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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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Related in: MedlinePlus

MiR-21 modulates cell growth via hTERT in HSFBs.A, the expression of miR-21 was analyzed by qRT-PCR and normalized to U6 in 15 pairs of NSFBs and HSFBs. The 15 paired HSFB/NSFB samples were cultured from 15 individuals. B, HSFBs were transfected with miR-21 mimic, miR-21 inhibitor or a scrambled oligonucleotide. Non-transfected cells (blank) were used as controls. At 24 h post-transfection, miR-21 expression levels in transfected HSFB and NSFB were evaluated by qRT-PCR analysis. C, MTT assays were performed on all four groups at 24 h intervals for 5 days. D, E, The four groups were transfected for 48 h. Apoptosis was measured by flow cytometric analysis of Annexin V and propidium iodide staining. Blank cells were used as control.
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pone-0097114-g001: MiR-21 modulates cell growth via hTERT in HSFBs.A, the expression of miR-21 was analyzed by qRT-PCR and normalized to U6 in 15 pairs of NSFBs and HSFBs. The 15 paired HSFB/NSFB samples were cultured from 15 individuals. B, HSFBs were transfected with miR-21 mimic, miR-21 inhibitor or a scrambled oligonucleotide. Non-transfected cells (blank) were used as controls. At 24 h post-transfection, miR-21 expression levels in transfected HSFB and NSFB were evaluated by qRT-PCR analysis. C, MTT assays were performed on all four groups at 24 h intervals for 5 days. D, E, The four groups were transfected for 48 h. Apoptosis was measured by flow cytometric analysis of Annexin V and propidium iodide staining. Blank cells were used as control.

Mentions: The specific regulation of miR-21 expression in NSFBs and HSFBs (15 samples of each type) was investigated by qRT-PCR analysis. As shown in Figure 1A, miR-21 was significantly overexpressed in HSFBs compared with NSFBs (pā€Š=ā€Š0.001). Aberrant miR-21 expression has also been demonstrated to be associated with a variety of cancers, affecting cells proliferation and invasion [19], [38], [39].


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)

MiR-21 modulates cell growth via hTERT in HSFBs.A, the expression of miR-21 was analyzed by qRT-PCR and normalized to U6 in 15 pairs of NSFBs and HSFBs. The 15 paired HSFB/NSFB samples were cultured from 15 individuals. B, HSFBs were transfected with miR-21 mimic, miR-21 inhibitor or a scrambled oligonucleotide. Non-transfected cells (blank) were used as controls. At 24 h post-transfection, miR-21 expression levels in transfected HSFB and NSFB were evaluated by qRT-PCR analysis. C, MTT assays were performed on all four groups at 24 h intervals for 5 days. D, E, The four groups were transfected for 48 h. Apoptosis was measured by flow cytometric analysis of Annexin V and propidium iodide staining. Blank cells were used as control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0097114-g001: MiR-21 modulates cell growth via hTERT in HSFBs.A, the expression of miR-21 was analyzed by qRT-PCR and normalized to U6 in 15 pairs of NSFBs and HSFBs. The 15 paired HSFB/NSFB samples were cultured from 15 individuals. B, HSFBs were transfected with miR-21 mimic, miR-21 inhibitor or a scrambled oligonucleotide. Non-transfected cells (blank) were used as controls. At 24 h post-transfection, miR-21 expression levels in transfected HSFB and NSFB were evaluated by qRT-PCR analysis. C, MTT assays were performed on all four groups at 24 h intervals for 5 days. D, E, The four groups were transfected for 48 h. Apoptosis was measured by flow cytometric analysis of Annexin V and propidium iodide staining. Blank cells were used as control.
Mentions: The specific regulation of miR-21 expression in NSFBs and HSFBs (15 samples of each type) was investigated by qRT-PCR analysis. As shown in Figure 1A, miR-21 was significantly overexpressed in HSFBs compared with NSFBs (pā€Š=ā€Š0.001). Aberrant miR-21 expression has also been demonstrated to be associated with a variety of cancers, affecting cells proliferation and invasion [19], [38], [39].

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