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. 2012 Oct 2;7(10):e46773. doi: 10.1371/journal.pone.0046773

Figure 2. MiR-103, miR-128 and miR-137 directly reduce NF1 reporter protein expression.

Figure 2

(A) Schematic of miRNA plasmids and their expression levels in HEK293 cells. Representative gel showing the products of RT-PCR reactions amplified with primers specific for mature miR-103, miR-128, and miR-137 in HEK293 cells transfected with scramble or miR-128 vectors. The amount of starting template for each condition was equilibrated relative to U6 RNA. Cycles were falling within the linear range of amplification for each primer pair. (B) Schematic of Nf1 3′-UTR reporter construct and miRNAs tested for Nf1 3′-UTR regulation of expression. HEK293 cells were co-transfected with both the reporter gene (0.2 µg/reaction) and miRNA expression vectors (pri-mir-103, pri-mir-128, pri-mir-137, pri-mir-27, pri-mir-182 and pri-mir-153) (0.8 µg/reaction) and luciferase activity was measured 48 hours later. The fold change from control values as well as the P-value for each miRNA condition is presented in the table. These assays demonstrated that only miR-103, miR-128 and miR-137 significantly reduce NF1 reporter protein levels. Subsequently, mutagenesis of the predicted miR-103, miR-128 and miR-137 binding sites alleviated this inhibitory effect indicating that miR-103, miR-128 and miR-137 directly suppress Nf1 expression by targeting the identified seed regions in the Nf1 3′-UTR. The average value of three single (scramble 1, scramble 2, miR-218) and two double (miR-101/181, miR-218/377) miRNA constructs expressing miRNA predicted not to bind Nf1 3′-UTR were used as controls for the single or double expression constructs, respectively. Data show the mean ± s.e.m from 6 independent transfections (*, P<0.05; **, P<0.01).