Abstract
BackgroundmicroRNAs (miRNAs) are small and non-coding RNAs which play critical roles in physiological and pathological processes. A number of methods have been established to detect and quantify miRNA expression. However, method for high-throughput miRNA function detection is still lacking.Principal FindingsWe describe an adeno-associated virus (AAV) vector-based microRNA (miRNA) sensor (Asensor) array for high-throughput functional miRNA profiling. Each Asensor contains a Gaussia luciferase (Gluc) and a firefly luciferase (Fluc) expression cassette to sense functional miRNA and to serve as an internal control respectively. Using this array, we acquired functional profiles of 115 miRNAs for 12 cell lines and found “functional miRNA signatures” for several specific cell lines. The activities of specific miRNAs including the let-7 family, miR-17-92 cluster, miR-221, and miR-222 in HEK 293 cells were compared with their expression levels determined by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). We also demonstrate two other practical applications of the array, including a comparison of the miRNA activity between HEK293 and HEK293T cells and the ability to monitor miRNA activity changes in K562 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA).Conclusions/SignificanceOur approach has potential applications in the identification of cell types, the characterization of biological and pathological processes, and the evaluation of responses to interventions.
Highlights
MicroRNAs are a species of non-coding RNAs that are 18-25 nucleotides in length [1] and regulate the expression of one third of the total genes in human cells through perfect or imperfect base pairing with specific sequences of mRNA [2]
The miRNA Asensor plasmid was constructed based on an associated virus (AAV) vector plasmid pAAV2neo [20] and contained two independent expression cassettes encoding firefly luciferase (Fluc) and Gaussia luciferase (Gluc), respectively (Fig. 1A)
The results showed that the expression of Fluc and Gluc exponentially correlated with the levels of loaded Asensor virus (Fig. S1A, B), indicating that the loaded Asensors were not saturated for reporter gene expression within the given range
Summary
MicroRNAs (miRNAs) are a species of non-coding RNAs that are 18-25 nucleotides (nt) in length [1] and regulate the expression of one third of the total genes in human cells through perfect or imperfect base pairing with specific sequences of mRNA [2]. The interaction of miRNAs with their target sequences leads to the modulation of gene expression through decreases in mRNA stability and the repression of mRNA translation [6]. These interactions play a critical role in the processes of cell differentiation and proliferation [7], cell death, including apoptosis [8], and tumorigenesis [9]. MiRNA expression levels do not always reflect the authentic activity of each miRNA [15,16] The latter correlates with mature miRNA functions and is often affected by multiple steps along the miRNA pathway, including the miRISC forming efficiency, the binding affinity of miRNA to the target sequences at the 39UTR, and the inhibition efficiency through miRISC binding [17]. Method for high-throughput miRNA function detection is still lacking
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