solution  (Chondrex Inc)

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: Epithelial cell-derived exosomes are transported to the primary fibroblasts, which promotes the myofibroblast differentiation phenotype. PBS-Exo (exosomes derived from epithelial cells treated with PBS), Arecoline-Exo (exosomes derived from epithelial cells treated with Arecoline), Arecoline+GW4869-Exo (exosomes derived from epithelial cells treated with Arecoline and GW4869). a Fibroblasts was incubated with DiI-labeled (red) exosomes from epithelial cells (50 μg) for 30 min and fixed for fluorescence staining. Nuclei were stained with DAPI (blue). Scale bars, 100 μm. b Immunostaining for fibrotic markers collagen type I and α-SMA in primary fibroblast with PBS-, Arecoline- and Arecoline+GW4869-Exo was evaluated by microscopy. DAPI (blue) was used for nuclear staining. Scale bar, 100 μm. c Immunoblotting of collagen type I and α-SMA by PBS-, Arecoline- and Arecoline+GW4869-Exo in primary fibroblasts. Sirius Red total collagen assay ( d ), collagen contraction assay ( e , f ) and transwell migration assay ( g , h ) were determined for fibroblast. n = 3–5 technical replicates, representative of two or three assays. Scale bars, 100 μm. Statistics: mean ± SEM, unpaired, one-way ANOVA ( d , f , h ), *** P < 0.001, **** P < 0.000 1

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Derivative Assay, Incubation, Labeling, Fluorescence, Staining, Immunostaining, Microscopy, Western Blot, Collagen Assay, Contraction Assay, Transwell Migration Assay

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: Arecoline induced the expression of miR-17-5p in epithelial cell exosomes. PBS-Exo (exosomes derived from epithelial cells treated with PBS), Arecoline-Exo (exosomes derived from epithelial cells treated with Arecoline), Arecoline+GW4869-Exo (exosomes derived from epithelial cells treated with Arecoline and GW4869), U6 was used as an internal control for qRT-PCR. The exosomes RNA–seq ( a ) and volcano plot ( b ) identified top 8 up-regulated miRNAs (change 1.5-fold) of 177 expressed differential genes in PBS-Exo and Arecoline-Exo. c qRT-PCR validation of exosomes miRNAs from PBS-Exo and Arecoline-Exo. The levels of miR-17-5p in epithelial cells ( d ) and exosomes ( e ) from its cell culture medium treated with PBS and Arecoline were determined by qRT-PCR. f The levels of miR-17-5p in epithelial cells treated with PBS, Arecoline and Arecoline+GW4869 were determined by qRT-PCR. g The levels of miR-17-5p in fibroblast after 48 h co-culturing with PBS-Exo, Arecoline-Exo and Arecoline+GW4869-Exo. h The levels of miR-17-5p in normal ( n = 12) or OSF ( n = 17) tissues. i miR-17-5p expression in normal ( n = 3) and cardiac fibrosis patients ( n = 3) from a public dataset (GSE196421). j miR-17 expression in normal ( n = 4) and diabetic nephropathy kidney fibrosis patients ( n = 12) from a public dataset (GSE51674). Statistics: mean ± SEM, unpaired, two-tailed Student’s t test ( c , h , i , j ) or one-way ANOVA ( d – g ), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Expressing, Derivative Assay, Control, Quantitative RT-PCR, RNA Sequencing Assay, Cell Culture, Two Tailed Test

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: Exosomes derived from epithelial cells transfer miR-17-5p to fibroblast, which promotes myofibroblast differentiation. Control (epithelial cells transfected negative control), miR-17-5p (epithelial cells transfected mimic), Anti-miR-17-5p (epithelial cells transfected inhibitor), U6 or GAPDH was used as an internal control for qRT-PCR. a A transwell co-culture cell model with transfected epithelial cells (top well) and fibroblasts (bottom well). A 0.4-μm porous membrane is between the 2 wells, allowing the transmission of exosomes, but inhibiting direct contact between cells. b A co-culture assay to study the miRNA cargo from epithelial cells to fibroblasts. The epithelial cells were transfected with a Cy3-labeled miR-17-5p (red) and then treated with PBS or GW4869. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. c The expression level of miR-17-5p in fibroblasts after 48 h co-culturing with epithelial cells transfected with miR-17-5p or anti-miR-17-5p. Immunostaining ( d ) and ( e ) Immunoblotting detection of collagen type I and α-SMA expression in fibroblast after 48 h co-culturing with epithelial cells. Scale bars, 100 μm. Sirius Red total collagen assay ( f ), collagen contraction assay ( g , h ) and transwell migration assay ( i , j ) were determined for fibroblast. n = 3–5 technical replicates, representative of two or three assays. Scale bars, 100 μm. Statistics: mean ± SEM, one-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Derivative Assay, Control, Transfection, Negative Control, Quantitative RT-PCR, Co-Culture Assay, Membrane, Transmission Assay, Co-culture Assay, Labeling, Expressing, Immunostaining, Western Blot, Collagen Assay, Contraction Assay, Transwell Migration Assay

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: Exosomal miR-17-5p directly targets Smad7 in fibroblasts. a Schematic miR-17-5p predicted binding sites in the 3’ UTRs of Smad7 and the sequence of mutant UTRs. b Luciferase reporter assay was performed on HEK293T after co-transfected with the miR-NC or miR-17-5p mimic and the Smad7-wt plasmid or Smad7-mut plasmid. c Luciferase reporter was carried out in HEK293T transfected with Smad7-wt plasmid or Smad7-mut plasmid, then incubated with exosomes (50 μg/mL) derived from PBS-, Arecoline- and Arecoline+GW4869- treated epithelial cells for 48 h. d After transfection miR-17-5p in fibroblast, Immunostaining detection of miR-17-5p (red) and Smad7 (green) expression. Scale bars, 100 μm. e The expression level of Smad7 in normal ( n = 12) and OSF ( n = 17) tissues detected by qRT-PCR. f The relationship between relative Smad7 expression normalized to GAPDH and miR-17-5p expression normalized to U6. g Immunoblotting of Smad7, fibrotic markers and TGF-β path pathway. Statistics: mean ± SEM, unpaired, two-tailed Student’s t test, * P < 0.05, *** P < 0.001

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Binding Assay, Sequencing, Mutagenesis, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Incubation, Derivative Assay, Immunostaining, Expressing, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: Exosomal miR-17-5p derived from Arecoline-treated epithelium confers fibroblast activation through Smad7. Fibroblasts were incubated with exosomes derived from PBS and Arecoline-treated epithelium, then transfection of Anti-15-5p or Smad7-siRNA for 48 h. Immunostaining ( a ) and Immunoblotting ( b ) detection of collagen type I and α-SMA expression in fibroblast. Scale bars, 100 μm. Collagen contraction assay ( c , d ), Sirius Red total collagen assay ( e ) and transwell migration assay ( f , g ) were determined for fibroblast. n = 3–5 technical replicates, representative of two or three assays. Statistics: mean ± SEM, one-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Derivative Assay, Activation Assay, Incubation, Transfection, Immunostaining, Western Blot, Expressing, Contraction Assay, Collagen Assay, Transwell Migration Assay

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: miR-17-5p inhibits the expression of E3 ubiquitin-protein ligase WWP1. a After transfected with miR-17-5p or anti-miR-17-5p for 48 h, the expression of miR-17-5p in fibroblasts was detected by qRT-PCR. Immunoblotting ( b ) and qRT-PCR ( c ) detection of TGFBR1 expression in fibroblasts transfected by miR-17-5p and anti-miR-17-5p. d UbiBrowser 2.0 online site predicts E3 ubiquitin ligase with TGFBR1 as substrate. e The annotation list for the predicted E3 ligases of TGFBR1. f qRT-PCR detection of E3 ubiquitin ligase (NEDD4L, SMURF1, SMURF2, TRAF6, VHL, WWP1) expression level in fibroblast transfected by miR-17-5p and anti-miR-17-5p. Statistics: mean ± SEM, one-way ANOVA, n.s.: no significance, **** P < 0.000 1

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: miR-17-5p inhibits WWP1-mediated degradation of TGFBR1 protein in fibroblasts. a Fibroblast transfected with miR-17-5p then incubated with cycloheximide (CHX, 100 μg/mL) for indicated time points. The protein levels of WWP1 and TGFBR1 were determined at indicated time points by Immunoblotting. b Fibroblasts transfected with miR-17-5p and anti-miR-17-5p then incubated with MG-132 (10 μM, 6 h). The protein levels of TGFBR1 were determined by Immunoblotting. c The expression localization of WWP1 (green) and TGFBR1 (red) in fibroblast was detected by Immunostaining. Scale bar: 50 mm. d , e Co-IP and Immunoblotting detected the interaction between WWP1 and TGFBR1 in fibroblast. f Co-IP and Immunoblotting detected the ubiquitination of TGFBR1 mediated by WWP1 and miR-17-5p overexpressed. g , h The expression of WWP1 and TGFBR1 protein levels in miR-17-5p overexpressed or inhibited in fibroblast was detected by Immunoblotting

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Transfection, Incubation, Western Blot, Expressing, Immunostaining, Co-Immunoprecipitation Assay

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: Down-regulated WWP1 expression can promote myofibroblast transformation. Fibroblasts were transfection of miR-17-5p, anti-15-5p or WWP1-siRNA for 48 h. Immunostaining ( a ) and Immunoblotting ( b ) detection of collagen type I and α-SMA expression in fibroblast. Scale bars, 100 μm. Collagen contraction assay ( c , d ), Sirius Red total collagen assay ( e ) and transwell migration assay ( f , g ) were determined for fibroblast. n = 3–5 technical replicates, representative of two or three assays. Statistics: mean ± SEM, one-way ANOVA, *** P < 0.001, **** P < 0.000 1

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Expressing, Transformation Assay, Transfection, Immunostaining, Western Blot, Contraction Assay, Collagen Assay, Transwell Migration Assay

Journal: International Journal of Oral Science

Article Title: Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

doi: 10.1038/s41368-024-00302-2

Figure Lengend Snippet: Working model showing that the Arecoline induces epithelial cells to release mir-17-5p enveloped exosomes, which could promote collagen contraction, secretion and migration after being absorbed by fibroblast. Mechanistically, it was demonstrated that miR-17-5p targets Smad7 and maintains TGFBR1 expression by inhibiting its E3 ubiquitination ligase WWP1, which promotes the expression of fibrosis hallmark genes

Article Snippet: Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight.

Techniques: Migration, Expressing