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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,B) Pearson’s correlation analysis of relative expression levels of lncRNA-ATB and those of miR-200c determined using qRT-PCR in keloid (K) tissue and keloid fibroblasts (KF) compared to levels normal skin (NS) tissue and normal fibroblasts (NF), respectively; (C) quantitative RT-PCR analysis of endogenous miR-200c in KF with lncRNA-ATB knockdown using an lncRNA-ATB specific shRNA (shRNA-ATB) compared to a control shRNA (shRNA-control); (D) relative expression levels of lncRNA-ATB in KF overexpressing lncRNA-ATB (Lv-ATB), Lv-Luc was used as an control; (E) relative expression levels of miR-200c in KF transfected with an empty vector (PC3), a vector carrying wild-type lncRNA-ATB (PC3-ATB), a vector carrying mutated lncRNA-ATB [PC3-ATB-mut(miR-200c)], or a vector carrying another lncRNA induced by TGF-β and carrying none miR-200c binding site (PC3-508851); (F) relative expression levels of lncRNA-ATB in KF overexpressing miR-200c compared with those overexpressing a control miRNA (miR-control) is measured at 96 h; (G) schematic representation of the predicted binding site of miR-200c on lncRNA-ATB transcript. The nucleotides in red are the seed sequences of miR-200c; (H) luciferase activity in KF cotransfected with miR-200c and luciferase reporters containing nothing (vector), lncRNA-ATB (lncRNA-ATB-wt), or mutant transcript (lncRNA-ATB-mut). Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.
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f5: Knockdown of lncRNA-ATB upregulates miR-200c expression in keloid fibroblasts.(A,B) Pearson’s correlation analysis of relative expression levels of lncRNA-ATB and those of miR-200c determined using qRT-PCR in keloid (K) tissue and keloid fibroblasts (KF) compared to levels normal skin (NS) tissue and normal fibroblasts (NF), respectively; (C) quantitative RT-PCR analysis of endogenous miR-200c in KF with lncRNA-ATB knockdown using an lncRNA-ATB specific shRNA (shRNA-ATB) compared to a control shRNA (shRNA-control); (D) relative expression levels of lncRNA-ATB in KF overexpressing lncRNA-ATB (Lv-ATB), Lv-Luc was used as an control; (E) relative expression levels of miR-200c in KF transfected with an empty vector (PC3), a vector carrying wild-type lncRNA-ATB (PC3-ATB), a vector carrying mutated lncRNA-ATB [PC3-ATB-mut(miR-200c)], or a vector carrying another lncRNA induced by TGF-β and carrying none miR-200c binding site (PC3-508851); (F) relative expression levels of lncRNA-ATB in KF overexpressing miR-200c compared with those overexpressing a control miRNA (miR-control) is measured at 96 h; (G) schematic representation of the predicted binding site of miR-200c on lncRNA-ATB transcript. The nucleotides in red are the seed sequences of miR-200c; (H) luciferase activity in KF cotransfected with miR-200c and luciferase reporters containing nothing (vector), lncRNA-ATB (lncRNA-ATB-wt), or mutant transcript (lncRNA-ATB-mut). Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.

Mentions: Because previous studies suggested that miR-200c may have a role in the pathogenesis of keloids719202122, we examined whether lncRNA-ATB is coexpressed with miR-200c in keloid tissue and keloid fibroblasts by measuring the expression levels of lncRNA-ATB and miR-200c by qRT-PCR. As shown in Fig. 5A,B, lncRNA-ATB transcript levels were significantly inversely correlated with miR-200c RNA levels in both keloid tissue and keloid fibroblasts.


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,B) Pearson’s correlation analysis of relative expression levels of lncRNA-ATB and those of miR-200c determined using qRT-PCR in keloid (K) tissue and keloid fibroblasts (KF) compared to levels normal skin (NS) tissue and normal fibroblasts (NF), respectively; (C) quantitative RT-PCR analysis of endogenous miR-200c in KF with lncRNA-ATB knockdown using an lncRNA-ATB specific shRNA (shRNA-ATB) compared to a control shRNA (shRNA-control); (D) relative expression levels of lncRNA-ATB in KF overexpressing lncRNA-ATB (Lv-ATB), Lv-Luc was used as an control; (E) relative expression levels of miR-200c in KF transfected with an empty vector (PC3), a vector carrying wild-type lncRNA-ATB (PC3-ATB), a vector carrying mutated lncRNA-ATB [PC3-ATB-mut(miR-200c)], or a vector carrying another lncRNA induced by TGF-β and carrying none miR-200c binding site (PC3-508851); (F) relative expression levels of lncRNA-ATB in KF overexpressing miR-200c compared with those overexpressing a control miRNA (miR-control) is measured at 96 h; (G) schematic representation of the predicted binding site of miR-200c on lncRNA-ATB transcript. The nucleotides in red are the seed sequences of miR-200c; (H) luciferase activity in KF cotransfected with miR-200c and luciferase reporters containing nothing (vector), lncRNA-ATB (lncRNA-ATB-wt), or mutant transcript (lncRNA-ATB-mut). Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.
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Related In: Results  -  Collection

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f5: Knockdown of lncRNA-ATB upregulates miR-200c expression in keloid fibroblasts.(A,B) Pearson’s correlation analysis of relative expression levels of lncRNA-ATB and those of miR-200c determined using qRT-PCR in keloid (K) tissue and keloid fibroblasts (KF) compared to levels normal skin (NS) tissue and normal fibroblasts (NF), respectively; (C) quantitative RT-PCR analysis of endogenous miR-200c in KF with lncRNA-ATB knockdown using an lncRNA-ATB specific shRNA (shRNA-ATB) compared to a control shRNA (shRNA-control); (D) relative expression levels of lncRNA-ATB in KF overexpressing lncRNA-ATB (Lv-ATB), Lv-Luc was used as an control; (E) relative expression levels of miR-200c in KF transfected with an empty vector (PC3), a vector carrying wild-type lncRNA-ATB (PC3-ATB), a vector carrying mutated lncRNA-ATB [PC3-ATB-mut(miR-200c)], or a vector carrying another lncRNA induced by TGF-β and carrying none miR-200c binding site (PC3-508851); (F) relative expression levels of lncRNA-ATB in KF overexpressing miR-200c compared with those overexpressing a control miRNA (miR-control) is measured at 96 h; (G) schematic representation of the predicted binding site of miR-200c on lncRNA-ATB transcript. The nucleotides in red are the seed sequences of miR-200c; (H) luciferase activity in KF cotransfected with miR-200c and luciferase reporters containing nothing (vector), lncRNA-ATB (lncRNA-ATB-wt), or mutant transcript (lncRNA-ATB-mut). Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity.
Mentions: Because previous studies suggested that miR-200c may have a role in the pathogenesis of keloids719202122, we examined whether lncRNA-ATB is coexpressed with miR-200c in keloid tissue and keloid fibroblasts by measuring the expression levels of lncRNA-ATB and miR-200c by qRT-PCR. As shown in Fig. 5A,B, lncRNA-ATB transcript levels were significantly inversely correlated with miR-200c RNA levels in both keloid tissue and keloid fibroblasts.

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