Fig. 2. Validation of miR-30a-5p predicted targets in an HNSCC line.

(A) Selected miR-30 target genes were validated by qRT-PCR measurement in UM-SCC-46 cells transfected with miRNA negative control (neg Con), miR-30a, or anti-miR-30a-5p oligonucleotide for 72 hrs. The mean of three independent experiments, ±SEM; * denotes p< 0.05 by Student’s t-test. (B) Base pairing of miR-30a-5p with 3’ UTR of target mRNAs was predicted by Mfold (http://unafold.rna.albany.edu/?q=mfold). Bases in red depict binding of miR-30a-5p seed sequence. Horizontal lines with delta symbols mark bases in the mRNA 3’ UTR that were deleted in cloned 3’-mutant UTR vectors to ablate miR-30-5p regulation. (C) Relative luciferase activity after co-transfection of UM-SCC-46 cells with miR-30a-5p or anti-miR30a-5p and vectors containing wild-type 3’-UTR (left) or 3’-mutant UTR (right) cloned behind a renilla luciferase reporter gene. Results for positive control vector (Pos Con) containing 5x miR-30-5p binding sites and a negative GAPDH 3’-UTR control are also displayed. All data represent the mean of three independent experiments and error bars represent SEM. * denotes p< 0.05 by Student’s t-test. (D) Protein expression for selected miR-30 targets and (E) downstream phospho- and total signal protein expression examined by Western blot using whole cell lysates isolated from Primary Human Oral Keratinocytes (HOK) and UM-SCC-46 cells 72 h after transfection with miR-30a-5p or anti-miR30a-5p oligonucleotides. β-tubulin is a loading control.