Figure 5.

PAX3 is subject to human–specific regulation of microRNA(s) during muscle differentiation. (a) Flowchart for narrowing down the microRNAs that specifically regulate human PAX3, but not mouse Pax3. (b) 16 candidates predicted by TargetScan were examined by luciferase reporter assays. Human PAX3, or mouse Pax3 3ʹUTR was cloned into the miR-Sens plasmid. These plasmids were transfected into HEK293T cells, and subsequently, microRNA mimics were transfected. Six microRNAs that showed suppression on the human construct, but not on the mouse construct are marked with stars. (c) Three microRNAs were further eliminated due to the absence of expression during the human MSC muscle differentiation. qRT-PCR was performed to quantify the level of microRNAs throughout the differentiation process. (d) Mutagenesis at the microRNAs target site. Both miR-495 and miR-382 downregulated the mouse Pax3 reporter when the sequence was changed into the seed sequence of humans. Both also caused upregulation of the human PAX3 reporter when the sequence was mutated to that of mice. miR-186 did not cause downregulation of mutated mouse reporter, even though it did not suppress the mutated human reporter. *, p < 0.05; **, p < 0.01; ***, p < 0.001.