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Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-β2 by targeting ZNF217 via miR-200c in keloid fibroblasts.

Zhu HY, Bai WD, Li C, Zheng Z, Guan H, Liu JQ, Yang XK, Han SC, Gao JX, Wang HT, Hu DH - Sci Rep (2016)

Bottom Line: Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-β2 and ZNF217 expression but upregulated expression of miR-200c in KFs.Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-β2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs.Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-β2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-β2.

View Article: PubMed Central - PubMed

Affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, Shaanxi, People's Republic of China.

ABSTRACT
Abnormally high activation of transforming growth factor-β (TGF-β) signaling has been demonstrated to be involved in the initiation and progression of keloids. However, the functional role of long non-coding RNA (lncRNA)-activated by TGF-β (lncRNA-ATB) in keloids has not been documented. Here we investigated the role of lncRNA-ATB in the autocrine secretion of TGF-β in keloid fibroblasts (KFs) and explored the underlying molecular mechanism. Using immunohistochemistry and quantitative RT-PCR analysis, we showed that lncRNA-ATB and ZNF217, a transcriptional activator of TGF-β, were overexpressed and miR-200c, which targets ZNF217, was under-expressed in keloid tissue and keloid fibroblasts. Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-β2 and ZNF217 expression but upregulated expression of miR-200c in KFs. Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-β2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs. Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-β2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-β2. These findings may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

No MeSH data available.


Related in: MedlinePlus

ZNF217 is a transcriptional activator of TGF-β2 in keloid fibroblasts.(A) Schematic representation of ZNF217 binding site in the TGF-β2 promoter region. TSS: transcription start site; (B) ChIP assays using anti-ZNF217 antibody or human IgG as a control on the TGF-β2 promoter region, followed by PCR using ZNF217 binding site-specific primers for measurement of TGF-β2 expression; (C) qRT-PCR determination of relative ZNF217 expression levels in keloid fibroblasts (KF) treated with a control siRNA (siRNA-control) or ZNF217 specific siRNAs (siRNA-ZNF217-1 and siRNA- ZNF217-2); (D,E) qRT-PCR and ELISA determination of relative TGF-β2 mRNA and protein expression levels in KF treated with ZNF217-specific siRNAs, respectively.
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f3: ZNF217 is a transcriptional activator of TGF-β2 in keloid fibroblasts.(A) Schematic representation of ZNF217 binding site in the TGF-β2 promoter region. TSS: transcription start site; (B) ChIP assays using anti-ZNF217 antibody or human IgG as a control on the TGF-β2 promoter region, followed by PCR using ZNF217 binding site-specific primers for measurement of TGF-β2 expression; (C) qRT-PCR determination of relative ZNF217 expression levels in keloid fibroblasts (KF) treated with a control siRNA (siRNA-control) or ZNF217 specific siRNAs (siRNA-ZNF217-1 and siRNA- ZNF217-2); (D,E) qRT-PCR and ELISA determination of relative TGF-β2 mRNA and protein expression levels in KF treated with ZNF217-specific siRNAs, respectively.

Mentions: To determine if TGF-β2 is regulated by ZNF217, we carried out chromatin immunoprecipitation (ChIP) experiments based on several ZNF217-binding sites23 found in the +3 kb to −1 kb promoter region of the TGF-β2 gene (Fig. 3A). Using ChIP assays followed by PCR with primers specific to the ZNF217 proximal promoter, we found that ZNF217 could associate with the TGF-β promoter and directly upregulate the transcription of TGF-β2 (Fig. 3B).


Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-β2 by targeting ZNF217 via miR-200c in keloid fibroblasts.

Zhu HY, Bai WD, Li C, Zheng Z, Guan H, Liu JQ, Yang XK, Han SC, Gao JX, Wang HT, Hu DH - Sci Rep (2016)

ZNF217 is a transcriptional activator of TGF-β2 in keloid fibroblasts.(A) Schematic representation of ZNF217 binding site in the TGF-β2 promoter region. TSS: transcription start site; (B) ChIP assays using anti-ZNF217 antibody or human IgG as a control on the TGF-β2 promoter region, followed by PCR using ZNF217 binding site-specific primers for measurement of TGF-β2 expression; (C) qRT-PCR determination of relative ZNF217 expression levels in keloid fibroblasts (KF) treated with a control siRNA (siRNA-control) or ZNF217 specific siRNAs (siRNA-ZNF217-1 and siRNA- ZNF217-2); (D,E) qRT-PCR and ELISA determination of relative TGF-β2 mRNA and protein expression levels in KF treated with ZNF217-specific siRNAs, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835760&req=5

f3: ZNF217 is a transcriptional activator of TGF-β2 in keloid fibroblasts.(A) Schematic representation of ZNF217 binding site in the TGF-β2 promoter region. TSS: transcription start site; (B) ChIP assays using anti-ZNF217 antibody or human IgG as a control on the TGF-β2 promoter region, followed by PCR using ZNF217 binding site-specific primers for measurement of TGF-β2 expression; (C) qRT-PCR determination of relative ZNF217 expression levels in keloid fibroblasts (KF) treated with a control siRNA (siRNA-control) or ZNF217 specific siRNAs (siRNA-ZNF217-1 and siRNA- ZNF217-2); (D,E) qRT-PCR and ELISA determination of relative TGF-β2 mRNA and protein expression levels in KF treated with ZNF217-specific siRNAs, respectively.
Mentions: To determine if TGF-β2 is regulated by ZNF217, we carried out chromatin immunoprecipitation (ChIP) experiments based on several ZNF217-binding sites23 found in the +3 kb to −1 kb promoter region of the TGF-β2 gene (Fig. 3A). Using ChIP assays followed by PCR with primers specific to the ZNF217 proximal promoter, we found that ZNF217 could associate with the TGF-β promoter and directly upregulate the transcription of TGF-β2 (Fig. 3B).

Bottom Line: Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-β2 and ZNF217 expression but upregulated expression of miR-200c in KFs.Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-β2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs.Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-β2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-β2.

View Article: PubMed Central - PubMed

Affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, Shaanxi, People's Republic of China.

ABSTRACT
Abnormally high activation of transforming growth factor-β (TGF-β) signaling has been demonstrated to be involved in the initiation and progression of keloids. However, the functional role of long non-coding RNA (lncRNA)-activated by TGF-β (lncRNA-ATB) in keloids has not been documented. Here we investigated the role of lncRNA-ATB in the autocrine secretion of TGF-β in keloid fibroblasts (KFs) and explored the underlying molecular mechanism. Using immunohistochemistry and quantitative RT-PCR analysis, we showed that lncRNA-ATB and ZNF217, a transcriptional activator of TGF-β, were overexpressed and miR-200c, which targets ZNF217, was under-expressed in keloid tissue and keloid fibroblasts. Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-β2 and ZNF217 expression but upregulated expression of miR-200c in KFs. Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-β2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs. Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-β2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-β2. These findings may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

No MeSH data available.


Related in: MedlinePlus