Fig. 4: Candidate target analysis of miR-181c-5p.

Targets of miR-181c-5p were predicted using the TargetScan, miRDB, DIANA, and miRSystem tools, and analyzed using Venn tools. Seventeen direct target genes of miR-181c-5p that may be involved in the formation and maturation of IPCs were found in the Venn diagram (a). Combining previous reports, smad7 and TGIF2 were selected to test the regulatory relationship after miR-181c-5p overexpression in hiPSCs. The location and sequences of the miR-181c-5p target site in the 3′UTR of human smad7 and TGIF2 (b). The sequence of human miR-181c-5p is indicated, along with mutations introduced in the target site (underlined nucleotides) for generating the mutated reporter construct (b). 293T cells were co-transfected with luciferase 3’UTR reporter vectors containing 3’UTR fragments of wild-type smad7/TGIF2 and mutated smad7/TGIF2 in the presence of miR-181c-5p mimic or negative control. Luciferase activity was assayed 24 h after transfection (c, n = 8). All luciferase values were normalized to Renilla luciferase. The data are expressed as the mean ± SD of three independent experiments. ^††p ≤ 0.001, relative to the negative control (NC). ns, not statistically significant. d, e Real-time PCR and western blot analysis of mRNA and proteins expressed by gene targets of miR-181c-5p following miR-181c-5p overexpression in 293T cells. The data are expressed as the mean ± SD of three independent experiments (n = 8). ^**p ≤ 0.001, relative to the hiPSCs control (Mock).