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MicroRNA-21 regulates hTERT via PTEN in hypertrophic scar fibroblasts.

Zhu HY, Li C, Bai WD, Su LL, Liu JQ, Li Y, Shi JH, Cai WX, Bai XZ, Jia YH, Zhao B, Wu X, Li J, Hu DH - PLoS ONE (2014)

Bottom Line: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases.Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation.In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN.

View Article: PubMed Central - PubMed

Affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, People's Republic of China.

ABSTRACT

Background: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated.

Methodology/principal findings: Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues.

Conclusions/significance: These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.

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Involvement of the PTEN/PI3K/AKT signaling pathway and hTERT in miR-21 mediated effects.A, Protein expression after transfection of HSFBs. B, PTEN and hTERT mRNA expression after transfection of HSFBs. C, Indirect immunofluorescence of PTEN (green) and hTERT (red) after transfection of HSFBs.
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pone-0097114-g003: Involvement of the PTEN/PI3K/AKT signaling pathway and hTERT in miR-21 mediated effects.A, Protein expression after transfection of HSFBs. B, PTEN and hTERT mRNA expression after transfection of HSFBs. C, Indirect immunofluorescence of PTEN (green) and hTERT (red) after transfection of HSFBs.

Mentions: We investigated the effects of miR-21 on gene expression and signaling pathways in HSFBs. In accordance with the results of the luciferase reporter assay results, transient transfection of HFSBs with the miR-21 mimic resulted in a significant reduction in PTEN expression at both the protein (Figure 3A and Figure S1) and mRNA (Figure 3B, p = 0.023) levels, while the miR-21 inhibitor-mediated upregulation of PTEN. The PI3K/AKT pathway is an important downstream target of PTEN and PTEN expression results in reduced levels of phosphorylated AKT [35]. Therefore, we investigated the levels of this protein in parallel. As shown in Figure 3A, the expression of phosphorylated AKT and PI3K were decreased by the miR-21 inhibitor. In contrast, transfection of HSFBs with the miR-21 mimic had the opposite effect on phosphorylated AKT and PI3K expression levels. Interestingly, we observed that hTERT, an important gene related to proliferation and apoptosis, was modulated positively by miR-21 at both the protein (Figure 3A and Figure S1) and mRNA levels (Figure 3B, p = 0.036). Indirect immunofluorescence staining of PTEN (green) and hTERT (red) displayed inverse levels in miR-21 mimic and miR-21 inhibitor-treated groups. Thus, we demonstrated that PI3K/AKT signaling was altered by targeting PTEN and that PI3K/AKT signaling is moderated by miR-21 at both the mRNA and protein levels.


MicroRNA-21 regulates hTERT via PTEN in hypertrophic scar fibroblasts.

Zhu HY, Li C, Bai WD, Su LL, Liu JQ, Li Y, Shi JH, Cai WX, Bai XZ, Jia YH, Zhao B, Wu X, Li J, Hu DH - PLoS ONE (2014)

Involvement of the PTEN/PI3K/AKT signaling pathway and hTERT in miR-21 mediated effects.A, Protein expression after transfection of HSFBs. B, PTEN and hTERT mRNA expression after transfection of HSFBs. C, Indirect immunofluorescence of PTEN (green) and hTERT (red) after transfection of HSFBs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016251&req=5

pone-0097114-g003: Involvement of the PTEN/PI3K/AKT signaling pathway and hTERT in miR-21 mediated effects.A, Protein expression after transfection of HSFBs. B, PTEN and hTERT mRNA expression after transfection of HSFBs. C, Indirect immunofluorescence of PTEN (green) and hTERT (red) after transfection of HSFBs.
Mentions: We investigated the effects of miR-21 on gene expression and signaling pathways in HSFBs. In accordance with the results of the luciferase reporter assay results, transient transfection of HFSBs with the miR-21 mimic resulted in a significant reduction in PTEN expression at both the protein (Figure 3A and Figure S1) and mRNA (Figure 3B, p = 0.023) levels, while the miR-21 inhibitor-mediated upregulation of PTEN. The PI3K/AKT pathway is an important downstream target of PTEN and PTEN expression results in reduced levels of phosphorylated AKT [35]. Therefore, we investigated the levels of this protein in parallel. As shown in Figure 3A, the expression of phosphorylated AKT and PI3K were decreased by the miR-21 inhibitor. In contrast, transfection of HSFBs with the miR-21 mimic had the opposite effect on phosphorylated AKT and PI3K expression levels. Interestingly, we observed that hTERT, an important gene related to proliferation and apoptosis, was modulated positively by miR-21 at both the protein (Figure 3A and Figure S1) and mRNA levels (Figure 3B, p = 0.036). Indirect immunofluorescence staining of PTEN (green) and hTERT (red) displayed inverse levels in miR-21 mimic and miR-21 inhibitor-treated groups. Thus, we demonstrated that PI3K/AKT signaling was altered by targeting PTEN and that PI3K/AKT signaling is moderated by miR-21 at both the mRNA and protein levels.

Bottom Line: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases.Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation.In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN.

View Article: PubMed Central - PubMed

Affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, People's Republic of China.

ABSTRACT

Background: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated.

Methodology/principal findings: Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues.

Conclusions/significance: These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.

Show MeSH
Related in: MedlinePlus