More than 200 miRNAs have been cloned and sequenced from human and mouse samples. Each miRNA apparently regulates multiple genes, and miRNAs are thought to regulate as many as 10% of human genes [1]. Several reports predict mRNA target sites for miRNAs [2,3]. A handful of these predicted sites have been verified by introducing miRNAs into cells and monitoring the expression of the putative target gene, but thousands of predicted sites remain to be verified. To facilitate these studies, Ambion has developed a series of precursor miRNAs (Pre-miR™ miRNA Precursors) and a series of miRNA inhibitors (Anti-miR™ miRNA Inhibitors).

In transfection experiments, the effects of introducing, increasing, or reducing the level of a given human or mouse miRNA in the cell can be assessed by measuring the expression level of the putative target gene. Reduced expression of the target gene in Pre-miR miRNA-transfected cells or increased expression of the target gene in Anti-miR miRNA Inhibitor-transfected cells is consistent with the prediction that the miRNA regulates the expression of a given gene. In addition to studying the impact of miRNAs on the expression of single genes, the Pre-miR miRNA Precursors and Anti-miR miRNA Inhibitors can be used to identify miRNAs that participate in various cellular processes.

Pre-miR miRNAs--Synthetic miRNAs

We investigated whether RNA molecules modeled after miRNA precursors could enter the miRNA pathway and regulate translation. Using an miRNA target containing a luciferase reporter to monitor miRNA activity, we tested three different miRNA precursor designs for five specific miRNAs. All three synthetic precursor miRNA designs resulted in decreased reporter activity, with Design #3 providing the strongest miRNA activity (i.e. inhibition of a target reporter construct) (Figure 1). Based on these and other similar experiments, Design #3 was used to develop Ambion's Pre-miR miRNAs.

Figure 1. Comparison of Synthetic Precursor miRNA Designs. In 24 well plates, ~40,000 HeLa cells were co-transfected with luciferase reporter constructs bearing targets sites for either miR-10, miR-33, miR-124, miR-19, or miR-130, and 10 nM of either a negative control precursor miRNA (not shown) or one of three different precursor miRNA designs for the appropriate miRNA (#1, #2, #3). The cells were assayed for luciferase activity 48 hours post-transfection. The reduction in luciferase activity in the Pre-miR miRNA-transfected cells was measured relative to the luciferase activity in the negative control-transfected cells. The data are averages of triplicate transfections.

We also wanted to determine whether Pre-miR miRNAs can regulate endogenous genes whose expression patterns are known to be modulated by miRNAs. Design #3 was used to create synthetic precursors of miR-1b, let-7a, and miR-196, known to regulate expression of G6PD [3], Caspase 3 [2], and HOXB8 [4], respectively. To find an informative cell model, we used an miRNA array protocol to screen miRNA expression levels in six common cell types (Figure 2) and chose three that had very low levels of miR-1b, let-7a, and miR-196. We then transfected the selected cells with the three Pre-miR miRNAs listed above and measured the expression of the target genes in miRNA- and negative control-transfected cells. Consistent with published reports, we found that cells transfected with the appropriate Pre-miR miRNAs exhibited reduced activity or expression of the target genes by as much as 80% (Figure 3).

Figure 2. Relative miRNA Expression Among Cell Types. The miRNA expression profiles of seven common cell types were assessed using the miRNA array procedure. miRNAs that generate very high signal on the microarray are denoted by ++++, high-signal miRNAs are denoted by +++, medium-signal miRNAs are denoted by ++, and low-signal miRNAs are denoted by +. A "0" indicates an miRNA with signal that does not exceed that of negative control elements on the microarray. Here a subset of miRNAs expressed in these cells is shown.

Figure 3. Reduction in Endogenous Gene Expression by Synthetic Pre-miR™ miRNA. HeLa, HepG2, and A549 were transfected with Pre-miR miRNAs for hsa-mir-1b (miR-1b), hsa-let-7a (let-7a), and hsa-mir-196 (miR-196), respectively, using siPORT™ NeoFX™ Transfection Agent (Ambion). Reduction in endogenous enzyme activity or gene expression by each of the Pre-miR miRNAs was expressed relative to cells transfected with a negative control Pre-miR. Expression of G6PD (miR-1b) and Caspase 3 (let-7a) were measured using enzymatic assays specific to the two target proteins. Expression of HOXB8 was monitored using real-time PCR as miR-196 has been shown to cleave the HOXB8 mRNA [4].

miRNA Inhibitors

Two recent publications describe the use of antisense RNA molecules to inhibit the activities of specific miRNAs [5, 6]. The activities of the endogenous miRNAs were measured after co-transfecting cells with individual reporter constructs that included an miRNA target site as described [6]. High miRNA activity would result in lower reporter gene activity, and inhibiting miRNA activity would increase reporter gene activity. On average, the Anti-miR miRNA Inhibitors induced an approximately 4-fold increase in the expression of the reporter relative to cells co-transfected with a negative control inhibitor and the various miRNA reporter plasmids (Figure 4). Two negative control samples were included in this experiment to emphasize the specificity of these results: a luciferase reporter construct that does not contain any miRNA target sites and a luciferase reporter construct containing a target site for a brain specific miRNA not expressed in HeLa cells. Both had high luciferase activity as expected.

Figure 4. Enhanced Expression of miRNA-regulated Genes by Anti-miR Inhibitors. HeLa cells (5 x 104 cells/well) were plated in 24 well plates. Duplicate samples were co-transfected with luciferase reporter constructs that contain target sites for miRNAs (e.g. Luc 23=luciferase reporter with miR-23 target site) and the corresponding Anti-miR inhibitors (NC=negative control Anti-miR Inhibitor). 24 hours post-transfection, cells were assayed for luciferase expression. Reduction in luciferase activity was measured relative to luciferase activity in the negative controls (Luc and Luc124). Error bars indicate the range of luciferase activity for 3 replicates.

Screening with miRNA Libraries

While establishing links between miRNAs and the genes that they regulate is important, perhaps a more interesting application for Ambion's Pre-miR miRNA Precursors and Anti-miR miRNA Inhibitors is in screening experiments. Transfecting cells with one of Ambion's Pre-miR miRNAs will create a cell population expressing relatively high levels of that specific miRNA. Conversely, cells can be depleted of a specific miRNA by transfection with one of the Anti-miR miRNA Inhibitors. The transfected cells can then be monitored for a phenotypic response making it possible to identify miRNAs that are involved in various biological processes.

As an example, we transfected HeLa cells with inhibitors targeting 90 different human miRNAs. Cell counting revealed that inhibiting miR-31, miR-190, or miR-218 resulted in reduced cell proliferation (bars below the horizontal shaded area), while inhibiting miR-21 and miR-24 increased cell proliferation (bars above the horizontal shaded area) (Figure 5). Interestingly, the cells transfected with the miR-31 inhibitor become elongated and display thin membrane protrusions similar in appearance to neurite outgrowths (Figure 5, left inset). The negative control data was identical to the nontransfected control (Figure 5, right inset). Additional screening experiments using groups of Pre-miR miRNA and Anti-miR miRNA Inhibitors will undoubtedly reveal miRNAs that are involved in a broad range of cellular processes.

Figure 5. Identification of miRNAs that Alter Cell Proliferation. HeLa cells (5 x 10 3) were transfected with individual Anti-miR miRNA Inhibitors in triplicate using siPORT™ NeoFX™ Transfection Agent (Ambion). 72 hr post-transfection, cells were fixed and stained with propidium iodide to count total cell number (TTP LabTech Acumen Explorer™). Cells were then stained for ß-actin using immunofluorescence.

The horizontal shaded area represents the normal range of cell number for this cell type, as exemplified by cells transfected with a negative control that does not affect cell proliferation (second bar from the right). The right inset shows the morphology of these control cells. The left inset shows the morphology of cells transfected with an Anti-miR Inhibitor to miR-31.

Pre-miR miRNA and Anti-miR miRNA Inhibitors

We have developed ready-to-use synthetic precursor miRNAs and antisense miRNAs for use in transfection experiments. These are valuable tools for functional analyses of miRNA activity in a wide variety of biological systems.