Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing

<p><b><i>Aim:</i></b> Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing. <b><i>Methods:</i></b> Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs. <b><i>Results:</i></b> Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, <i>RNU6B</i> was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to <i>Gapdh</i> the normalization to multiple reference genes gave a similar outcome. <b><i>Conclusion:</i></b> <i>RNU6B</i> is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.</p

Genome-wide gene expression analysis reveals the induction of pro-inflammatory genes in growth plates of 13-day-old <i>Ahsg</i>^<i>-/-</i> mice.

<p>(A) Heatmap representation of the normalized gene expression in individual growth plates shows consistently strong induction of large gene clusters in fetuin-A deficient <i>Ahsg</i>^<i>-/-</i> mice compared to wildtype <i>Ahsg</i>^<i>+/+</i> mice. Gene expression in growth plates of <i>Ahsg</i>^<i>+/-</i> was heterogeneous and had partial overlap with both <i>Ahsg</i>^<i>-/-</i> and <i>Ahsg</i>^<i>+/+</i> genotypes. (B) Volcano plot comparing the normalized growth plate gene expression between <i>Ahsg</i>^<i>-/-</i> and <i>Ahsg</i>^<i>+/+</i> mice. The plot shows that more significantly differentially expressed genes were induced (red), and few genes were significantly repressed (blue). Genes marked with red circles were used for validation of microarray data with qRT-PCR. (C) The seven most highly differentially induced genes were validated using qRT-PCR. The graph represents the log₂ fold changes in expression in <i>Ahsg</i>^<i>-/-</i> compared to <i>Ahsg</i>^<i>+/+</i> samples. Data was analyzed using Student’s t-test: **p<0.005, ***p<0.001.</p

Immunofluorescent localization of the vasculature in distal femoral growth plates.

<p>Thick cryo-sections of femora from 13-day-old mice were stained with anti-CD31 antibody (red) and nuclei were counterstained with DAPI (blue) to visualize the vascular network. We examined 5 <i>Ahsg</i>^<i>+/+</i> samples, 6 <i>Ahsg</i>^<i>+/-</i> samples and 8 <i>Ahsg</i>^<i>-/-</i> samples. Upper panel shows overviews representing overlays of blue, red and bright-field channels. Lower panel shows a magnified view of the red channel (CD31). Fewer capillary loops reaching the chondro-osseous junction were found in <i>Ahsg</i>^<i>-/-</i> mice compared to <i>Ahsg</i>^<i>+/+</i> and <i>Ahsg</i>^<i>+/-</i> littermates. Scale bar upper panel is 500 μm, scale bar lower panel is 250 μm.</p

Expression of perivascular and endothelial cell-specific markers in wound repair.

<p>Analysis of PECAM1, desmin, α-SMA and Sca1 expression in the wounded skin. (A) Dorsal view of full thickness wounds on the back skin of mice one (D1), seven (D7) and 14 (D14) days post injury. Representative H&E-stained cryosections of selected wounds (arrowhead) during inflammation (D1), granulation (D7) and remodeling (D14) are shown. (B-D) Immunostaining of (B) PECAM1/desmin, (C) PECAM1/α-SMA or (D) PECAM1/Sca1 expression at the different stages of wound healing. The individual monochrome signals for PECAM1, desmin, α-SMA and Sca1 are shown in overviews. Squares within the images represent closeups of overlays for the PECAM1/desmin, PECAM1/α-SMA PECAM1/Sca1 stainings (B-D). Bars 1 cm (A, top), 1 mm (A, lower panel), 100 µm (B).</p

Growth plate histology and immunofluorescent localization of fetuin-A in distal femoral growth plates.

<p>(A) Decalcified femur paraffin sections from mice of different ages were stained with an anti-fetuin-A antibody (red) and nuclei were counterstained with DAPI (blue). In the growth plate, fetuin-A was localized in hypertrophic chondrocytes. <i>Ahsg</i>^<i>-/-</i> mice served as negative control (lower panel). (B) Similar to wildtype mice, fetuin-A was detected in hypertrophic chondrocytes from eight-week-old <i>Ahsg</i>^<i>+/-</i> mice. Fetuin-A staining was negative in growth plates containing a lesion (green arrows). (C) A magnified view of the marked area in (A) shows the cytoplasmic localization of fetuin-A in hypertrophic chondrocytes. Scale bars are 75 μm.</p

Distribution of PECAM1 and Sca1 protein on skin- and wound-derived cells.

<p>(A) Flow cytometry analysis of Sca1 and PECAM1 expression in CD45^- non-hematopoietic cells isolated from newborn (nb), three weeks (3 wk) and three months (3 mo) old dermis. Sca1⁺ (1, red box), PECAM1⁺ (2, green ellipse) and PECAM1⁺/Sca1⁺ (3, black ellipse) cell populations are highlighted. (B) Dot plots of Sca1 and PECAM1 expression in cell suspensions isolated from full thickness wounds one (D1), seven (D7) and 14 days (D14) post injury of eight weeks old mice. Percentage of positive cell populations at the different time points of flow cytometry analysis (lower panel) is given with standard deviation and significant changes were determined using the unpaired two-tailed student’s T-test (n≥3, **p≤0.01, n.s = not significant).</p

Immunofluorescent localization of STAT1 and phospho-STAT1 (pSTAT1) in distal femoral growth plates.

<p>Decalcified paraffin sections were stained with antibodies for STAT1 and pSTAT1 (red) and nuclei were counterstained with DAPI (blue). The figure shows representative micrographs for each time point and each genotype recorded with a 20-fold (uppercase letter) and a 63-fold lens (lowercase letter). STAT1 and pSTAT1 signal was mainly localized in the hypertrophic zone. The signal was increased in <i>Ahsg</i>^<i>+/-</i> and <i>Ahsg</i>^<i>-/-</i> mice of all ages. Scale bars are 150 μm for micrographs taken at lower and 20 μm for micrographs taken at a higher magnification.</p

<i>In situ</i> detection of the CD38 receptor in the maturing skin and wound.

<p>(A) Expression of PECAM1 and CD38 in cryosections of newborn (nb), three weeks (3 wk) and three months (3 mo) old skin was detected by immunofluorescence microscopy. The fluorescence signal for CD38 (top row) and the overlay with PECAM1 is shown. (B) Confocal microscopy analysis of PECAM1/CD38 expression in three weeks old skin. PECAM1⁺ vessels lacking CD38 expression are indicated (arrowheads). (C) Localization of PECAM1 and CD38 expression in cryosections of wounds one (D1), seven (D7) and 14 days (D14) post injury. The fluorescence signal for CD38 (top row), the overlay with PECAM1 and higher magnifications of the wounded area are shown (A, C, squares). Bars 100 µm (A, C), 50 µm (B).</p

Bone dysplasia in fetuin-A deficient mice.

<p>(A) Growth curves of femora from both, male and female <i>Ahsg</i>^<i>+/+</i>, <i>Ahsg</i>^<i>+/-</i> and <i>Ahsg</i>^<i>-/-</i> mice reveal that femora of <i>Ahsg</i>^<i>-/-</i> mice were significantly shortened compared to their littermates at the age of four weeks and older. Error bars show SD. Data was analyzed by One-Way ANOVA: *p<0.05, **p<0.01, ***p<0.001. (B) Measurement of distal femur width shows a widening of the distal femoral epiphysis in <i>Ahsg</i>^<i>-/-</i> mice at the age of four weeks and older. (C) Three-dimensional reconstructions from μCT measurements of eight-week-old male mice and (D) matching 2D μCT cross-sections. Micro-CT analysis shows a posterior rotation of the distal femoral epiphysis in <i>Ahsg</i>^<i>-/-</i> mice. (E) The angle of the distal femoral epiphysis relative to the shaft (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187030#pone.0187030.s001" target="_blank">S1 Fig</a>) was significantly decreased in male <i>Ahsg</i>^<i>-/-</i> mice. Error bars show SD. Data was analyzed by Student’s t-test: ***p<0.001. (F) Prevalence of the dysplasia in male and female C57BL/6 <i>Ahsg</i>^<i>-/-</i> mice. Mice at the age of four weeks and older were evaluated for dysplasia by either bone length measurements from isolated bones or from radiographic images. Out of 62 <i>Ahsg</i>^<i>-/-</i> mice, 44 showed dysplasia in their distal femur in one or both legs, 18 mice had no obvious phenotype anomaly.</p

Growth plate morphology in 13-day-old mice.

<p>(A) Histological staining with safranin O/fast green revealed an elongated hypertrophic zone in 13-day-old <i>Ahsg</i>^<i>-/-</i> mice. Scale bar is 500 μm. (B) Length measurements of reserve (RZ), proliferative (PZ) and hypertrophic zones (HZ) from 13-day-old male <i>Ahsg</i>^<i>+/+</i>, <i>Ahsg</i>^<i>+/-</i> and <i>Ahsg</i>^<i>-/-</i> mice were performed on paraffin sections. Error bars show SD. Data was analyzed by One-Way ANOVA: *p<0.05, ***p<0.001. (C) The percentage of EdU⁺ cells indicated that proliferation was essentially identical in the proliferative zone of 13-day-old mice of all genotypes.</p