MicroRNA-146a modulates TGF-ß1-induced phenotypic differentiation in human dermal fibroblasts by targeting SMAD4.

During wound healing and tissue repair the dermal fibroblast-to-myofibroblast transdifferentiation plays an important role, transforming growth factor-ß1 (TGF-ß1) is considered to be the main stimuli factor of transdifferentiation. MicroRNAs (miRNAs) have recently emerged as key post-transcriptional regulators of gene expression. The involvement of miRNAs and their roles in TGF-ß1-induced myofibroblast transdifferentiation remains to be determined in detail. The current study found that the expression of miR-146a was upregulated in human dermal fibroblasts cells in response to TGF-ß1 stimulation in dose-dependent manner by quantitative RT-PCR. Bioinformatic analyses predict that signaling effectors mothers against decapentaplegic protein 4 (SMAD4) is a miR-146a target gene. Luciferase assay demonstrated that miR-146a mimics suppressed SMAD4 3'-UTR reporter construct activity. Furthermore, miR-146a overexpression in dermal fibroblast did not decrease target mRNA levels, but significantly reduced target protein expression. In addition, dermal fibroblasts transfected with miR-146a mimics exhibited attenuated TGF-ß1 -induced α-smooth muscle actin (α-SMA) expression compared with the control. This study demonstrated that miR-146a may function as a novel negative regulator to modulate myofibroblast transdifferentiation during TGF-ß1 induction by targeting SMAD4.

[Effect of skin autograft with different thickness on the transdifferentiation of fibroblasts after deep partial thickness burn in rats].

OBJECTIVE: To investigate the effect of thin split-thickness skin, inter-mediate thickness skin and full-thickness skin autograft on the differentiation of fibroblasts into myofibroblasts in rats after deep partial thickness burn. METHODS: A total of 40 SD rats were divided randomly into two groups (Group A & Group B, n = 20 each). In Group A, tissue samples were collected at Day 2 after skin-grafting while Day 7 in Group B. In each group, every rat was scalded to cause deep partial thickness wound with an area of 10% of total body surface. The wounds received eschar shaving instantly coupled with skin-autograft, covering with thin split-thickness skin, inter-mediate thickness skin and full-thickness skin respectively. Meanwhile the control wound on the same rat was scalded only. Then the expression of α-SMA was detected by immunohistochemistry in each wound. And the numbers of myofibroblasts (α-SMA positive cells) and fibroblasts (negative cells) were counted to calculate the conversion ratio of myofibroblasts. RESULTS: In Group A, the conversion ratios of myofibroblasts of control, thin split-thickness skin autograft, inter-mediate thickness skin and full-thickness skin groups were (76.3 ± 3.3)%, (69.8 ± 1.6)%, (57.5 ± 1.6)% and (44.7 ± 1.7)% respectively. In Group B, the ratios were (72.9 ± 6.1)%, (63.6 ± 4.7)%, (50.2 ± 1.6)% and (32.3 ± 1.2)% respectively. The ratio was higher in control group than that in any other one (P < 0.01). There was statistic difference between thin split-thickness skin, inter-mediate thickness skin and full-thickness skin autograft groups (P < 0.05). CONCLUSION: A direct association may exist between the conversion ratio of myofibroblasts and the application of skin-grafting in rats after deep partial thickness scalding. It is probably related with varying degrees of scar contracture in the long-term.

[The effect of RhoA/Rho kinase signal pathway on TGF-beta1-induced phenotypic differentiation of human dermal fibroblasts].

OBJECTIVE: To examine the effect of RhoA/Rho kinase signal pathway on TGF-beta1-induced phenotypic differentiation of human dermal fibroblasts. METHODS: The 4th generation of primary cultured human dermal fibroblasts were stimulated with TGF-beta1, (10 ng/ml). The expression of alpha-SMA was detected after treatment with TGF-beta1, for 0, 3, 6, and 24 h. The expression of alpha-SMA was also detected after treatment with different concentration of TGF-beta1 (0, 2, 10, 50 ng/ml). Then the human dermal fibroblasts (4th generation) were stimulated with TGF-beta1, (10 ng/ml) after being treated with the RhoA/Rho kinase signaling pathway inhibitor Y-27632 (10 umol/ml). The fibroblasts were treated with nothing as sham control, or with Y-27632 (10 umol/L) only as negative control group, or with TGF-beta1 (10 ng/ml) only as positive control group. The expression of alpha-SMA was detected in all the groups. Protein expression was analyzed with ANOVA statistical method. RESULTS: alpha-SMA expression in fibroblasts with 10 ng/ml TGF-beta1 stimulation for 0, 3, 6, 24 h was 1.0, 1.9 0.2, 2.1 +/- 0. 1, 3. 1 +/- 0.1, respectively. Alpha-SMA expression in 24 h group was significantly higher than that in other three groups (n = 4, P < 0.05). alpha-SMA expression in human dermal fibroblasts after stimulation with different concentration of TGF-beta1 (0, 2, 10, 50 ng/ml) was 1.0, 1.4 +/- 0.2, 3.2 + 0.1, 3.1 +/- 0.2, respectively. alpha-SMA expression in 10 ng/ ml group was significantly higher than that in 2 ng/ml group and control group (n = 4, P < 0.05). There was no statistical difference in alpha-SMA expression between 10 ng/ml group and 50 ng/ml group (n = 4, P > 0.05). With both Y-27632 (10 micromol/L) and TGF-beta1 stimulation, the cell phenotype differentiation was inhibited. Alpha-SMA expression in experimental group (1.2 +/- 0.2) was significantly reduced, when compared with that in positive control group (2.9 +/- 0.1) (n = 5, P < 0.05). There was no significant difference (n = 5, P > 0.05) in alpha-SMA expression between control group (1.0) and negative control group (1.1 +/- 0.1). CONCLUSIONS: RhoA/Rho kinase signaling pathway should be involved in TGF-beta1-induced phenotypic differentiation of human dermal fibroblasts.