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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

MiR-23b suppressed the proliferation of heat-denatured fibroblasts.(A) miR-23b expression level was examined by quantitative RT-PCR after transfection with mimics and inhibitor. (B) Cell proliferation was measured by CCK-8 assay after treatment with the miR-23b mimics, inhibitor or empty vector. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control.
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pone.0131867.g003: MiR-23b suppressed the proliferation of heat-denatured fibroblasts.(A) miR-23b expression level was examined by quantitative RT-PCR after transfection with mimics and inhibitor. (B) Cell proliferation was measured by CCK-8 assay after treatment with the miR-23b mimics, inhibitor or empty vector. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control.

Mentions: To investigate the biological function of miR-23b in heat-denatured fibroblasts, human fibroblasts were transfected with miR-23b mimics to increase the endogenous level of miR-23b, or miR-23b inhibitor to decrease the level of miR-23b. qRT-PCR was performed to validate the level of miR-23b after transfection. Compared with miR-23b mimics and inhibitor negative controls, transfection of miR-23b mimics led to a dramatic increase of miR-23b, and transfection of miR-23b inhibitor suppressed endogenous miR-23b expression (Fig 3A). Next, fibroblasts were subjected to heat damage 12 h after transfection of miR-23b mimics or inhibitor and cell proliferation was assessed by CCK-8 assay at 24, 48 and 72 h after transfection. After 48 h, the proliferation of cells transfected with miR-23b inhibitor significantly increased compared with cells transfected with scramble mimics or untreated cells. However, there was no significant difference in the viability of cells transfected with miR-23b mimics compared with control cells (Fig 3B). To investigate whether miR-23b regulates fibroblast migration following heat damage, in vitro scratch and Transwell migration assays were performed on heat-denatured fibroblasts following miR-23b transfection. Results showed that the migration capacity of cells transfected with miR-23b inhibitor was dramatically increased compared with the scramble and control group and the migration capacity of cells transfected with miR-23b mimics was dramatically decreased compared with the scramble and control group cells (Fig 4). These results clearly demonstrate that suppression of miR-23 promotes the proliferation and migration of heat-denatured fibroblasts, suggesting that downregulation of miR-23b might be required for the recovery of heat-denatured dermis and 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)

MiR-23b suppressed the proliferation of heat-denatured fibroblasts.(A) miR-23b expression level was examined by quantitative RT-PCR after transfection with mimics and inhibitor. (B) Cell proliferation was measured by CCK-8 assay after treatment with the miR-23b mimics, inhibitor or empty vector. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496062&req=5

pone.0131867.g003: MiR-23b suppressed the proliferation of heat-denatured fibroblasts.(A) miR-23b expression level was examined by quantitative RT-PCR after transfection with mimics and inhibitor. (B) Cell proliferation was measured by CCK-8 assay after treatment with the miR-23b mimics, inhibitor or empty vector. *P < 0.05, **P < 0.01, mimics versus control, inhibitor versus control.
Mentions: To investigate the biological function of miR-23b in heat-denatured fibroblasts, human fibroblasts were transfected with miR-23b mimics to increase the endogenous level of miR-23b, or miR-23b inhibitor to decrease the level of miR-23b. qRT-PCR was performed to validate the level of miR-23b after transfection. Compared with miR-23b mimics and inhibitor negative controls, transfection of miR-23b mimics led to a dramatic increase of miR-23b, and transfection of miR-23b inhibitor suppressed endogenous miR-23b expression (Fig 3A). Next, fibroblasts were subjected to heat damage 12 h after transfection of miR-23b mimics or inhibitor and cell proliferation was assessed by CCK-8 assay at 24, 48 and 72 h after transfection. After 48 h, the proliferation of cells transfected with miR-23b inhibitor significantly increased compared with cells transfected with scramble mimics or untreated cells. However, there was no significant difference in the viability of cells transfected with miR-23b mimics compared with control cells (Fig 3B). To investigate whether miR-23b regulates fibroblast migration following heat damage, in vitro scratch and Transwell migration assays were performed on heat-denatured fibroblasts following miR-23b transfection. Results showed that the migration capacity of cells transfected with miR-23b inhibitor was dramatically increased compared with the scramble and control group and the migration capacity of cells transfected with miR-23b mimics was dramatically decreased compared with the scramble and control group cells (Fig 4). These results clearly demonstrate that suppression of miR-23 promotes the proliferation and migration of heat-denatured fibroblasts, suggesting that downregulation of miR-23b might be required for the recovery of heat-denatured dermis and 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