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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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The 3′-UTR of PTEN is a target for miR-21.A, Predicted miR-21 binding sites within the 3′-UTR of PTEN mRNA. The arrows display the mutational nucleotides. B, The wt or mutant reporter plasmid was cotransfected into HSBFs with miR-21 inhibitor or negative control (NC). The normalized luciferase activity in the control group was set as relative luciferase activity. Luciferase activity of pGL3-PTEN was increased significantly by miR-21 inhibitor. However, luciferase activity of pGL3-PTEN-mut was not affected by the miR-21 inhibitor. All data are representative of three independent experiments.
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pone-0097114-g002: The 3′-UTR of PTEN is a target for miR-21.A, Predicted miR-21 binding sites within the 3′-UTR of PTEN mRNA. The arrows display the mutational nucleotides. B, The wt or mutant reporter plasmid was cotransfected into HSBFs with miR-21 inhibitor or negative control (NC). The normalized luciferase activity in the control group was set as relative luciferase activity. Luciferase activity of pGL3-PTEN was increased significantly by miR-21 inhibitor. However, luciferase activity of pGL3-PTEN-mut was not affected by the miR-21 inhibitor. All data are representative of three independent experiments.

Mentions: MiRNAs post-transcriptionally silence specific genes via binding to the target mRNAs. We predicted potential targets using the computer-aided algorithms in Targetscan and miRbase Targets. PTEN, a widely expressed tumor suppressor, was identified as a potential miR-21 target gene. The 3′-UTR of PTEN containing the potential miR-21 binding site was cloned for use in a firefly luciferase reporter assay (Figure 2A). The PTEN-UTR or PTEN-UTR-mutant(mut) reporter plasmids were cotransfected into HSFBs along with the miR-21 inhibitor or scramble control [27]. Compared with the scramble control, the miR-21 inhibitor increased the relative luciferase activity significantly when cotransfected with the PTEN-UTR reporter plasmid (p = 0.018). However, the mutant reporter plasmid abolished the miR-21 inhibitor-mediated increase in luciferase activity (Figure 2B, p = 0.536). These findings suggest that miR-21 suppresses PTEN by direct binding to the 3′-UTR of PTEN.


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)

The 3′-UTR of PTEN is a target for miR-21.A, Predicted miR-21 binding sites within the 3′-UTR of PTEN mRNA. The arrows display the mutational nucleotides. B, The wt or mutant reporter plasmid was cotransfected into HSBFs with miR-21 inhibitor or negative control (NC). The normalized luciferase activity in the control group was set as relative luciferase activity. Luciferase activity of pGL3-PTEN was increased significantly by miR-21 inhibitor. However, luciferase activity of pGL3-PTEN-mut was not affected by the miR-21 inhibitor. All data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0097114-g002: The 3′-UTR of PTEN is a target for miR-21.A, Predicted miR-21 binding sites within the 3′-UTR of PTEN mRNA. The arrows display the mutational nucleotides. B, The wt or mutant reporter plasmid was cotransfected into HSBFs with miR-21 inhibitor or negative control (NC). The normalized luciferase activity in the control group was set as relative luciferase activity. Luciferase activity of pGL3-PTEN was increased significantly by miR-21 inhibitor. However, luciferase activity of pGL3-PTEN-mut was not affected by the miR-21 inhibitor. All data are representative of three independent experiments.
Mentions: MiRNAs post-transcriptionally silence specific genes via binding to the target mRNAs. We predicted potential targets using the computer-aided algorithms in Targetscan and miRbase Targets. PTEN, a widely expressed tumor suppressor, was identified as a potential miR-21 target gene. The 3′-UTR of PTEN containing the potential miR-21 binding site was cloned for use in a firefly luciferase reporter assay (Figure 2A). The PTEN-UTR or PTEN-UTR-mutant(mut) reporter plasmids were cotransfected into HSFBs along with the miR-21 inhibitor or scramble control [27]. Compared with the scramble control, the miR-21 inhibitor increased the relative luciferase activity significantly when cotransfected with the PTEN-UTR reporter plasmid (p = 0.018). However, the mutant reporter plasmid abolished the miR-21 inhibitor-mediated increase in luciferase activity (Figure 2B, p = 0.536). These findings suggest that miR-21 suppresses PTEN by direct binding to the 3′-UTR of PTEN.

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