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To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell-cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, beta-catenin, beta1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell-cell adhesion.

Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.

5 páginas, 4 figuras.-- Letter., The tumour microenvironment can be a potent carcinogen, not only by facilitating cancer progression and activating dormant cancer cells, but also by stimulating tumour formation1. We have previously investigated stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours2, and found that MMP-3 can cause epithelial–mesenchymal transition (EMT) and malignant transformation in cultured cells3, 4, 5, and genomically unstable mammary carcinomas in transgenic mice3. Here we explain the molecular pathways by which MMP-3 exerts these effects: exposure of mouse mammary epithelial cells to MMP-3 induces the expression of an alternatively spliced form of Rac1, which causes an increase in cellular reactive oxygen species (ROS). The ROS stimulate the expression of the transcription factor Snail and EMT, and cause oxidative damage to DNA and genomic instability. These findings identify a previously undescribed pathway in which a component of the breast tumour microenvironment alters cellular structure in culture and tissue structure in vivo, leading to malignant transformation., This work was supported by grants from the OBER office of the Department of Energy and an Innovator award from the Department of Defense (to M.J.B.) and from the National Institutes of Health (to M.J.B. and Z.W.), and by fellowships from the American Cancer Society (D.C.R.), the National Cancer Institute (L.E.L.), the Department of Defense (H.L. and C.M.N.) and the California Breast Cancer Research Program (J.E.F).

Background Micro(mi)RNAs regulate gene expression through translational attenuation and messenger (m)RNA degradation, and are associated with differentiation, homeostasis and disease. Natural miRNA target recognition is determined primarily by perfect complementarity in a seed region (nucleotide positions 2 to 7) with additional interactions contributing in a sequence- and target-specific manner. Synthetic miRNA target analogs, which are fully complementary, chemically modified oligonucleotides, have been used successfully to inhibit miRNA function. Results In this paper, we present a first systematic study to evaluate the effect of mismatches in the target site on synthetic inhibitor activity. Panels of miRNA inhibitors containing two-nucleotide mismatches across the target site were tested against three miRNAs (miR-21, miR-22 and miR-122). The results showed that the function of inhibitors vary as mismatch positions in the inhibitors change. Conclusions The data indicate that features important for natural miRNA target recognition (such as seed region complementarity) are also important for inhibitor functionality. In addition, base pairing at a second, more 3' region appears to be equally important in determining the efficacy of synthetic inhibitors. Considering the importance of these inhibitor regions and the expression of closely related miRNA sequences will enable researchers to interpret results more accurately in future experiments.

Nat. Methods 3, 199–204 (2006). In Figure 3 of the original article, the genes in panels a–c were incorrectly identified. The correct gene names are: (a) Ppyr/LUC siRNA A, (b) ALPPL2 and (c) Ppyr/LUC siRNA B. The figure legend should read: Figure 3 | Systematic single base pair–mismatch analysis of siRNA functionality.

Addendum: 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets