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

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

<div><p>Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.</p></div

Enhanced expression of eGFP.

<p>HEK293 cells were transiently transfected with eGFP alone (basic) or fused to different expression tags. (A) All proteins contained a signal peptide sequence for secretion. The eGFP signals were visualized by fluorescence microscopy. (B) To quantify the secreted eGFP, the supernatants from the HEK293 cells were excited with a laser at 488 nm and the emission signal was detected at 509 nm (top). Densitometric analysis of secreted eGFP from HEK293 cells by western blot analysis using a Strep-Tactin®-HRP conjugate to detect Strep II tagged eGFP fusion proteins (bottom). (C) Without signal peptide, eGFP was transiently expressed intracellularly with or without fused MBP. eGFP signals were visualized by fluorescence microscopy. (a.u.: arbitrary unit).</p

Global application of the MBP tag as an expression enhancing tag.

<p>(A) Protein expression levels in HEK293 cells of intracellular proteins (HSP90α and Gli1) with and without MBP. (B) Western blot analysis of transmembrane proteins (MEGF9 and MMP14) cloned into the basic and the MBP vector system. (C) Comparison of the expression levels of netrin-4 delta in the basic and the MBP vector transfected into COS-7, CHO-K1, and HEK293 cells. (D) The N-terminal laminin β2LN-LEa1-4 fragment and the respective R246Q mutant version causing congenital nephrotic syndrome were expressed and western blot analysis was performed.</p

Biophysical analysis of MBP as well as MBP fusion proteins and cell attachment studies.

<p>(A) Light scattering profile obtained for a 1.80 mg/mL solution of MBP (dotted line) and for a 3.28 mg/mL solution of MBP-netrin-4 delta (solid line) in the TBS buffer display the respective hydrodynamic radius. (B) Concentration dependence of the hydrodynamic radius of MBP (dotted line) and MBP-netrin-4 delta (solid line), respectively, deduced from the peaks of DLS profiles. (C-D) B16-F1 cells were allowed to adhere to surface coated with MBP-netrin-4 delta. (C) Dependence of cell attachment on the coating concentration of MBP-netrin-4 delta. (D) Cell attachment at E₅₀ coating concentration (MBP-netrin-4 delta) for different netrin-4 delta proteins (with and without tag).</p

Expression analysis in HEK293 cells of commonly used expression tags.

<p>(A-D) The expression levels were compared by western blot analysis detecting the double Strep II tag, which is present as an N-terminal tag on all proteins. (A) Different short arm laminin chains and netrins were transiently expressed and analyzed by western blot. (B) Different expression tags were transiently expressed and visualized by western blot. (C) Netrin-1 delta was fused to different expression tags. (D) Expression of netrin-4 delta in different vector systems was analyzed by western blot analysis. (Isom.: modified SlyD; mFc: Fc part from the mouse IgG protein; mFc-opt: codon optimized Fc part from the mouse IgG protein; ser alb: serum albumin; MBP: maltose binding protein; basic: without fusion protein).</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

Growth plate histology.

<p>Paraffin sections of decalcified bone were stained with safranin O and fast green. (A-H) Sections of distal femora from eight-week-old <i>Ahsg</i>^<i>-/-</i> mice show growth plate anomalies. (A-D) show entire growth plates (scale bar 500 μm), while (E-H) shows higher magnifications (scale bar 200 μm) of each corresponding growth plate above. (I-L) Bone section of an eight-week-old heterozygous <i>Ahsg</i>^<i>+/-</i> mouse showing an extended lesion spanning half the width of the growth plate (scale bar 500 μm). (J-L) show details of I with arrows pointing to lesion-associated cells. (M) TUNEL staining of an <i>Ahsg</i>^<i>+/-</i> growth plate. (PZ, proliferative zone; L, lesion; HZ, hypertrophic zone; scale bar 100 μm). Cells in the lesion stained TUNEL-negative, while few cells in the bone marrow were TUNEL-positive (green). (N-S) Sections of distal femora from three-week-old mice. (N-P) show entire growth plates (scale bar 500 μm), while (Q-S) show higher magnifications (scale bar 200 μm) of each corresponding growth plate above. Lesions with lesion-associated cells were never found in wildtype <i>Ahsg</i>^<i>+/+</i> growth plates, but were present in both heterozygous <i>Ahsg</i>^<i>+/-</i> (O, R) and in homozygous fetuin-A deficient <i>Ahsg</i>^<i>-/-</i> (P, S) growth plates (arrows). Diminished safranin O staining around lesion-associated cells indicated cartilage matrix degradation.</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 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