Experimental validation of miRNA targets

Abstract

MicroRNAs are natural, single-stranded, small RNA molecules that regulate gene expression by binding to target mRNAs and suppress its translation or initiate its degradation. In contrast to the identification and validation of many miRNA genes is the lack of experimental evidence identifying their corresponding mRNA targets. The most fundamental challenge in miRNA biology is to define the rules of miRNA target recognition. This is critical since the biological role of individual miRNAs will be dictated by the mRNAs that they regulate. Therefore, only as target mRNAs are validated will it be possible to establish commonalities that will enable more precise predictions of miRNA/mRNA interactions. Currently there is no clear agreement as to what experimental procedures should be followed to demonstrate that a given mRNA is a target of a specific miRNA. Therefore, this review outlines several methods by which to validate miRNA targets. Additionally, we propose that multiple criteria should be met before miRNA target validation should be considered "confirmed."

Figure 1

Proposed flow diagram for microRNA validation.

Figure 2

miR-155 can specifically inhibit luciferase reporter activity. CHO cells were cotransfected with pMIR/883/5′→3′, pMIR/883/3′→5′, pMIR/883/5′→3′/ΔmiR-155, pRL-CMV and 50 nM of a given miRNA mimic. Forty-eight hours after transfection luciferase activities were measured. F-luc activity was normalized to r-luc expression and the mean activities ± S.E. from five independent experiments are shown. (*p<0.01 versus CHO cells transfected with miR-control mimic)

Figure 3

Mature miR-155 and the hAT₁R are co-expressed in VSMC. The TaqMan® miR-155 and TaqMan® AT₁R gene expression assay kits were utilized to quantify the expression of miR-155 and hAT₁R levels in VSM and CHO cells. The expression of mature miR-155 or hAT₁R relative to 18S rRNA was determined using SYBR green real-time quantitative PCR assay as described [21].

Figure 4

miR-155 is expressed in endothelial and VSM cells. A. Representative example of the distribution of mature miR-155 after in situ hybridization analysis with an LNA miR-155-specific antisense probe. In the arteriole, the endothelial (small arrow) and VSM (large arrow) cells abundantly express miR-155. B. No signal was evident in the arteriole when the serial section was probed with a scrambled probe.

Figure 5

miR-155 regulates the endogenous hAT₁R expression in VSMCs. VSMCs were either mock transfected or transfected with the miRNA mimics (A) or antisense miRNAs (B) as indicated. 48 hrs after transfection, the cells were utilized for AT₁R radioreceptor binding assays as described [21]. The data have been normalized for protein and transfection differences and represent specific binding. The values are shown as percent of maximal specific binding of mock transfected hPFBs and represent the mean ± S.E. from four independent experiments (*p<0.001 vs. mock transfected cells).

Figure 6

Anti-miR-155 enhances Ang II-induced signaling in VSMCs. VSMCs were transfected with the anti-miRNA oligonucleotides as indicated. A. Ang II-induced phosphoERK1/2 experiments were performed utilizing serum-starved, transiently-transfected cells as described [21,22]. A representative immunoblot is shown. Results are representative of four independent experiments. B. The quantitation of Ang II-(1 μM for 5 min) induced ERK1/2 phosphorylation was determined by densitometry. Values are expressed as a percent of the maximal phosphorylation of ERK1/2 in response to Ang II in mock transfected cells and represents the mean ± S.E. from four independent transfection experiments (*p<0.01).