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MicroRNA-23b Inhibits the Proliferation and Migration of Heat-Denatured Fibroblasts by Targeting Smad3.

Zhang X, Yang J, Zhao J, Zhang P, Huang X - PLoS ONE (2015)

Bottom Line: MicroRNAs (miRNA) are small noncoding RNAs and regulate normal physiology as well as disease development.In addition, miR-23b modulated denatured dermis by activating the Notch1 and TGF-β signaling pathways.Our findings suggest that downregulation of miR-23b contributes to the recovery of denatured dermis, which may be valuable for treatment of skin burns.

View Article: PubMed Central - PubMed

Affiliation: Department of Burns and Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China.

ABSTRACT

Background: Skin grafting with the preservation of denatured dermis is a novel strategy for the treatment of burn-injured skin. Denatured dermis has the ability to restore to the morphology and function of normal skin, but the underlying molecular mechanism is elusive. MicroRNAs (miRNA) are small noncoding RNAs and regulate normal physiology as well as disease development. In this study, we assessed the potential role of miRNA-23b (miR-23b) in the regulation of cell proliferation and migration of heat-denatured fibroblasts and identified the underlying mechanism.

Methods: The expression of miR-23b in denatured dermis and heat-denatured fibroblasts was detected by quantitative real-time polymerase chain reaction (RT-PCR). The effects of miR-23b on cell proliferation and migration of heat-denatured fibroblasts were assessed by transient transfection of miR-23b mimics and inhibitor. The target gene of miR-23b and the downstream pathway were further investigated.

Results: miR-23b was downregulated in denatured dermis and heat-denatured fibroblasts. Downregulation of miR-23b dramatically promoted the proliferation and migration of heat-denatured fibroblasts. Subsequent analyses demonstrated that Smad3 was a direct and functional target of miR-23b in heat-denatured fibroblasts, which was validated by the dual luciferase reporter assay. Moreover, immunohistochemistry analysis showed that denatured dermis from rats displayed enhanced staining of Smad3. In addition, miR-23b modulated denatured dermis by activating the Notch1 and TGF-β signaling pathways.

Conclusions: Our findings suggest that downregulation of miR-23b contributes to the recovery of denatured dermis, which may be valuable for treatment of skin burns.

No MeSH data available.


Related in: MedlinePlus

Smad3 is a direct target of miR-23b.(A) Western blotting analysis of Smad3 expression after transfection of miR-23b mimics, inhibitor and control in fibroblasts. (B) The intensity of Smad3 staining in denatured dermis of rats was examined by immunohistochemistry three days after burning. Brown-yellow granules in nucleus or cytoplasm were considered positive staining. Scale bar: 100um. (C) Western blotting analysis of Smad3 expression in normal skin and denatured dermis of rats. (D) Dual luciferase assays showed an increase after transfection of miR-23b inhibitor or a decrease after transfection of miR-23b mimics. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control, and denatured dermis versus normal skin.
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pone.0131867.g005: Smad3 is a direct target of miR-23b.(A) Western blotting analysis of Smad3 expression after transfection of miR-23b mimics, inhibitor and control in fibroblasts. (B) The intensity of Smad3 staining in denatured dermis of rats was examined by immunohistochemistry three days after burning. Brown-yellow granules in nucleus or cytoplasm were considered positive staining. Scale bar: 100um. (C) Western blotting analysis of Smad3 expression in normal skin and denatured dermis of rats. (D) Dual luciferase assays showed an increase after transfection of miR-23b inhibitor or a decrease after transfection of miR-23b mimics. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control, and denatured dermis versus normal skin.

Mentions: To well underlying molecular mechanism of miR-23b suppresses the proliferation and migration of heat-denatured fibroblasts, bioinformatic analysis of three publicly available algorithms (PicTar, TargetScan and miRBase) was performed to explore the target gene of miR-23b. Results revealed complementarity between Smad3 3’UTR and miR-23b. To test whether Smad3 can be regulated by miR-23b, the protein level of Smad3 in heat-denatured human fibroblasts transfected with miR-23b mimics or inhibitor was determined by Western blotting. The results showed that miR-23b mimics dramatically decreased the protein level of Smad3, while miR-23b inhibitor increased the Smad3 protein level in heat-denatured human fibroblasts (Fig 5A). Furthermore, in agreement with the decreased expression of miR-23b (Fig 1B), immunohistochemistry of denatured dermis showed that the staining of Smad3 in denatured dermis three days after burning was much stronger than that in normal skin (Fig 5B). In addition, immunoblotting also revealed a marked increase of Smad3 in denatured dermis compared to normal skin from rats (Fig 5C).


MicroRNA-23b Inhibits the Proliferation and Migration of Heat-Denatured Fibroblasts by Targeting Smad3.

Zhang X, Yang J, Zhao J, Zhang P, Huang X - PLoS ONE (2015)

Smad3 is a direct target of miR-23b.(A) Western blotting analysis of Smad3 expression after transfection of miR-23b mimics, inhibitor and control in fibroblasts. (B) The intensity of Smad3 staining in denatured dermis of rats was examined by immunohistochemistry three days after burning. Brown-yellow granules in nucleus or cytoplasm were considered positive staining. Scale bar: 100um. (C) Western blotting analysis of Smad3 expression in normal skin and denatured dermis of rats. (D) Dual luciferase assays showed an increase after transfection of miR-23b inhibitor or a decrease after transfection of miR-23b mimics. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control, and denatured dermis versus normal skin.
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pone.0131867.g005: Smad3 is a direct target of miR-23b.(A) Western blotting analysis of Smad3 expression after transfection of miR-23b mimics, inhibitor and control in fibroblasts. (B) The intensity of Smad3 staining in denatured dermis of rats was examined by immunohistochemistry three days after burning. Brown-yellow granules in nucleus or cytoplasm were considered positive staining. Scale bar: 100um. (C) Western blotting analysis of Smad3 expression in normal skin and denatured dermis of rats. (D) Dual luciferase assays showed an increase after transfection of miR-23b inhibitor or a decrease after transfection of miR-23b mimics. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control, and denatured dermis versus normal skin.
Mentions: To well underlying molecular mechanism of miR-23b suppresses the proliferation and migration of heat-denatured fibroblasts, bioinformatic analysis of three publicly available algorithms (PicTar, TargetScan and miRBase) was performed to explore the target gene of miR-23b. Results revealed complementarity between Smad3 3’UTR and miR-23b. To test whether Smad3 can be regulated by miR-23b, the protein level of Smad3 in heat-denatured human fibroblasts transfected with miR-23b mimics or inhibitor was determined by Western blotting. The results showed that miR-23b mimics dramatically decreased the protein level of Smad3, while miR-23b inhibitor increased the Smad3 protein level in heat-denatured human fibroblasts (Fig 5A). Furthermore, in agreement with the decreased expression of miR-23b (Fig 1B), immunohistochemistry of denatured dermis showed that the staining of Smad3 in denatured dermis three days after burning was much stronger than that in normal skin (Fig 5B). In addition, immunoblotting also revealed a marked increase of Smad3 in denatured dermis compared to normal skin from rats (Fig 5C).

Bottom Line: MicroRNAs (miRNA) are small noncoding RNAs and regulate normal physiology as well as disease development.In addition, miR-23b modulated denatured dermis by activating the Notch1 and TGF-β signaling pathways.Our findings suggest that downregulation of miR-23b contributes to the recovery of denatured dermis, which may be valuable for treatment of skin burns.

View Article: PubMed Central - PubMed

Affiliation: Department of Burns and Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China.

ABSTRACT

Background: Skin grafting with the preservation of denatured dermis is a novel strategy for the treatment of burn-injured skin. Denatured dermis has the ability to restore to the morphology and function of normal skin, but the underlying molecular mechanism is elusive. MicroRNAs (miRNA) are small noncoding RNAs and regulate normal physiology as well as disease development. In this study, we assessed the potential role of miRNA-23b (miR-23b) in the regulation of cell proliferation and migration of heat-denatured fibroblasts and identified the underlying mechanism.

Methods: The expression of miR-23b in denatured dermis and heat-denatured fibroblasts was detected by quantitative real-time polymerase chain reaction (RT-PCR). The effects of miR-23b on cell proliferation and migration of heat-denatured fibroblasts were assessed by transient transfection of miR-23b mimics and inhibitor. The target gene of miR-23b and the downstream pathway were further investigated.

Results: miR-23b was downregulated in denatured dermis and heat-denatured fibroblasts. Downregulation of miR-23b dramatically promoted the proliferation and migration of heat-denatured fibroblasts. Subsequent analyses demonstrated that Smad3 was a direct and functional target of miR-23b in heat-denatured fibroblasts, which was validated by the dual luciferase reporter assay. Moreover, immunohistochemistry analysis showed that denatured dermis from rats displayed enhanced staining of Smad3. In addition, miR-23b modulated denatured dermis by activating the Notch1 and TGF-β signaling pathways.

Conclusions: Our findings suggest that downregulation of miR-23b contributes to the recovery of denatured dermis, which may be valuable for treatment of skin burns.

No MeSH data available.


Related in: MedlinePlus