One way ANOVA analysis was used to identify differentially expressed miRNAs between each group of cell lines. Using stringent criteria, 19 of the 847 miRNA genes were found to be differentially expressed with statistical significance between the two groups of cell lines. 11 of the 19 miRNAs read this have an average fold change above 10 between the invasive and less invasive groups. Among Inhibitors,Modulators,Libraries the 11 most differentially expressed miRNAs, miR 141, miR 183, miR 200c, miR 205, miR 203, miR 34a, and miR 375 were down regulated in invasive cell lines when com pared to the normal and less invasive lines conversely, miR 100, miR 125b1, miR 138, and miR 146a were found to be up regulated. Supervised cluster analysis using the miRNA expression values demon strated Inhibitors,Modulators,Libraries that the three groups of breast cancer cell lines can be clearly separated by the expression profiles of the 11 miRNAs.

Quantitative RT PCR analysis of individual miRNAs confirmed that miR 200c, miR 205, miR 375, and miR 146a were indeed differentially expressed between the two groups of breast cancer cell lines. In particular, miR 200c, miR 205, and miR 375 were found to be down regulated more than 100 fold on average. Transient transfection of miR 200c, 205, and 375 inhibits Inhibitors,Modulators,Libraries cell migration and invasion Because miR 200c, miR 205, and miR 375 have been previously reported to affect cell migration or invasion, and were all found to be down regulated in the invasive cell lines, we investigated the effects of transient transfection of these three miRNAs on breast cancer cell migration and invasion.

As shown in Figure 2, transfec tion of miR 200c into the invasive breast cancer cell line MDA MB 231 had the greatest impact on both cell migration and invasion, both of which decreased by ap proximately 50%. Transfection of miR 205 into the MDA MB 231 cells was less effective than miR 200c, but cell migration and invasion were still reduced Inhibitors,Modulators,Libraries by about Inhibitors,Modulators,Libraries 20%. miR 375 was found to play the opposite role in regulating migration and invasion in MDA MB 231 cells as the migration and invasion changed in the op posite direction. The results from these experiments confirmed that these three miRNAs were capable of regulating cell migration and invasion and therefore ultimately affect the invasiveness of breast can cer cells. Differential mRNA expression between invasive and less invasive breast cancer cell lines We performed gene expression microarray analysis using the same set of breast cancer cell lines with the aim of identifying differentially expressed genes that might be target gene candidates for the differentially expressed miRNA. The Affymetrix GeneChip analysis showed that 2412 genes were differentially expressed between normal and less invasive cell inhibitor lines vs. invasive cell lines.