Analysis of the role of let-7 miRNAs in cell proliferation using miScript miRNA Mimics and Cignal Reporter Assays

Introduction 
The let-7 family of miRNAs is widely viewed as playing a role in tumor suppression. In support of this theory, it has been observed that the expression of let-7 family members is downregulated in many cancer types and during tumor progression in comparison to normal tissue (1). It has also been shown that let-7 overexpression causes human cancer cells to decrease cell cycle progression (2). Humans have 10 mature let-7 family sequences (let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, miR-98, and miR-202) that are produced from 13 precursor sequences (3). Interestingly, certain let-7 species are also known to be upregulated in some cancer types (4, 5). These data on the expression of let-7 in various cancers indicate that individual let-7 family members can have different activities.
Members of the E2F family of transcription factors are key regulators of cell-cycle checkpoints in mammalian cells and play important roles in cell proliferation. During cell proliferation, when not bound to pRb (a cell-cycle regulation protein), E2F (along with its binding partner DP-1) mediates the transactivation of E2F target genes that facilitate cell progression to the G1/S transition and S-phase. When cells are not proliferating, E2F family members contribute to transcriptional repression of their target genes. Therefore, the intracellular activity of the E2F family of transcription factors determines the proliferation status of the cell.

Materials and methods
In this study, we used miScript miRNA Mimics and the Cignal Cell Cycle Reporter Assay to characterize the function of individual let-7 family members (let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, and miR-98) during the cell cycle. miScript miRNA Mimics are chemically synthesized, double-stranded RNAs which mimic mature endogenous miRNAs after transfection into cells. Cignal Reporter Assays are reporter constructs that consist of transcription factor responsive elements fused to a reporter gene. Transfection of a miScript miRNA Mimic for an miRNA of interest, followed by reporter analysis, can help to elucidate the target and role of the miRNA. Reduced reporter gene activity after transfection of a mimic provides evidence that the miRNA under study is involved in regulation of the pathway under study. In this study, transfection of a miScript miRNA Mimic for a let-7 miRNA was followed by transfection of the Cignal Cell Cycle Reporter Assay, which contains the firefly luciferase reporter gene under the control of a minimal promoter and tandem repeats of an E2F transcriptional response element. Increased luciferase expression indicates upregulation of the E2F protein and consequently upregulation of E2F target proteins which promote cell proliferation. Decreased luciferase expression indicates downregulation of E2F and E2F target proteins, which results in repression of cell proliferation. 

Results
Our results showed that individual let-7 family members have different activities during cell proliferation (see figure Function of let-7 family members in cell proliferation). Consistent with a role in tumor suppression, let-7a, let-7d, and let-7e inhibit the activity of E2F transcription factors. In contrast, other members of the let-7 family have no significant effect on E2F transcription factors. 

Conclusions 

• These data indicate that members of the let-7 family of miRNAs play differing roles in cell proliferation.
• The combination of synthetic miRNAs and cell-based reporter assays provide a powerful technique for miRNA research.

 

References
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2. Johnson, C. D. et al. (2007) The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res. 67, 7713.
3. Roush, S. and Slack, F. J. (2008) The let-7 family of microRNAs. Trends Cell Biol. 18, 505.
4. Guled, M. et al. (2009) CDKN2A, NF2, and JUN are dysregulated among other genes by miRNAs in malignant mesothelioma -A miRNA microarray analysis. Genes Chromosomes Cancer 48, 615.
5. Lawrie, C.H. et al. (2008) Expression of microRNAs in diffuse large B cell lymphoma is associated with immunophenotype, survival and transformation from follicular lymphoma. J. Cell Mol. Med. 13, 1248.