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The let-7 microRNA Family Regulates RAS: Implications for Development and Oncogenesis

he luciferase reporter gene. The control plasmid did not contain any insert. HeLa cells were transfected in 12 well plates using Lipofectamine 2000 (Invitrogen) with the plasmids described. 24 h post-transfection, cells were harvested and assayed using the Dual-Luciferase assay (Promega). (A) Fold induction of reporter gene activity was expressed relative to the negative control vector. (B) To show specificity of the interaction between let-7 and RAS 3' UTRs, similar experiments were performed with an additional cotransfection of let-7 miRNA inhibitors (let-7a Anti-miR miRNA Inhibitors [Ambion] or a negative control miRNA inhibitor [Ambion]). Modified with permission from Cell 120: 635647.


To verify that this result is related to let-7 expression in HeLa cells, the luciferase reporter vectors were co-transfected with the let-7 miRNA inhibitor or a negative control miRNA inhibitor. As seen in Figure 2B, decreasing let-7 activity in HeLa cells de-repressed expression of the reporter gene. In these assays, luciferase activity increased by 2- to 2.5-fold compared to cells transfected with the negative control inhibitor. Along with the identification of several potential LCSs in the RAS 3'UTR, the data presented in Figure 2 indicate that let-7 miRNA can mediate RAS expression through the RAS 3'UTR.


let-7 in Human Lung Cancer

In a related research project, Ambion's miRNA microarray technology (167 miRNA prob
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