Research Paper Volume 13, Issue 23 pp 25342—25364

Figure 5. Hsa-miR-4535 targets Smad4. (A) MicroRNA expression in HS68 cells treated with galangin was assessed using microarrays. (B) Venn diagram depicting potential mRNA candidates that may be regulated by hsa-miR-4535 based on miRDB and TargetScanHuman databases. Sequence analysis of putative miRNA-binding sites in Smad4-3′-UTR mRNA for hsa-miR-4535. Matches are indicated by straight lines. (C) HS68 cells were co-transfected with Smad4-3′-UTR-WT (wild-type; 0.5 μg/mL) plus hsa-miR-4535 mimic (20 nM) or Smad4-3′-UTR-MT (mutant; 0.5 μg/mL) plus hsa-miR-4535 mimic (20 nM) for 24 h. Luciferase activity was determined and normalized to that of Renilla. Quantification of the results is shown (n = 3); ^***P < 0.001, versus the Smad4-3′-UTR-MT plus hsa-miR-4535 mimic group. G1 = control, G2 = galangin (10 μM), G3 = galangin (30 μM).