This study was conducted in accordance with institutional guidelines and approved by the Ethical Review Committee, Guanghua School of Stomatology, Sun Yat-sen University (Approval number ERC2012-16). All surgery was performed under chloral hydrate anesthesia, and all efforts were made to minimize suffering.

Adult Sprague-Dawley male rats (6-8 weeks of age, 260-300 g) were obtained from the Experimental Animal Center of Sun Yat-sen University and randomly assigned to group (5/cage) or individual housing for 4 weeks prior to, and throughout this study. All rats were given free access to food and water. The facility was maintained on a 12/12-h light/dark cycle (lights on at 7 AM).

Rats were weighed every 3 days from the beginning of the study until sacrificed. At wounding, each rat was intraperitoneally anesthetized with 10% chloral hydrate (0.3 ml/100 g), and one full-thickness wound was placed on the oral hard palate using a sterile 3.5-mm biopsy punch (Miltex Instrument Company, York, PA, USA). On a daily basis, wounds were visually inspected under a 10× magnifier by the same investigator until considered healed. During these assessments, rats were briefly anesthetized by inhaling ether. The investigator was kept blind to the housing condition when wounds were inspected. Wounds were considered fully healed when they were completely closed, and there were no signs of erosions or ulcers. To determine intra-rater reliability for wound closure, the rater visually assessed all subjects at two different time points at day 7 and these data were evaluated using weighted Kappa statistics. The Kw value was 0.82, suggesting there was a high intra-rater reliability.

Blood samples were obtained at 10: 00 AM before isolation (baseline), on the second day of isolation, and on days 1, 3, 5, 8 and 10 post-wounding. Rats were briefly restrained (less than 2 min) in polystyrene tubes and blood was taken from the tail vein. Approximately 80 µl of whole blood was collected from each rat and serum samples were stored at -80^° C for corticosterone analysis by ELISA.

In separate animals, the entire wound was harvested using a sterile 6.0-mm biopsy punch (Miltex Instrument Company) on day 1, 3, or 5 post-wounding. The excised tissue was immediately placed in 1 ml of Trizol (Invitrogen, Carlsbad, CA, USA), flash frozen in liquid nitrogen, and stored at -80^° C for qRT-PCR analysis. Animals were euthanized afterwards.

HEK293 cells were maintained in DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO, Carlsbad, CA, USA) at 37° C in a humidified incubator containing 5% CO2. For functional analysis, miR-29 (a, b, c) mimics, miR-203 mimics and non-targeting miRNA mimics (Dharmacon, Lafayette, CO, USA) were transfected into cells using DharmaFECT Transfection Reagent 1 (Dharmacon) per the manufacturer’s instructions.

Corticosterone levels in serum were measured using ELISA kits (IB79175, IBL-America, Minneapolis, MN, USA) according to the manufacturer’s instructions. OD values were determined at 450 nm on an ELISA plate reader. Corticosterone concentrations were quantified by comparison to the standard curves. Samples were analyzed in triplicate.

For gene expression, total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using a Transcriptor First Strand cDNA Synthesis kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Real-time PCR was performed using a Roche 480 System (Roche) with 2 µl of 1:5 diluted cDNA, which was mixed with a TaqMan Universal PCR Master Mix, No AmpErase (Applied Biosystems, Foster City, CA, USA), primers and probe for GAPDH and the target gene, and RNase-free water to yield a 20-µl reaction volume. All of the real-time primers and probes (Table 1) were designed and produced by Bioperfectus Technologies (Jiangsu, China). The suppressor of cytokine signaling 3 (SOCS3) Taqman gene expression assay (cat. # 4331182) was purchased from Applied Biosystems. Amplification was performed in duplicate under the following conditions: 50^oC (2 min), 95^oC (10 min), 40 alternating cycles of 95^oC (15 s) and 60^oC (1 min). Relative mRNA levels of target genes were assessed by normalizing transcript Ct values to those for transcription of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ΔCT) and comparing the results (2^-ΔCT) directly. For miRNA expression, a quantitative 2-step RT-PCR assay using mirVanaTM qRT-PCR microRNA Detection Kit was used as per the manufacturer’s protocol (Ambion, Austin, TX, USA). Specific primer sets for miR-29a, miR-29b, miR-29c, miR-203 and U6 were obtained from Ambion. The relative expression level of miRs was determined using the 2^-ΔΔCT analysis method, where U6 was used as an internal reference.

Luciferase-expressing vectors were constructed as follows. A BglII-to-NheI fragment containing the HSV thymidine kinase (TK) promoter was inserted between BglII and HindIII sites of pGL3-Basic (Promega, Madison, WI, USA) to create pGL-TK, in which luciferase cDNA, followed by a SV40-derived polyadenylation signal, is expressed from the TK promoter. Regions covering the entire rat VEGFA 3’ UTR were amplified by PCR and inserted between XbaI and BamHI sites of pGL3-TK to replace the SV40 polyadenylation signal and create pGL-VEGFA. The constructs containing mutated microRNA binding sites were established by amplifying the whole vector excluding the 8-bp seed region from a pair of primers containing an additional restriction enzyme site. Thereafter, the product was digested using that restriction enzyme and ligated to recircularize the plasmid such that the seed region was replaced with the restriction enzyme site. Using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), cells were transfected with the reporter constructs containing either the targeting sequence from the VEGFA 3’ UTR (named WT) or its mutant. The pRL-TK vector (Promega) was co-transfected as a control for transfection efficiency. The luciferase activities were then determined using a Lumat LB 9507 Luminometer (Berthold Technologies, Bad Wildbad, Germany).

Western blots were performed as standard protocol using antibodies specific to VEGFA (Novus Biologicals, Littleton, CO, USA) and β-actin (Sigma, St. Louis, MO, USA). For semi- quantification, the western blot bands were quantified using a ChemiDoc XRS System (Bio-Rad, Laboratories, Hercules, CA) equipped with Quantity One software (version 4.6.3).

Statistical comparisons of differences in serum corticosterone levels were analyzed using repeated measures, treating Day (eight time points) as a within-subjects measure and Group (control, isolation) as a between-subjects measure. Analyses of mRNA levels were performed by analyte using ANOVA, treating both Day (1,3,5) and Group as between-subject measures. Post-hoc tests were performed only when significant main effects or interactions were evident that involved Group. Chi-square tests were used to assess differences in the healing rates between groups (i.e., healed vs. not healed). All hypothesis tests were 2-tailed, and the data were determined to be statistically significant when p < 0.05. Error bars represent the standard error of the mean (SEM). SPSS 18.0 (Chicago, IL, USA) was used for all analyses.

The overall rate of wound closure was markedly slower in isolated rats than in controls (p < 0.01). It took the isolated rats 31.2% longer than controls to be considered healed (6.4 vs. 8.4 days). As a result, on days 7 and 8 a significantly lower proportion of isolated animals were considered healed compared to non-stressed controls (Figure 1A).

There was no significant effect of isolation on body weight compared to controls over the 6-week experimental period (data not shown). When examining serum corticosterone levels, a significant Day x Group interaction was apparent [F(7,56)=3.50, p<0.01)]. Post-hoc analysis revealed that corticosterone levels were significantly higher in isolated rats as compared to controls for the first five days post-wounding (Figure 1B).

Using quantitative real-time PCR to examine mRNA levels, we found Day x Group interactions for IL1β [F(2,59)=3.36, p<0.05], TNFα [F(2,59)=4.04, p<0.05], MIP1α [F(2,59)=10.88, p<0.001], and FGF7 [F(2,59)=9.47, p<0.001]. In addition, there was a main effect of Group for VEGF [F(1,59)=16.47, p<0.001]. Post-hoc analyses revealed markedly lower mRNA level of IL1β in the wounded mucosa of isolated rats (Figure 2A) on day 1. A reduced level of MIP1α mRNA was observed on day 1 in isolated rats; conversely, MIP1α levels on days 3 and 5 were higher in isolated animals. mRNA levels of FGF7 were markedly lower in isolated rats on day 1 and day 3, and by day 5 an increased expression of FGF7 was observed in the isolated rats (Figure 2C). In particular, elevated gene expression for FGF7 and MIP1α appeared to be delayed in isolated rats compared with controls. mRNA levels for VEGFA was lower in the stress condition on all days (1 through 5) (Figure 2E). We also measured IL6, αSMA, KC and MCP1 mRNA levels in the wounded mucosa. These analytes did not differ between controls and isolated rats (not shown).

Compared to group housed controls, isolated rats persistently exhibited higher levels of miR-29a and miR-29c on all days in wounded mucosa (Figure 3A). Overexpression of miR-29b was detected on days 3 and 5. Expression level of miR-203 was also markedly higher on days 3 and 5 (4.2-fold and 2.6-fold, respectively) (Figure 3B). Corresponding to the higher miR-203 levels, mRNA levels of SOCS3, which is a known direct target of miR-203, were found to be lower in isolated rats on days 3 and 5 (0.32 ± 0.04 and 0.75 ± 0.09, respectively) compared to control rats.

Based on bioinformatics analysis, a highly conserved miR-29 isoform-targeting sequence and a miR-203-targeting sequence were identified in the VEGFA mRNA 3’ UTR (Figure 4A). To identify miRNAs capable of directly targeting these sequences, a dual-luciferase reporter assay was performed. This assay quantified the ability of overexpressed miRNAs to reduce firefly luciferase activity from an mRNA bearing the VEGFA 3′ UTR. The whole 3′ UTR of rat VEGFA was cloned downstream of the firefly luciferase coding sequence. miR-29a, miR-29c (Figure 4B) and miR-203 (Figure 4C) significantly reduced activity of firefly luciferase by directly targeting the rat VEGFA 3′ UTR. MiR-29c exerted the largest effect, decreasing luciferase activity by at least 60% (p<0.001) (Figure 4B). Both miR-29a and miR-203 reduced luciferase activity by ≥ 40% (p<0.001) (Figure 4B, C). To provide further confirmation of the specificity of the direct interactions between the miRNAs and VEGFA 3′ UTR, we mutated the predicted binding sites and repeated the luciferase assays. In both the putative miR-29 and miR-203 binding sites, the 6 to 8 nucleotides of the “seed region” were replaced with a restriction enzyme site with minimal complementarity to the miRNA sequence (see Materials and Methods). As expected, 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 (Figure 4B). Similarly, mutation of the putative miR-29 site led to abolishing the action of the miR-29 isoforms (Figure 4C).

We further evaluated the ability of each of the above miRNAs to target endogenous VEGFA. The miRNA-induced change in VEGFA mRNA expression was confirmed by qRT-PCR. As shown in Figure 4D, ectopic transfection of miR-29a, miR-29c and miR-203 in HEK293 cells led to a significant decrease in VEGFA protein expression. Furthermore, co-transfection of miR-29c and miR-203 mimics produced an additive effect on reducing VEGFA expression in vitro.