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MicroRNAs have emerged in recent years as important regulators of cell function in both normal and diseased cells. MiRNAs coordinately regulate large suites of target genes by mRNA degradation and/or translational inhibition. The mRNA target specificities of miRNAs in animals are primarily encoded within a 7 nt “seed region” mapping to positions 2–8 at the molecule's 5′ end. We here combine computational analyses with experimental studies to explore the functional significance of sequence variation within the seed region of human miRNAs. The results indicate that a substitution of even a single nucleotide within the seed region changes the spectrum of mRNA targets by >50%. The high functional cost of even single nucleotide changes within seed regions is consistent with their high sequence conservation among miRNA families both within and between species and suggests processes that may underlie the evolution of miRNA regulatory control.

BackgroundMicroRNAs (miRNAs) are a class of small RNAs that have been linked to a number of diseases including cancer. The potential application of miRNAs in the diagnostics and therapeutics of ovarian and other cancers is an area of intense interest. A current challenge is the inability to accurately predict the functional consequences of exogenous modulations in the levels of potentially therapeutic miRNAs.MethodsIn an initial effort to systematically address this issue, we conducted miRNA transfection experiments using two miRNAs (miR-7, miR-128). We monitored the consequent changes in global patterns of gene expression by microarray and quantitative (real-time) polymerase chain reaction. Network analysis of the expression data was used to predict the consequence of each transfection on cellular function and these predictions were experimentally tested.ResultsWhile ~20% of the changes in expression patterns of hundreds to thousands of genes could be attributed to direct miRNA-mRNA interactions, the majority of the changes are indirect, involving the downstream consequences of miRNA-mediated changes in regulatory gene expression. The changes in gene expression induced by individual miRNAs are functionally coordinated but distinct between the two miRNAs. MiR-7 transfection into ovarian cancer cells induces changes in cell adhesion and other developmental networks previously associated with epithelial-mesenchymal transitions (EMT) and other processes linked with metastasis. In contrast, miR-128 transfection induces changes in cell cycle control and other processes commonly linked with cellular replication.ConclusionsThe functionally coordinated patterns of gene expression displayed by different families of miRNAs have the potential to provide clinicians with a strategy to treat cancers from a systems rather than a single gene perspective.

BackgroundDocumented changes in levels of microRNAs (miRNA) in a variety of diseases including cancer are leading to their development as early indicators of disease, and as a potential new class of therapeutic agents. A significant hurdle to the rational application of miRNAs as therapeutics is our current inability to reliably predict the range of molecular and cellular consequences of perturbations in the levels of specific miRNAs on targeted cells. While the direct gene (mRNA) targets of individual miRNAs can be computationally predicted with reasonable degrees of accuracy, reliable predictions of the indirect molecular effects of perturbations in miRNA levels remain a major challenge in molecular systems biology.ResultsChanges in gene (mRNA) and miRNA expression levels between normal precursor and ovarian cancer cells isolated from patient tissue samples were measured by microarray. Expression of 31 miRNAs was significantly elevated in the cancer samples. Consistent with previous reports, the expected decrease in expression of the mRNA targets of upregulated miRNAs was observed in only 20-30% of the cancer samples. We present and provide experimental support for a network model (The Transcriptional Override Model; TOM) to account for the unexpected regulatory consequences of modulations in the expression of miRNAs on expression levels of their target mRNAs in ovarian cancer.ConclusionsThe direct and indirect regulatory effects of changes in miRNA expression levels in vivo are interactive and complex but amenable to systems level modeling. Although TOM has been developed and validated within the context of ovarian cancer, it may be applicable in other biological contexts as well, including of potential future use in the rational design of miRNA-based strategies for the treatment of cancers and other diseases.

The molecular basis of cancer is not merely the consequence of structural and/or regulatory mutations in genes, but additionally to disruptions in networks of regulatory interactions existing among these genes and other components of the genome. Disruptions in network relationships may manifest as the loss, gain or reversal of functionally significant interactive gene relationships in cancer cells. In this study, we first employ an unsupervised (Pearson correlation) approach to quantitatively estimate the overall change in network relationships between precursor (control) ovarian surface epithelial cells and ovarian cancer epithelial cells. We find that ovarian cancer cells display a significant overall reduction in correlated gene network interactions relative to normal precursor cells reflective of an overall loss of regulatory control.  We next focus on gene relationships that qualitatively change between normal and cancer samples. We find that biological processes significantly over represented among differentially expressed genes are substantially different from those associated with genes involved in qualitatively disrupted network interactions. Our findings provide novel insights into the processes underlying ovarian cancer and identify a potential new class of genes for targeted therapy.