MicroRNAs (miRNAs) have demonstrated great promise as a novel class of biomarkers for early detection of various cancers, including breast cancer. However, due to technical difficulties in detecting these small molecules, miRNAs have not been adopted into routine clinical practice for early diagnostics. Thus, it is important to develop alternative detection strategies that could offer more advantages over conventional methods. Here, we demonstrate the application of a “turn-on” SERS sensing technology, referred to as “inverse Molecular Sentinel (iMS)” nanoprobes, as a homogeneous assay for multiplexed detection of miRNAs. This SERS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform. The “OFF-to-ON” signal switch is based on a nonenzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. This technique was previously used to detect a synthetic DNA sequence of interest. In this study, we modified the design of the nanoprobe to be used for the detection of short (22-nt) miRNA sequences. The demonstration of using iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of short miRNAs molecules.

Methylation of the retinoic acid receptor-B2 (RARB2) P2 promoter is hypothesized to be an important mechanism for loss of RARB2 function during early mammary carcinogenesis. The frequency of RARB2 P2 methylation was tested in (a) 16 early stage breast cancers and (b) 67 random periareolar fine needle aspiration (RPFNA) samples obtained from 38 asymptomatic women who were at increased risk for breast cancer. Risk was defined as either (a) 5-year Gail risk calculation z z1.7%; (b) prior biopsy exhibiting atypical hyperplasia, lobular carcinoma in situ, or ductal carcinoma in situ; or (c) known BRCA1/2 mutation carrier. RARB2 P2 promoter methylation was assessed at two regions, M3 (À À51 to 162 bp) and M4 (104-251 bp). In early stage cancers, M4 methylation was observed in 11 of 16 (69%) cases; in RPFNA samples, methylation was present at M3 and M4 in 28 of 56 (50%) and 19 of 56 (38%) cases, respectively. RPFNAs were stratified for cytologic atypia using the Masood cytology index. The distribution of RARB2 P2 promoter methylation was reported as a function of increased cytologic abnormality. Methylation at both M3 and M4 was observed in (a) 0 of 10 (0%)

Unlike estrogen receptor-positive (ER( þ )) breast cancers, normal human mammary epithelial cells (HMECs) typically express low nuclear levels of ER (ER poor). We previously demonstrated that 1.0 lM tamoxifen (Tam) promotes apoptosis in acutely damaged ER-poor HMECs through a rapid, 'nonclassic' signaling pathway. Interferon-regulatory factor-1 (IRF-1), a target of signal transducer and activator of transcription-1 transcriptional regulation, has been shown to promote apoptosis following DNA damage. Here we show that 1.0 lM Tam promotes apoptosis in acutely damaged ER-poor HMECs through IRF-1 induction and caspase-1/3 activation. Treatment of acutely damaged HMEC-E6 cells with 1.0 lM Tam resulted in recruitment of CBP to the c-IFN-activated sequence element of the IRF-1 promoter, induction of IRF-1, and sequential activation of caspase-1 and -3. The effects of Tam were blocked by expression of siRNA directed against IRF-1 and caspase-1 inhibitors. These data indicate that Tam induces apoptosis in HMEC-E6 cells through a novel IRF-1-mediated signaling pathway that results in activated caspase-1 and -3.

No abstract