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

Tumors alter absolute myeloid cell numbers in wounds examined 1 or 5 days after wounding.

Representative flow cytometry bivariate dot plots of Ly6C and Ly6G labeling of CD11b+ wound cells from mice (A) without tumors and with tumors 1 or 5 days post-wounding. Gates: R1 = Ly6Chigh Mo/MΦ, R2 = Ly6ClowMo/MΦ, and R3 = Neutrophils. Average (±SEM) (B) neutrophil (Cd11b/Ly6G+), (C) Ly6Chigh Mo/ MΦ, and (D) Ly6Clow Mo /MΦ isolated from wound tissues and quantified using flow cytometry. (E) Average (±SEM) percent of these Ly6Chigh or Ly6Clow Mo/ MΦ expressing IL-4Rα protein. n = 2-3/group; *p#p<0.05 between treatments.</p

Tumor cell-conditioned media reduces murine fibroblast migration and proliferation.

(A) Schematic of conditioned media paradigm and in vitro “wounding” scratch assay. (B) Average (±SEM) percent decrease in scratch “wound” width of adult murine fibroblasts 18 and 26 h post-scratching cultured with fibroblast-conditioned control media or tumor cell-conditioned media. n = 6 wells/group; *p≤0.05 between treatments (C) Representative photographs of scratch assay. Black bars represent margins of scratch.</p

Tumors reduce relative gene expression of inflammatory markers in wounds.

<p>Average (±SEM) quantitative expression of (A) <i>Tlr4</i>, (B) <i>IL-1β</i>, (C) <i>Ccl2</i>, (D) <i>IL-10</i>, (E) <i>Ccl3</i>, and (F) <i>Cxcl1</i> mRNA extracted from wound tissue 1 or 5 days post-wounding. n = 8-10/group; *p<0.05 between cancer treatments.</p

Tumors Alter Inflammation and Impair Dermal Wound Healing in Female Mice - Fig 5

<p><b>Absolute circulating myeloid cell concentrations (CBC) with and without wounding.</b> Average (±SEM) circulating (A) total white blood cells, (B) neutrophils, and (C) monocytes in tumor-bearing and -free mice with and without dermal wounding. n = 8-10/group; *p<0.05 within the same cancer treatment between wounding treatments; #p<0.05 between cancer treatments with same wound treatment; &p<0.05 within the tumor groups between Days 1 & 5.</p

Tumors delay dermal wound closure.

<p>(A) Average percent (±SEM) of original 3.5 mm dermal excisional wound size analyzed over 5 days using digital photography. (B) Representative photographs of wounds over time. Unhealed skin is circled. n = 10/group; *p<0.05.</p

Tumors alter gene expression pattern of select inflammatory/growth factors in wounds over time.

<p>Average (±SEM) quantitative expression of (A) <i>Tnfα</i>, (B) <i>Cxcl2</i>, (C) <i>Cxcl10</i>, (D) <i>Tgf-β</i>, and (E) <i>Igf-1</i>mRNA extracted from wound tissue 1 or 5 days post-wounding. n = 8-10/group; *p<0.05 between cancer treatments.</p