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. 2013 Sep 24;12(22):3537–3546. doi: 10.4161/cc.26592

Figure 2.

Figure 2.

MiR-141 posttranslationally targets BMI1 and upregulates p16 expression at the mRNA level. (A) miR-141 targets PcG proteins BMI1 and EZH2, and PRC activities as shown by western blot analysis of EZH2 and BMI1, and H3K27Me3 and H2AK119Ub in miR-141 overexpressing and control MRC5 cells. The BMI1 and EZH2 expression was normalized to β-actin while H2AK119Ub and H3K27Me3 levels were normalized to total H2A and H3, respectively. (B) MiR-141 targets BMI1 via 3′UTR sequences. Wild-type and mutant miR-141 target site was cloned in pLS-3′UTR reporter vector and the normalized activity of 3′UTR reporters was determined as described in the “Materials and Methods”. Result shows dose-dependent decrease in the luciferase activity of wild-type but not the mutant 3′UTR reporters. The error bars represent the means ± SD of 3 independent experiments. (C) Relative mRNA levels of BMI1 and p16INK4a were analyzed by qRT-PCR from MRC5-derived control and miR-141 overexpressing cells as indicated. (D) Binding of BMI1 and H3K27Me3 to p16INK4a promoter was determined using a ChIP assay in the indicated set of MRC5 stable cell lines. Error bars represent ± SD *P < 0.05.