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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 promoted the proliferation of heat-denatured fibroblasts.(A) Western blotting analyses confirmed upregulation and downregulation of Smad3 in fibroblasts by pcDNA-Smad3 plasmid or siRNA, respectively. (B) Cell proliferation was measured by CCK-8 assay after transfection with pcDNA-Smad3 or siRNA. *P < 0.05, **P < 0.01, siRNA group versus control group, pcDNA group versus control group.
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pone.0131867.g006: Smad3 promoted the proliferation of heat-denatured fibroblasts.(A) Western blotting analyses confirmed upregulation and downregulation of Smad3 in fibroblasts by pcDNA-Smad3 plasmid or siRNA, respectively. (B) Cell proliferation was measured by CCK-8 assay after transfection with pcDNA-Smad3 or siRNA. *P < 0.05, **P < 0.01, siRNA group versus control group, pcDNA group versus control group.

Mentions: Our above observations suggest that upregulation of Smad3 might be required for the recovery of heat-denatured dermis and fibroblasts. To test this hypothesis, we assessed the cell growth and the migratory capability of heat-denatured fibroblasts after up or down-regulation of Smad3. Western blotting was performed to validate the level of Smad3 in fibroblasts after transfection with plasmid or siRNA (Fig 6A). The CCK-8 proliferation assay showed that the growth rate was increased in fibroblasts transfected with pcDNA-Smad3 compared with cells transfected with control. Meanwhile, the growth rate was reduced while transfecting with Smad3 siRNA compared with control. (Fig 6B). Moreover, the migratory capacity of cells transfected with pcDNA-Smad3 was dramatically increased compared with the control group and the migratory capacity of cells transfected with siRNA was decreased compared with the control group cells. (Fig 7). These results suggested that up-and down-regulation of Smad3 mimicked the effect of decreasing and increasing miR-23b expression in heat-denatured fibroblasts.


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 promoted the proliferation of heat-denatured fibroblasts.(A) Western blotting analyses confirmed upregulation and downregulation of Smad3 in fibroblasts by pcDNA-Smad3 plasmid or siRNA, respectively. (B) Cell proliferation was measured by CCK-8 assay after transfection with pcDNA-Smad3 or siRNA. *P < 0.05, **P < 0.01, siRNA group versus control group, pcDNA group versus control group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496062&req=5

pone.0131867.g006: Smad3 promoted the proliferation of heat-denatured fibroblasts.(A) Western blotting analyses confirmed upregulation and downregulation of Smad3 in fibroblasts by pcDNA-Smad3 plasmid or siRNA, respectively. (B) Cell proliferation was measured by CCK-8 assay after transfection with pcDNA-Smad3 or siRNA. *P < 0.05, **P < 0.01, siRNA group versus control group, pcDNA group versus control group.
Mentions: Our above observations suggest that upregulation of Smad3 might be required for the recovery of heat-denatured dermis and fibroblasts. To test this hypothesis, we assessed the cell growth and the migratory capability of heat-denatured fibroblasts after up or down-regulation of Smad3. Western blotting was performed to validate the level of Smad3 in fibroblasts after transfection with plasmid or siRNA (Fig 6A). The CCK-8 proliferation assay showed that the growth rate was increased in fibroblasts transfected with pcDNA-Smad3 compared with cells transfected with control. Meanwhile, the growth rate was reduced while transfecting with Smad3 siRNA compared with control. (Fig 6B). Moreover, the migratory capacity of cells transfected with pcDNA-Smad3 was dramatically increased compared with the control group and the migratory capacity of cells transfected with siRNA was decreased compared with the control group cells. (Fig 7). These results suggested that up-and down-regulation of Smad3 mimicked the effect of decreasing and increasing miR-23b expression in heat-denatured fibroblasts.

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