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MicroRNAs are post-transcriptional regulators that control mRNA stability and the translation efficiency of their target genes. Mature microRNAs are approximately 22-nucleotide in length. They mediate post-transcriptional gene regulation by binding to the imperfect complementary sequences (a.k.a. microRNA regulatory elements, MRE) in the target mRNAs. It is estimated that more than one-third of the protein-coding genes in the human genome are regulated by microRNAs. The experimental methods to examine the interaction between the microRNA and its targeting site(s) in the mRNA are important for understanding microRNA functions. The luciferase reporter gene assay has recently been adapted to test the effect of microRNAs. In this chapter, we use a previously identified miR-138 targeting site in the 3′-untranslated region (3′-UTR) of the RhoC mRNA as an example to describe a quick method for testing the interaction of microRNA and mRNA.
MicroRNAs (miRNAs) are endogenously expressed, single-stranded noncoding RNAs (approximately 20–24 nucleotides in length) found in almost all eukaryotic cells. miRNAs have been shown to regulate many developmental and physiological processes, and the deregulation of miRNAs has been linked to a number of disease processes (1, 2). miRNAs constitute an important class of fine-tuning gene expression regulators referred to as “dimmer switches” because of their ability to repress gene expression without completely silencing it. They are post-transcriptional regulators that bind to imperfect complementary sequences (a.k.a. miRNA regulatory element, MRE) on the target messenger RNA transcripts (mRNAs) and usually result in translational repression and gene silencing (2). Animal miRNAs usually bind to sites in the 3′-untranslated region (3′-UTR), whereas plant miRNAs usually bind to coding regions of mRNAs. A number of bioinformatics tools are available to predict the miRNA targeting sequences (3). However, to understand the roles of microRNA in complex biological processes, it is important to experimentally assess the functional relevance of the predicted miRNA targeting site(s).
MicroRNA-138 (miR-138) has been shown to regulate a number of essential biological processes, including the development of the mammary gland (4), regulation of dendritic spine morphogenesis (5), modulation of cardiac patterning during embryonic development (6), and thermo-tolerance acquisition (7). The deregulation of miR-138 has been frequently observed in a number of cancer types, including thyroid cancer (8), lung cancer (9), leukemia (10), and head and neck/oral cancers (11–15). Down-regulation of miR-138 is associated with enhanced RhoC expression and cell migration and invasion in oral cancer cells (12, 16). A targeting sequence for miR-138 has recently been identi fied in the 3′-UTR of the RhoC mRNA (Fig. 1) (12). In this chapter, for illustration, we will test the interaction of miR-138 and its targeting sequence in the 3′-UTR of the RhoC mRNA.
Firefly luciferase is commonly used as a reporter to assess the transcriptional activity in intact cells. The most common application of luciferase reporter gene assay is to examine the regulation of transcriptional activities by promoters and transcription factors. Recently, this assay has also been adapted for testing the effect of miRNA-mediated, post-transcriptional regulation on target genes. This is achieved by engineering a luciferase gene construct containing the predicted miRNA targeting sequence from the target gene (often located in the 3′-UTR). For many human genes, luciferase constructs containing the entire 3′-UTR can be obtained from a number of commercial sources (e.g., OriGene Technologies, Inc, GeneCopoeia, Inc, and SwitchGear Genomics). However, the 3′-UTRs may contain multiple targeting sequences and other regulatory elements. Specific assays to test each miRNA targeting sequences are needed. In this chapter, we describe a quick method to test the interaction of miRNA and the specific target sequence. We also present a simple strategy for creating mutant construct as the negative control.
There are a number of available luciferase reporter vectors, including recently introduced ones that are designed for testing miRNA-mediated gene silencing. In this chapter, we use pGL3-Control Vector (Promega), one of the most commonly used luciferase reporter vectors.
The pRL-TK vector provides constitutive expression of Renilla luciferase. pRL-TK vector co-transfected with firefly luciferase vector provides an internal control for normalization of the transfection efficiency.
pGL3 luciferase reporter constructs are created by cloning the specific miRNA binding sequence (wild-type/mutants) into the XbaI site located at 3′-UTR of pGL3-Control vector.
HeLa is an immortal cell line used for transfection experiments. Other comparable cell lines can also be used based on experimental design.
Dual-luciferase Reporter Assay kit provides an optimized system and all the necessary reagents for the sequential assay of firefly and Renilla luciferase activity.
Other comparable luminometer can also be used.
Chemical-based oligonucleotide synthesis provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence (up to several hundred nucleotide residues). To simplify our cloning strategy, we will take the advantage of vastly available resources for synthesizing oligonucleotides (e.g., Integrated DNA Technologies, Sigma-Genosys). To illustrate, we will design a set of oligonucleotides to test the previously described hsa-miR-138 targeting sequence in the 3′-UTR of the RhoC mRNA (12). Sense and antisense sequences corresponding to a 62-bp fragment from the 3′-UTR of RhoC mRNA (position 1210–1271, NM_175744) will be used. Partial sequences for the XbaI site are appended to the ends of the oligo for creating sticky ends upon annealing. The 5′phosphorylation is also required to facilitate the ligation. The sequences of these oligonucleotides are listed below. The XbaI sites are indicated by bold font, and the seed regions of the hsa-miR-138 targeting site are indicated by underlining.
Insert synthesized oligos into pGL3-Control vector at XbaI site.
The ligation products are ready for transformation. Many different transformation methods could be used. We use XL1-blue super-competent cells from Stratagene to perform transformation. We brie fl y describe the procedure here:
All volumes are multiplied by 3.5 to account for the triplicate samples and loss during pipetting.
This will make the final working concentrations at 100 nM miRNA mimic, 2.5 ng/ μL pGL3 plasmid, and 12.5 pg/ μL pRL-TK.
This work was supported in part by NIH PHS grants (CA135992, CA139596, DE014847) and supplementary funding from UIC CCTS (UL1RR029879). Y.J. is supported by PHS T32DE018381 from NIDCR. We thank Ms. Katherine Long for her editorial assistance.