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

C. elegans.


let-7 regulates RAS expression in human cells

To confirm that let-7 regulates the expression of the human RAS genes, cultured cells were transfected with let-7 miRNA precursors and inhibitors, and RAS protein levels were monitored. HepG2 cells, which do not express let-7 at levels detectable by microarray analysis, were transfected with a let-7a Pre-miR miRNA Precursor Molecule using siPORT NeoFX Transfection Agent. The let-7a Pre-miR miRNA Precursor enters the miRNA pathway and modifies the expression of genes that are regulated by the endogenous miRNA. In three separate experiments, increasing let-7 miRNA levels decreased RAS expression by ~70% compared to cells transfected with a negative control (Pre-miR Negative Control #1 miRNA) (Figures 1AB), while GAPDH and p21 protein levels did not change (data not shown).

Figure 1. let-7 Influences the Expression of RAS in Human Cells. HepG2 cells were transfected in 24 well plates using siPORT NeoFX (Ambion) with either 30 nM let-7a Pre-miR miRNA Precursor Molecule (Ambion) or a negative control miRNA inhibitor (AB). HeLa cells were transfected in 24 well plates using Lipofectamine 2000 (Invitrogen) with either 30 nM let-7a Anti-miR miRNA Inhibitors (Ambion) or a negative control miRNA inhibitor (CD). Three days post-transfectio
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