QRT-PCR verification of miRNAs expression results from microarray data.

<p>Blue bars represent the results from microarray, while red bars indicate the results from qRT-PCR. The error bars are the standard error of mean (SEM) for each analysis. QRT-PCR results are largely consistent with our microarray data. Five representative miRNAs (miR-21, miR-183, miR-141, and miR-200b/c) were observed up-regulated during the Normal-ADH transition, and their high expression levels were maintained throughout the tumor developmental stages. miR-557 was found to be down-regulated specifically in the DCIS stage.</p

Microdissected samples from breast cancer FFPE blocks.

*<p>The letters (A, B, C …) represent each patient and the numbers, 1, 2, 3, 4 indicate “Normal”, “ADH”, “DCIS”, “IDC” respectively in each patient's FFPE tissue. “X” means that no histological samples were obtained from an individual FFPE sample.</p

Unsupervised clustering results on ANOVA identified miRNAs and conditions of the 23 samples.

<p>Each solid color box represents a certain condition. The clustering result indicates the significantly altered miRNA entities identified by ANOVA test have more potential to distinguish different stages of breast cancer than broad-wide miRNAs. Seven individual clusters were clearly discerned by the clustering algorithms and the miRNAs circled by red rectangles representing their discrete clusters.</p

Knockdown of miR-21 restores the expression of SMAD7 and MSH2 in MCF-7 and Hs578T breast cancer cell lines.

<p>MCF-7 and Hs578T cells were transfected with miR-21 inhibitor and a negative mock control using the Lipofectamine 2000 kit (Invitrogen). After 48 hrs, miR-21 expression level was knocked down by ∼10 fold as compared to the mock controls in both MCF-7 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054213#pone-0054213-g006" target="_blank">Fig. 6A</a>) and Hs578T (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054213#pone-0054213-g006" target="_blank">Fig. 6B</a>) cell lines using the Invitrogen SYBR green qRT-PCR kit. Untransfected cells were also included in the analysis (WT). With down-regulated miR-21 in both MCF-7 and Hs578T cells, MSH2 and SMAD7 mRNA expression was up-regulated by ∼1.67 and ∼3.6 fold, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054213#pone-0054213-g006" target="_blank">Fig. 6A and 6B</a>), while the protein level was increased by ∼35–43% for MSH2 and ∼80–133% for SMAD7 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054213#pone-0054213-g006" target="_blank">Fig. 6C</a>).</p

Venn diagram of ANOVA test results from paired and unpaired miRNA expression analysis.

<p>ANOVA test on the paired miRNA microarray data analysis resulted in 35 deregulated miRNAs, while ANOVA test on the unpaired analysis showed 98 deregulated miRNAs. There are 10 overlapping miRNAs (miR-1268, mir-130a, miR-141, miR-193b, miR-200b, miR-21, miR-320a, miR-370, miR-557 and kshv-mir-K12-3).</p

A representative list of deregulated miRNA entities during the breast lesion transition.

<p>A set of samples diagnosed with Normal, ADH, DCIS, and IDC (4 of each) were subject to the microarray analysis as we performed for the microdissected groups. In both paired and un-paired analyses, there were more deregulated miRNAs during the Normal-ADH transition compared to other processes. Deregulated miRNAs that appeared in both analyses are bolded.</p