 that demonstrated negative correlations were identified. Twelve candidate targets for mir-146a, mir-200c, mir-203, mir-205 and mir-375, including IRAK1, TRAF6, FOXF1, SHOX2, CDH11, ZEB1, FYN, ETS1, CSF1, ACSL4, SNAIL2 and BMI1, were selected for functional analysis. Quantitative RT-PCR analysis confirmed the differential expression patterns of the 5 miRNAs and their 12 target genes in the above-mentioned 12 cell lines. We have further established stable clones overexpressing individual mir-200c, mir-203, mir-205 and mir-375 in MDA-MB-231 cell line. The change in the aggressiveness of miRNA-overexpressing cells was evaluated by migration, invasion and soft agar colony formation assays. qRT-PCR and western blot validation for the potential miRNA-mRNA target pairs and 3′UTR reporter assays in miRNA-overexpressing clones are currently underway. In summary, we have identified a group of negatively correlated miRNA-mRNA target pairs that are differentially expressed in aggressive breast cancer cells. The results obtained from this study may advance our understanding of the role of these candidate miRNAs and their target genes in breast cancer aggressiveness and lead to identification of novel biomarkers associated with prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2011-4937