Research Paper Volume 9, Issue 11 pp 2376—2396

An expression screen for aged-dependent microRNAs identifies miR-30a as a key regulator of aging features in human epidermis

Figure 5. AVEN, IDH1 and LOX are direct targets of miR-30a in keratinocytes. (A) The expression levels of AVEN, IDH1 and LOX proteins were evaluated by western blotting in cultured keratinocytes transduced by the miR-30a lentivirus construction after doxycycline treatment. (B) Immuno-fluorescent staining of AVEN and IDH1 and immunohistochemical staining of LOX in reconstructed epidermis overexpressing or not miR-30a after doxycycline treatment. Counterstaining was performed with DAPI and polycarbonate membrane position is indicated by a dotted line. Representative photographs of 3 independent replicates were shown. Scale bar = 25 μm. Right panels: quantification of the IDH1, LOX or AVEN labeled area in the REs Results are mean +/- SD from three independent samples. ****P<0,0001, ***P<0,001 (C) The alignment of miR-30a putative binding sites in human AVEN, IDH1 or LOX 3’-UTR region have been schematized to show complementary pairing of miR-30a with AVEN, IDH1 or LOX wild-type (WT) and mutant (Mut) 3’-UTR constructs. Transduced keratinocytes were transfected with WT or Mut reporter constructs. Luciferase intensities were normalized to β-galactosidase level. Results are mean +/- SD from three independent samples. *P<0,05, **P<0,01, ***P<0,001, ****P<0,0001.