*, P < 0.05. Discussion In the present study, we identify increased expression of miR-21 in 78% (25/32) of breast cancer samples analyzed, as compared to patient-matched normal breast epithelium. Further, we identify that the invasive ability of breast cancer cell lines closely correlates JPH203 nmr with miR-21 expression, as incidence of lymph node metastases increases with miR-21 expression. These data are consistent with reports indicating that miR-21 expression increased with advanced clinical stage and shortened survival of the patients [19], and that miR-21

expression is associated with poor disease-free survival in early stage patients [20]. Greater than 50% of miRNA are located at genomic regions implicated in human cancers, emphasizing the potential importance of miRNA in cancer progression [21]. Specifically, the miR-21 gene is located on chromosome 17q23.2, which is located within the common fragile site FRA17B. This region is frequently found amplified in breast, colon, and lung cancer, consistent with the fact that miR-21 overexpression is widespread in many types of cancer, including the breast [22]. Despite the link of miR-21 to carcinogenesis,

little is known regarding the specific mechanism how miR-21 may impact cancer progression. The correlation of miR-21 expression with tumor metastasis, and VRT752271 order supportive evidence that miR-21 regulates cell invasion in vitro, raises the question how miR-21 may impact a cell’s metastatic potential. Several factors suggest that miR-21 may be impacting matrix YH25448 metalloproteinases inhibitors, such as TIMP3, that play a crucial role in cancer invasion and metastasis[23] Tyrosine-protein kinase BLK including recent studies that identified TIMP3 as a functional target of miR-21 in cell invasion and metastasis in glioma and cholangiocarcinoma[15, 16]. As TIMP3 expression is down-regulated or lost in several tumor types [24–26], and adenoviral transfer of TIMP3 into HeLa, HT1080 fibrosarcoma, and melanoma cells reduces their invasiveness and stimulates apoptosis[27, 28], we tested whether miR-21 may be impacting TIMP3 expression in primary breast cancer specimen as well as four breast cancer-derived cell lines. Our findings report for the

first time that microRNA-21 negatively regulates TIMP3 in breast cancer, and suggests that TIMP3 may be negatively regulated by miR-21 at the transcriptional level via binding of the 3′UTR of TIMP3 mRNA. Further, we provide evidence that it is this regulation of TIMP3 expression that impacts cell invasion in vitro. These compelling data support miR-21 regulation of TIMP3 expression as a novel mechanism impacting breast cancer invasion. Our studies suggest that miR-21 regulation of TIMP3 may represent a novel target for therapeutic intervention to prevent breast cancer metastasis, and warrant further investigation. Conclusion Our data identify that miRNA-21 is overexpressed in breast cancer tissues and breast cancer cell lines, promoting breast cancer invasion in multiple cell lines in vitro.