Journal: Febs Letters

Article Title: miRNA ‐29 regulates epidermal and mesenchymal functions in skin repair

doi: 10.1002/1873-3468.70051

Figure Lengend Snippet: miRNA‐CLIP successfully identifies miRNA‐29 targetome in primary skin cells. (A) Venn diagram of an overlap between the miRNA‐29 targetomes (identified using miRNA‐CLIP) of skin cells. (B) Percentage of all predicted targets of the miRNA‐29 family members captured by the miRNA‐29 probe in miRNA‐29‐CLIPs. (C) Empirical Cumulative Distribution Function plotting the distribution of all miRNA‐29‐CLIP targets (blue) with predicted targets (red) against log2FC with the distance and P value for each of the cell types. This shows target enrichment achieved by the CLIP method vs. the target prediction. (D) Radar plot showing selected Gene Ontology (GO) terms represented in miRNA‐29 targetomes in fibroblasts and keratinocytes. (E) Top : Venn diagram showing the overlap of direct miRNA‐29‐CLIP targets with cell adhesion GO terms (punctate oval), ECM (square), and with the mRNAs upregulated by miRNA‐29 inhibition in keratinocytes. Bottom : the overlap of targets in GO term clusters for cell adhesion, proliferation, and ECM, with direct miRNA‐29‐CLIP targets in fibroblasts. Consistent with our data, proliferation function is absent in keratinocytes and the inhibition of miRNA‐29 is used to select upregulated targets of miRNA‐29 in fast adhering cells. Error bars indicate standard deviation of the mean.

Article Snippet: DF were maintained in fibroblast growth medium kit (PeloBiotech) at 37 °C, 5% CO 2 .

Techniques: Inhibition, Standard Deviation

Journal: Febs Letters

Article Title: miRNA ‐29 regulates epidermal and mesenchymal functions in skin repair

doi: 10.1002/1873-3468.70051

Figure Lengend Snippet: Functional analysis of miRNA‐29‐CLIP targetome in primary skin cells using DAVID. Functional analysis of the miRNA‐29‐CLIP targetome using the DAVID online tool for (A) human follicular keratinocytes (HFK) and interfollicular keratinocytes (IFK), and (B) dermal fibroblasts (DF). Proportion of miRNA‐29 targets represented in each of the clusters is on the x axis, and the color of the bars represents the cluster enrichment score.

Article Snippet: DF were maintained in fibroblast growth medium kit (PeloBiotech) at 37 °C, 5% CO 2 .

Techniques: Functional Assay

Journal: Febs Letters

Article Title: miRNA ‐29 regulates epidermal and mesenchymal functions in skin repair

doi: 10.1002/1873-3468.70051

Figure Lengend Snippet: miRNA‐29 regulates proliferation and ECM deposition in dermal fibroblasts. (A) Expression of αSMA was assessed by immunofluorescence in dermal fibroblasts (DF) transfected with antisense miRNA‐29 (abc) or non‐specific (nsa) and treated with 1 ng·mL −1 of TGFβ1. Representative images are shown for nuclei staining with control (nsa) or miRNA‐29 inhibitors (abc) in red (antisense oligonucleotides, ASO), DAPI in blue, and αSMA in green. Arrows indicate localization of miRNA‐29 inhibitors inside the round‐shaped fibroblasts. Arrowheads point to a spindle‐shaped cell apparently not transfected with miRNA‐29 inhibitors. Untransfected cells are treated with TGFβ1 as a control for fibroblast into myofibroblast differentiation. Scale bar is 25 μm. (B) Expression of αSMA and fibronectin was assessed by the western blot in transfected fibroblasts after the treatment with TGF‐β1. GAPDH was used as a loading control. (C) Growth rate of DF was determined following miRNA‐29 inhibition (abc) compared to a non‐specific inhibitor (nsa). (D) DFs were transfected with miRNA‐29 inhibitors (abc) or non‐specific inhibitors (nsa) and (E) miRNA‐29ab mimic (abm) or non‐specific mimics (nsm) and the extracellular matrix (ECM) deposited by the cells was extracted by pepsin and quantified using BCA at 1‐, 3‐, and 5‐days post‐transfection. (F) Relative levels of miRNA‐29 were measured by TaqMan assays to confirm overexpression and the inhibition in DF. Punctate line indicates miRNA‐29 levels in nsm and in nsa control samples (both set to 1). Two‐way ANOVA followed by Šídák's multiple comparison test. * P < 0.05 or ** P < 0.01 or *** P < 0.001 or **** P < 0.0001. Error bars indicate standard deviation of the mean.

Article Snippet: DF were maintained in fibroblast growth medium kit (PeloBiotech) at 37 °C, 5% CO 2 .

Techniques: Expressing, Immunofluorescence, Transfection, Staining, Control, Western Blot, Inhibition, Over Expression, Comparison, Standard Deviation

Journal: Febs Letters

Article Title: miRNA ‐29 regulates epidermal and mesenchymal functions in skin repair

doi: 10.1002/1873-3468.70051

Figure Lengend Snippet: miRNA‐29 regulates ECM, proliferation, and adhesion through SPARC, FERMT2, and COL4. (A) Selected miRNA‐29 target genes represented in ECM and cell–cell adhesion cluster were confirmed by qPCR in human primary DF following inhibition of miRNA‐29 (magenta) vs. control (blue). (B) Expression of SPARC and FERMT2 was assessed by the western blot with tubulin as a loading control in DF transfected with miRNA‐29 inhibitors (abc) or control oligo (nsa). (C) Representative images for FERMT2 (green), miRNA‐29 inhibitors (abc) or control (nsa) antisense oligos in red (ASO) and nuclei (DAPI, blue). Scale bar = 25 μm. (D) Quantification of FERMT2 expression from western blots at 5, 48, and 72 h post‐transfection, N = 3, unpaired t ‐test. * P < 0.05 or ** P < 0.01. (E and F) Representative images (E) and quantification of collagen IV (F) using 10 images per condition. Unpaired t ‐test, ** P < 0.01. Scale bar = 35 μm. (G) Soluble SPARC was quantified by ELISA in the medium conditioned by miRNA‐29 inhibitors (abc) and control (nsa) inhibitors. Graph shows direct ELISA results where medium samples were diluted for precise quantification. The actual levels of SPARC are shown as numbers next to the graph. Both one‐way and two‐way ANOVA followed by Šídák's multiple comparison tests were performed, showing a highly significant increase in secreted SPARC, **** P < 0.0001. (H) Adhesion of keratinocytes was quantified on the increasing concentrations of conditioned media from miRNA‐29 inhibitors (abc) or control (nsa)‐treated fibroblasts. N = 3, unpaired t ‐test, * P < 0.05 or ** P < 0.01. The absorbance value coming from the PrestoBlue reagent corresponds to the number of alive adherent cells. (I) Interaction between proposed molecular players of enhanced keratinocyte adhesion regulated through miRNA‐29. BK – basal keratinocyte, BM – basal membrane, ECM – extracellular matrix, f – fibroblasts, and miRNA‐29 inhibitors – antisense oligonucleotides (ASO). Error bars indicate standard deviation of the mean.

Article Snippet: DF were maintained in fibroblast growth medium kit (PeloBiotech) at 37 °C, 5% CO 2 .

Techniques: Inhibition, Control, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Direct ELISA, Comparison, Membrane, Standard Deviation