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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

Knockdown of lncRNA-ATB upregulates miR-200c expression in keloid fibroblasts.(A) Correlation between miR-200c and ZNF217 mRNA expression in keloid tissue; (B) the predicted binding sequences of human miR-200c to the wild-type and mutant 3′-UTR of ZNF217; (C) relative luciferase activity in lncRNA-ATB-knockdown keloid fibroblasts (KF) co-transfected with plasmid containing wild-type (ZNF217-UTR-wt) or mutant 3′-UTR of ZNF217 (ZNF217-UTR-mut) and scrambled (control) or miR-200c inhibitor (inh 200c). Luciferase activity was measured 48 h after transfection; (D,E) the mRNA and protein expression of ZNF217 in stable KFs transfected with different shRNAs as assessed in the presence or absence of an miR-200c inhibitor (inh 200c); (F) luciferase activity in stable KFs transfected with luciferase reporters containing ZNF217 3′-UTR, inhibitor, or control. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.
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f6: Knockdown of lncRNA-ATB upregulates miR-200c expression in keloid fibroblasts.(A) Correlation between miR-200c and ZNF217 mRNA expression in keloid tissue; (B) the predicted binding sequences of human miR-200c to the wild-type and mutant 3′-UTR of ZNF217; (C) relative luciferase activity in lncRNA-ATB-knockdown keloid fibroblasts (KF) co-transfected with plasmid containing wild-type (ZNF217-UTR-wt) or mutant 3′-UTR of ZNF217 (ZNF217-UTR-mut) and scrambled (control) or miR-200c inhibitor (inh 200c). Luciferase activity was measured 48 h after transfection; (D,E) the mRNA and protein expression of ZNF217 in stable KFs transfected with different shRNAs as assessed in the presence or absence of an miR-200c inhibitor (inh 200c); (F) luciferase activity in stable KFs transfected with luciferase reporters containing ZNF217 3′-UTR, inhibitor, or control. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.

Mentions: Consistent with our previous finding that ZNF217 is a target of miR-200c in breast cancer cells8, here we found that the mRNA levels of ZNF217 were inversely correlated with miR-200c levels in keloid fibroblasts (Fig. 6A).


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)

Knockdown of lncRNA-ATB upregulates miR-200c expression in keloid fibroblasts.(A) Correlation between miR-200c and ZNF217 mRNA expression in keloid tissue; (B) the predicted binding sequences of human miR-200c to the wild-type and mutant 3′-UTR of ZNF217; (C) relative luciferase activity in lncRNA-ATB-knockdown keloid fibroblasts (KF) co-transfected with plasmid containing wild-type (ZNF217-UTR-wt) or mutant 3′-UTR of ZNF217 (ZNF217-UTR-mut) and scrambled (control) or miR-200c inhibitor (inh 200c). Luciferase activity was measured 48 h after transfection; (D,E) the mRNA and protein expression of ZNF217 in stable KFs transfected with different shRNAs as assessed in the presence or absence of an miR-200c inhibitor (inh 200c); (F) luciferase activity in stable KFs transfected with luciferase reporters containing ZNF217 3′-UTR, inhibitor, or control. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Knockdown of lncRNA-ATB upregulates miR-200c expression in keloid fibroblasts.(A) Correlation between miR-200c and ZNF217 mRNA expression in keloid tissue; (B) the predicted binding sequences of human miR-200c to the wild-type and mutant 3′-UTR of ZNF217; (C) relative luciferase activity in lncRNA-ATB-knockdown keloid fibroblasts (KF) co-transfected with plasmid containing wild-type (ZNF217-UTR-wt) or mutant 3′-UTR of ZNF217 (ZNF217-UTR-mut) and scrambled (control) or miR-200c inhibitor (inh 200c). Luciferase activity was measured 48 h after transfection; (D,E) the mRNA and protein expression of ZNF217 in stable KFs transfected with different shRNAs as assessed in the presence or absence of an miR-200c inhibitor (inh 200c); (F) luciferase activity in stable KFs transfected with luciferase reporters containing ZNF217 3′-UTR, inhibitor, or control. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.
Mentions: Consistent with our previous finding that ZNF217 is a target of miR-200c in breast cancer cells8, here we found that the mRNA levels of ZNF217 were inversely correlated with miR-200c levels in keloid fibroblasts (Fig. 6A).

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