Although the role of miRNAs and other non-coding RNAs in the regulation of cellular function is only beginning to be understood, the diagnostic and therapeutic potential of these regulatory RNAs is already widely recognized and accepted [30
]. Indeed, a number of pre-clinical trials of miRNAs are currently in progress [10
]. The initial results of these studies appear to be generally supportive of the potential utility of miRNAs in the early diagnosis of cancer [31
], but significant questions remain with respect to their potential therapeutic value.
One of the challenges to the therapeutic use of miRNAs is technical in nature and deals with the problems of packaging and delivering miRNAs to cancer cells. Significant progress has been made in this area in recent years and all indications are that these technical challenges are not insurmountable [33
]. The second challenge is more scientific in nature and centers around our current inability to precisely predict the molecular consequences of modulating miRNA levels on cell function. While the exogenous administration of miRNAs (or “antagomirs”) have been shown to be capable of effectively regulating targeted oncogenes and tumor suppressor genes in specific cellular contexts [30
], the indirect effects of perturbing miRNA levels remains difficult to predict. Since this molecular unpredictability could translate into clinically unanticipated side-effects, it may significantly mitigate the intended therapeutic benefits [11
]. It is generally agreed that increased therapeutic predictability of miRNAs and other regulatory RNAs will require a better understanding of the processes underlying the molecular function of these molecules in vivo
In an initial effort to systematically dissect the molecular consequences of exogenously modulating levels of miRNA in ovarian cancer cells, we transfected two distinct human miRNAs, miR-7 and miR-128, into a well-characterized ovarian cancer cell line and monitored the consequent changes on endogenous levels of gene expression. These specific miRNAs were chosen because modulations in their levels of expression have both been previously correlated with ovarian cancer onset/progression [14
We found that transfection of miR-7 and miR-128 resulted in significant changes in expression of hundreds to thousands of genes. Consistent with the fact that miR-7 is predicted to target fewer genes than miR-128 (using miRanda, TargetScan or PicTar), we found that the number of genes differentially expressed after miR-7 transfection was substantially less (approximately one-third) than that after miR-128 transfection. To determine if the large number of genes differentially expressed after the miRNA transfections were likely due to the direct regulatory action of the miRNAs, we computed the proportions of differentially expressed genes that are direct targets of miR-7 or miR-128 regulation as predicted by three different target prediction algorithms. On average, the proportion of differentially expressed genes predicted to be directly regulated by either miRNA was less than one-fifth indicating that most of the changes in gene expression induced by the miRNA transfections are indirect.
One recently described model to account for indirect effects of miRNA transfection postulates that transfected miRNAs may out-compete endogenous miRNAs for available RISCs [20
] and consequently lead to up-regulation of targets of these endogenous miRNAs. This model may be relevant with respect to our miR-128 transfection experiment where the majority (70%) of differentially expressed genes displayed a significant increase in gene expression.
Consistent with the predictions of the RISC-competition model, we found that ~60% of the mRNAs enriched for binding sites of those miRNAs most highly expressed in pre-transfected HEY cells were significantly over-represented among mRNAs up-regulated after miR-128 transfection. While these results do not exclude alternative explanations, they are consistent with the hypothesis that at least some of the genes displaying increased expression after miR-128 transfection may be explained by the RISC-competition model. The model does not explain the >700 genes down regulated after miR-128 transfection or the fact that the vast majority of genes differentially expressed after miR-7 transfection were down-regulated. It is also not immediately apparent why the changes in global patterns of gene expression induced by the displacement of endogenous miRNAs should be functionally coordinated.
Perhaps the most commonly observed mechanism underlying coordinated changes in global patterns of gene expression involves the modulation of centralized or “hub” regulatory genes [35
]. Hub genes have the potential to exert control on suites of downstream genes thereby inducing cascades of regulatory changes in patterns of gene expression [36
]. Network analyses revealed that a substantial number of genes differentially expressed after miR-7 or miR-128 transfection are regulatory hubs capable of controlling many of the down-stream genes that were differentially expressed after miR-7 and miR-128 transfection. These findings indicate that miRNAs can both directly and indirectly induce changes in cellular regulatory networks.
In general, our experiments demonstrate that exogenous expression of miRNAs in ovarian cancer cells induces regulatory changes far in excess of those on the predicted targets. From the clinical perspective, this begs the question as to whether miRNAs are appropriate agents for ovarian cancer therapy. Clearly, if the clinical intent is to precisely silence a specific gene and if the collateral indirect effects induced by miRNAs are counter-therapeutic, the answer would be “no”. However, it is important to keep in mind that miRNAs have been evolving as essential components of the eukaryotic regulatory system for millions of years. Thus, the network of regulatory effects exerted by miRNAs is unlikely to be random and indeed may eventually provide clinicians with a strategy to treat ovarian cancer cells from a systems rather than a single gene perspective.
Our pathway enrichment analysis of the pathways most significantly affected by miR-7 and miR-128 transfection predicts a significant difference in the functional consequence of exogenous perturbations in levels of these two miRNAs. The pathways most significantly affected by miR-7 transfection are predicted to be involved with G1/S-phase cell cycle control as well as cell adhesion and other developmental networks previously associated with epithelial-mesenchymal transitions (EMT) and other processes linked with metastasis [37
]. In contrast, the pathways most significantly affected by miR-128 transfection are more focused on mitotic-phase cell cycle control and other processes commonly linked with cellular replication [38
]. Experimental analyses of cell adhesion and cell cycle control of cells transfected by miR-7 and miR-128 respectively were found to be remarkably consistent with these computational predictions. Although both sets of functions are generally characteristic of ovarian cancer cells, particular pathways may be relatively more important in particular tumors.
The therapeutic impact of individual miRNA treatments will ultimately be evaluated at the clinical level. However, the more we can learn about the processes underlying functional specialization of different families of miRNAs in experimentally controlled conditions, the better positioned we will be to interpret the significance of changes of these regulatory RNAs in individual tumors and to rationally select specific miRNAs for possible use in targeted cancer therapy.