MiR-100 direct targeting HOXA1 mRNA.

<p>(<b>A</b>) Two predicted miR-99 family targeting sequences (TS1 and TS2) are located in the coding region and the 3′-UTR of HOXA1 mRNA, respectively. The base-pairing (green: microRNA sequence; red: mRNA sequence) and the minimum free energy (mfe) for the binding of miR-100 to the targeting sequences were predicted using the RNAhybrid program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080625#pone.0080625-Kruger1" target="_blank">[26]</a>. (<b>B</b>) Dual luciferase reporter assays were performed to test the interaction of miR-100 and its targeting sequences in the HOXA1 mRNA using constructs containing the predicted targeting sequences (pGL-TS1 and pGL-TS2) and mutated targeting sequences (pGL-TS1m and pGL-TS2m) cloned into the 3′-UTR of the reporter gene. (<b>C</b>) RIP-IP assays were performed to co-IP the Ago2 complexes from cells transfected with either miR-100 mimic or negative control mimic. qRT-PCR assays were performed on RNA samples isolated from the Ago2 co-IP fractions to measure the relative enrichment of the HOXA1 mRNA. Data represent at least 3 independent experiments with similar results. *: p<0.05.