MiR-203 and miR-29 directly target VEGFA mRNA.

<p>(A) The predicted miR-203 and miR-29 targeting sequences located in the 3’-untranslated region (3’-UTR) of VEGFA mRNA. (B–C) Dual luciferase reporter assays were performed as described in <i>Materials and Methods</i> section. Cells were co-transfected with constructs containing the predicted targeting sequence (WT) or mutated targeting sequence (Mutant) cloned into the 3’-UTR of the reporter gene, along with miRNA mimics of miR-29 or miR-203. Mutation of the putative miR-203 binding site abolished the effect of miR-203 while leaving the action of all three miR-29 isoforms unaffected (B). Similarly, mutation of the putative miR-29 binding site abolished the action of the miR-29 isoforms (C). (D) Western blot analyses (upper panel) and qRT-PCR (lower) were performed to examine the effects of miRNA on VEGFA protein and gene expression in cells that were treated with miRNA mimics or control mimics. * Indicates significant differences between WT and Mutant group, ^# indicates significant differences between vector and each miRNA in WT cells (*, ^#. <i>p</i> < 0.05; ***, ^##<i>p</i> < 0.001). Data represent at least three independent experiments with similar results.</p

Oral palatal wound closure and serum corticosterone levels.

<p>(A) Closure of palatal wounds was significantly slower in isolated rats (ISO) than in group-housed rats (GRO). Compared with group-housed rats, a lower proportion of wounds were closed on days 7 and 8 in isolated rats (n=10/group; * <i>p</i><0.05, ** <i>p</i><0.01). (B) Serum corticosterone levels were significantly higher in isolated rats on days 1, 3 and 5 as compared to controls (n=5/group; * <i>p</i><0.05). BL: baseline.</p

Expression levels of healing-associated genes in isolated (ISO) and group-housed (GRO) rats on Days 1, 3 and 5 post-wounding.

<p>(A) IL1β; (B) MIP1α; (C) FGF7; (D) TNFα; and (E) VEGFA. Error bars represent SEM. * indicates a significant difference between stress conditions. ^# and ⁺ indicate significant differences between days within group-housed and isolated rats, respectively (n=10/group; *, ^#, ⁺, <i>p</i><0.05; **, ^{+ +}, ^##<i>p</i><0.01; ^###<i>p</i><0.001).</p

Expression of miR-29 family members, miR-203 and SOCS3 mRNA in wounded tissues by qRT-PCR.

<p>Relative expression levels of miR-29 family members (A) and miR-203 (B) in isolated (ISO) rats are in comparison to control levels expressed as 1 on the graph. U6 was used as an internal reference. (C) SOCS3 mRNA relative expression, GAPDH was used as a reference gene. (n=10/group [ISO&GRO], * indicates a significant difference between stress conditions. * <i>p</i><0.05; ** <i>p</i><0.01).</p