Journal: bioRxiv

Article Title: miRNA-29-CLIP uncovers new targets and functions to improve skin repair

doi: 10.1101/2024.05.02.591622

Figure Lengend Snippet: Functional analysis of the miR-29-CLIP targetome using DAVID online tool for human follicular keratinocytes (HFK), interfollicular keratinocytes (IFK), and dermal fibroblasts (DF). Number of miR-29 targets represented in each of the cluster is on the x axis and the colour of the bars represent the cluster enrichment score.

Article Snippet: DF were maintained in fibroblasts growth medium kit (PeloBiotech) at 37 °C, 5% CO2.

Techniques: Functional Assay

Journal: bioRxiv

Article Title: miRNA-29-CLIP uncovers new targets and functions to improve skin repair

doi: 10.1101/2024.05.02.591622

Figure Lengend Snippet: Enrichment of predicted targets (as predicted in TargetScan tool) of miRNA-29 through miRNA-CLIP using the 409 probe in the first step of immunoprecipitation (IP1) and in the final step of miRNA-CLIP protocol, determined by qPCR in human follicular (HFK), interfollicular keratinocytes (IFK) and dermal fibroblasts (DF) using qPCR (A-C) and TargetScan (D) analyses.). *P < 0.05 or **P < 0.01 or *** P < 0.001 or **** P < 0.0001, two-way ANOVA, Šídák’s multiple comparisons test. Error bars indicate bidirectional standard deviation.

Article Snippet: DF were maintained in fibroblasts growth medium kit (PeloBiotech) at 37 °C, 5% CO2.

Techniques: Immunoprecipitation, Standard Deviation

Journal: bioRxiv

Article Title: miRNA-29-CLIP uncovers new targets and functions to improve skin repair

doi: 10.1101/2024.05.02.591622

Figure Lengend Snippet: (A) Venn diagram of overlap between the miR-29 targetomes (identified using miRNA-CLIP) of skin cells. (B) Percentage of all miRNA-29 family predicted targets captured by the miRNA-29 probe in all 3 miRNA-CLIPs. (C) Empirical Cumulative Distribution Function (ECDF) plotted the distribution of all miRNA-29-CLIP targets (blue) with predicted targets (red) against log2FC with distance and the p value for each of the cell types. This shows target enrichment achieved by the CLIP method vs. all target prediction. (D) Radar plot with the percentage representation of keratinocytes (HFK+IFK) and fibroblasts (DF) targetome in selected GO terms. (E) Top: Venn diagram showing the overlap of targets in GO term cluster for cell adhesion and ECM, with miRNA-29-CLIP targets (direct) and mRNAs rescued by miRNA-29 inhibition in keratinocytes. Bottom: the overlap of targets in GO term clusters for cell adhesion, proliferation, and ECM, with miR-29-CLIP targets in fibroblasts.

Article Snippet: DF were maintained in fibroblasts growth medium kit (PeloBiotech) at 37 °C, 5% CO2.

Techniques: Inhibition

Journal: bioRxiv

Article Title: miRNA-29-CLIP uncovers new targets and functions to improve skin repair

doi: 10.1101/2024.05.02.591622

Figure Lengend Snippet: (A) Expression of αSMA was assessed by immunofluorescence in human primary DF transfected with anti-sense miRNA-29 (abc) or non-specific (nsa) and treated with 1ng/ml TGF-β1. Representative images are shown for nuclei staining with ASO in red, DAPI in blue, and αSMA in green. Arrows indicate localization of ASO inside the round-shaped fibroblasts with miRNA-29 LOF. Arrowheads point to a spindle-shaped cell not transfected with ASO. Scale bar is 25μm. (B) Expression of αSMA and fibronectin was carried out by western blot in transfected fibroblasts after the treatment with TGF-β1. GAPDH was used as a loading control. (C) Growth rate of fibroblasts was determined following miRNA-29 knockdown compared to non-specific oligo. (D and E) DF were double transfected anti-sense miR-29 (abc) or non-specific (nsa) or miR-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 miR-29 was measured after the second transfection to assess the knock-down/up levels. 2-way ANOVA followed by Šídák’s multiple comparison test. *P < 0.05 or **P < 0.01 or *** P < 0.001 or **** P < 0.0001.

Article Snippet: DF were maintained in fibroblasts growth medium kit (PeloBiotech) at 37 °C, 5% CO2.

Techniques: Expressing, Immunofluorescence, Transfection, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: miRNA-29-CLIP uncovers new targets and functions to improve skin repair

doi: 10.1101/2024.05.02.591622

Figure Lengend Snippet: (A) Selected miR-29 target genes represented in ECM and cell-cell adhesion cluster were confirmed by qPCR in human primary dermal fibroblasts. (B) Expression of SPARC and FERMT2 was assessed by western blot with tubulin as a loading control. (C) Representative images for FERMT2 (green), anti-miR-29 oligo (red) and nuclei (DAPI). Scale bar = 25μm. (D) Quantification of FERMT2 expression from western blots at 5 hr, 48hr and 72 hr 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 ten images per condition. Unpaired t-test, **P < 0.01. Scale bar = 35μm. (G) Soluble SPARC was quantified by ELISA in the medium conditioned by miRNA-29 ASO (abc) and control (nsa) ASO. 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 ANOWA followed by Šídák’s multiple comparison tests were performed, showing 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 ASO (abc) or control fibroblasts (nsa). N=3, unpaired t-test, *P < 0.05 or **P < 0.01. Please note the absorbance value coming from the PrestoBlue reagent reduced by alive cells and thus, corresponding to their number. (I) Schematics of the interaction between proposed players of enhanced keratinocyte adhesion regulated through miRNA-29. BK – basal keratinocyte, BM - basal membrane, ECM – extracellular matrix, f – fibroblasts, and ASO - antisense oligonucleotides.

Article Snippet: DF were maintained in fibroblasts growth medium kit (PeloBiotech) at 37 °C, 5% CO2.

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