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. 2016 Aug 24:6:32231.
doi: 10.1038/srep32231.

TGF-β1 promotes scar fibroblasts proliferation and transdifferentiation via up-regulating MicroRNA-21

Ying Liu  1 , Yue Li  2 , Ning Li  1 , Wen Teng  1 , Min Wang  1 , Yingbo Zhang  1 , Zhibo Xiao  1
Affiliations

TGF-β1 promotes scar fibroblasts proliferation and transdifferentiation via up-regulating MicroRNA-21

Ying Liu et al. Sci Rep. .

Abstract

TGF-β1, upregulated in keloid tissue, promotes the proliferation, collagen formation and differentiation of dermal fibroblasts. miR-21 is one of microRNAs first found in human genome. The aim of our study is to explore the mechanisms of miR-21 in TGF-β1-induced scar fibroblasts proliferation and transdifferentiation. In the present study, first we found that TGF-β1 promoted scar fibroblasts proliferation and transdifferentiation via up-regulating miR-21 expression, which could be attenuated when miR-21 was inhibited. Overexpression of miR-21 had similar effect as TGF-β1 on proliferation and transdifferentiation. Additionally, TGF-β1 increased the expressions and activities of MMP2 and MMP9 in keloid fibroblasts, which was suppressed by miR-21 inhibition. Finally, the results demonstrated that PTEN/AKT signaling pathway played important role in TGF-β1-induced transdifferentiation. In conclusion, our study suggests that TGF-β1 promotes keloid fibroblasts proliferation and transdifferentiation via up-regulation of miR-21 and PTEN/AKT signalling pathway plays important role in this process, which provides a potential theoretical basis for clinical treatment of skin scars.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Up-regulating of miR-21 was induced by TGF-β1 in human keloid fibroblasts.
The expressions of pre- (A) and mature miR-21 (B) in human keloid fibroblasts after treatment with TGF-β1 for different time were detected by real-time PCR. Each result represents at least three independent experiments. Data represents the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, versus the control group.
Figure 2
Figure 2. Overexpression of miR-21 promoted proliferation and transdifferentiation in human keloid fibroblasts.
Cell proliferation was determined by MTT (A) and BrdU (B) assays. (C,D) α-SMA was up-regulated and E-cadherin was down-regulated by miR-21 overexpression assayed by western blot. β-actin was used as a loading control. (E) The protein expressions of α-SMA and E-cadherin were detected by immunofluorescence staining. Each result represents at least three independent experiments. Data represents the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, versus the parental group.
Figure 3
Figure 3. TGF-β1 induced-proliferation and transdifferentiation in human keloid fibroblasts was attenuated by miR-21 inhibition.
Cell proliferation was determined by MTT (A) and BrdU (B) assays. (C) The protein expressions of α-SMA and E-cadherin were determined by western blot assay. (D) The protein quantification histogram was shown. β-actin was used as a loading control. (E) The protein expressions of α-SMA and E-cadherin were determined by immunofluorescence staining. Each result represents at least three independent experiments. Data represents the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, versus the parental group. ##P < 0.01, ###P < 0.001, versus the TGF-β1 group.
Figure 4
Figure 4. TGF-β1 induced-expressions and activities of MMP2 and MMP9 in human keloid fibroblasts was attenuated by miR-21 inhibition.
(A) The protein expressions of MMP9 and MMP2 were determined by western blot. The protein quantification histogram was shown. β-actin was used as a loading control. (B) The mRNA expressions of MMP9 and MMP2 were determined by real-time PCR. (C) The activities of MMP9 and MMP2 were detected by gelatin zymography assay. Each result represents at least three independent experiments. Data represents the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, versus the parental group. #P < 0.05, ###P < 0.001, versus the TGF-β1 group.
Figure 5
Figure 5. PTEN/AKT signaling pathway was regulated by TGF-β1 in human keloid fibroblasts, which could be weakened by miR-21 inhibition.
The protein expressions of PTEN (A) and AKT (B) after treatment with TGF-β1 for different time were determined by western blot. β-actin was used as a loading control. (C) Inhibition of miR-21 attenuated the down-regulation of PTEN induced by TGF-β1. β-actin was used as a loading control. Each result represents at least three independent experiments. Data represents the mean ± SD (n = 3). *P < 0.05, ***P < 0.001, versus the parental group. ##P < 0.01, versus the TGF-β1 group.
Figure 6
Figure 6. Inhibiting the expression of AKT could restrain the transdifferentiation induced by miR-21 overexpression in keloid fibroblasts.
(A) The protein expressions of α-SMA and E-cadherin were determined by western blot assay. The protein quantification histogram was shown. β-actin was used as a loading control. (B) The protein expressions of α-SMA and E-cadherin were determined by immunofluorescence staining. Each result represents at least three independent experiments. Data represents the mean ± SD (n = 3). ***P < 0.001, versus the parental group. ###P < 0.001, versus the TGF-β1 group.
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