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MicroRNA 181b regulates decorin production by dermal fibroblasts and may be a potential therapy for hypertrophic scar.

Kwan P, Ding J, Tredget EE - PLoS ONE (2015)

Bottom Line: Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis.By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts.This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing.

View Article: PubMed Central - PubMed

Affiliation: Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; Wound Healing Research Group, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Hypertrophic scarring is a frequent fibroproliferative complication following deep dermal burns leading to impaired function and lifelong disfigurement. Decorin reduces fibrosis and induces regeneration in many tissues, and is significantly downregulated in hypertrophic scar and normal deep dermal fibroblasts. It was hypothesized that microRNAs in these fibroblasts downregulate decorin and blocking them would increase decorin and may prevent hypertrophic scarring. Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis. A decorin 3' un-translated region reporter assay demonstrated microRNA decreased decorin in deep dermal fibroblasts, and microRNA screening predicted miR- 24, 181b, 421, 526b, or 543 as candidates. After finding increased levels of mir-181b in deep dermal fibroblasts, it was demonstrated that TGF-β1 stimulation decreased miR-24 but increased miR-181b and that hypertrophic scar and deep dermis contained increased levels of miR-181b. By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts. This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing. Furthermore, blocking miR-181b reversed TGF-β1 induced decorin downregulation and myofibroblast differentiation in hypertrophic scar fibroblasts, suggesting a potential therapy for hypertrophic scar.

No MeSH data available.


Related in: MedlinePlus

Regulation of DCN by miR-181b.HEK293A were cultured in DMEM + 2% FBS and transfected with pmirGLO constructs containing various miRNA binding sites and (a) relative fluorescence quantitated using a luminometer to determine relative knockdown by miR-181b (mean ± SEM, n = 4, *** P ≤ 0.01). SF were cultured in DMEM + 2% FBS and transfected with miR-control, synthetic miR-181b or siRNA-DCN and (b) DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, ** P < 0.03), and (c) DCN mRNA was measured using RT-qPCR on total RNA (mean ± SEM, n = 3, * P < 0.05). (d) DF were cultured in DMEM + 2% FBS and transfected with antagomiR-control (amiR-control) or antagomiR-181b (amiR-181b) and DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, *** P < 0.01).
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pone.0123054.g005: Regulation of DCN by miR-181b.HEK293A were cultured in DMEM + 2% FBS and transfected with pmirGLO constructs containing various miRNA binding sites and (a) relative fluorescence quantitated using a luminometer to determine relative knockdown by miR-181b (mean ± SEM, n = 4, *** P ≤ 0.01). SF were cultured in DMEM + 2% FBS and transfected with miR-control, synthetic miR-181b or siRNA-DCN and (b) DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, ** P < 0.03), and (c) DCN mRNA was measured using RT-qPCR on total RNA (mean ± SEM, n = 3, * P < 0.05). (d) DF were cultured in DMEM + 2% FBS and transfected with antagomiR-control (amiR-control) or antagomiR-181b (amiR-181b) and DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, *** P < 0.01).

Mentions: To confirm predicted miR-181b binding sites from the DCN 3’UTR a series of dual luciferase reporter vectors based on pmirGLO were created and transfected into HEK293A cells (Fig 5A). There was no difference in regulation by miR-181b of reporters with no binding site or a scramble site (P = 0.96), however reporters with a perfect miR-181b site or one of three predicted miR-181b binding sites from the DCN 3’UTR (S2 Fig) were all significantly downregulated by miR-181b (P ≤ 0.01). One method to confirm miRNA regulation is to modulate miRNA levels and observe effects on its putative target [47]. Therefore, to confirm that miR-181b regulates DCN, synthetic miR-181b and antagomiR-181b were used to change miR-181b levels and changes in DCN measured in dermal fibroblasts. SF were transfected with a synthetic miR-181b mimic which significantly reduced DCN protein by ELISA (P < 0.03) (Fig 5B), similar to DCN siRNA (P < 0.02), but not DCN mRNA by RT-qPCR (Fig 5C). When DF were transfected with antagomiR-181b, DCN protein by ELISA was significantly increased (P < 0.01) (Fig 5D). DCN protein levels are expressed as fold changes to allow comparison despite variation between fibroblasts from different individuals.


MicroRNA 181b regulates decorin production by dermal fibroblasts and may be a potential therapy for hypertrophic scar.

Kwan P, Ding J, Tredget EE - PLoS ONE (2015)

Regulation of DCN by miR-181b.HEK293A were cultured in DMEM + 2% FBS and transfected with pmirGLO constructs containing various miRNA binding sites and (a) relative fluorescence quantitated using a luminometer to determine relative knockdown by miR-181b (mean ± SEM, n = 4, *** P ≤ 0.01). SF were cultured in DMEM + 2% FBS and transfected with miR-control, synthetic miR-181b or siRNA-DCN and (b) DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, ** P < 0.03), and (c) DCN mRNA was measured using RT-qPCR on total RNA (mean ± SEM, n = 3, * P < 0.05). (d) DF were cultured in DMEM + 2% FBS and transfected with antagomiR-control (amiR-control) or antagomiR-181b (amiR-181b) and DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, *** P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383602&req=5

pone.0123054.g005: Regulation of DCN by miR-181b.HEK293A were cultured in DMEM + 2% FBS and transfected with pmirGLO constructs containing various miRNA binding sites and (a) relative fluorescence quantitated using a luminometer to determine relative knockdown by miR-181b (mean ± SEM, n = 4, *** P ≤ 0.01). SF were cultured in DMEM + 2% FBS and transfected with miR-control, synthetic miR-181b or siRNA-DCN and (b) DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, ** P < 0.03), and (c) DCN mRNA was measured using RT-qPCR on total RNA (mean ± SEM, n = 3, * P < 0.05). (d) DF were cultured in DMEM + 2% FBS and transfected with antagomiR-control (amiR-control) or antagomiR-181b (amiR-181b) and DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, *** P < 0.01).
Mentions: To confirm predicted miR-181b binding sites from the DCN 3’UTR a series of dual luciferase reporter vectors based on pmirGLO were created and transfected into HEK293A cells (Fig 5A). There was no difference in regulation by miR-181b of reporters with no binding site or a scramble site (P = 0.96), however reporters with a perfect miR-181b site or one of three predicted miR-181b binding sites from the DCN 3’UTR (S2 Fig) were all significantly downregulated by miR-181b (P ≤ 0.01). One method to confirm miRNA regulation is to modulate miRNA levels and observe effects on its putative target [47]. Therefore, to confirm that miR-181b regulates DCN, synthetic miR-181b and antagomiR-181b were used to change miR-181b levels and changes in DCN measured in dermal fibroblasts. SF were transfected with a synthetic miR-181b mimic which significantly reduced DCN protein by ELISA (P < 0.03) (Fig 5B), similar to DCN siRNA (P < 0.02), but not DCN mRNA by RT-qPCR (Fig 5C). When DF were transfected with antagomiR-181b, DCN protein by ELISA was significantly increased (P < 0.01) (Fig 5D). DCN protein levels are expressed as fold changes to allow comparison despite variation between fibroblasts from different individuals.

Bottom Line: Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis.By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts.This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing.

View Article: PubMed Central - PubMed

Affiliation: Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; Wound Healing Research Group, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Hypertrophic scarring is a frequent fibroproliferative complication following deep dermal burns leading to impaired function and lifelong disfigurement. Decorin reduces fibrosis and induces regeneration in many tissues, and is significantly downregulated in hypertrophic scar and normal deep dermal fibroblasts. It was hypothesized that microRNAs in these fibroblasts downregulate decorin and blocking them would increase decorin and may prevent hypertrophic scarring. Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis. A decorin 3' un-translated region reporter assay demonstrated microRNA decreased decorin in deep dermal fibroblasts, and microRNA screening predicted miR- 24, 181b, 421, 526b, or 543 as candidates. After finding increased levels of mir-181b in deep dermal fibroblasts, it was demonstrated that TGF-β1 stimulation decreased miR-24 but increased miR-181b and that hypertrophic scar and deep dermis contained increased levels of miR-181b. By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts. This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing. Furthermore, blocking miR-181b reversed TGF-β1 induced decorin downregulation and myofibroblast differentiation in hypertrophic scar fibroblasts, suggesting a potential therapy for hypertrophic scar.

No MeSH data available.


Related in: MedlinePlus