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

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.

Scientific RepoRts | 6:24728 | DOI: 10.1038/srep24728 signaling has been shown to be required for the initiation and progression of keloids. These findings necessitate a better understanding of the special downstream effectors of TGF-β and a search for specific inhibitors of different TGF-β -dependent pathways for keloid treatment.
Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides with limited protein coding potential 16 . Studies have shown that lncRNAs play an important role in the development, growth, and progression of human carcinomas, acting as oncogenic drivers through diverse mechanisms 17,18 . Recently, Yuan et al. 7 discovered that the lncRNA activated by TGF-β (lncRNA-ATB), an lncRNA located on chromosome 14 (ENST00000493038, http://genome.ucsc.edu/), promotes tumor cell invasion and mediates distant metastasis, exhibiting oncogenic functions. Given the fundamental biological processes regulated by TGF-β signaling and the knowledge that many of these processes are altered in keloids, we surmised that lncRNA-ATB may be involved in the pathogenesis of this benign tumor. However, until now, the functional role of lncRNA-ATB in keloids has not been documented.
MicroRNAs (miRNAs) have emerged as critical regulators of cell signaling including TGF-β signaling 19 . A previous study demonstrated that lncRNA-ATB is physically associated with the miR-200 family 7 , of which miR-200c is a well-known tumor-suppressive miRNA that is frequently downregulated in multiple cancers 20,21 . In addition, miR-200c showed significant downregulation in keloid tissue compared to matched normal skin tissue, suggesting that loss of miR-200c may have a role in the pathogenesis of keloids 22 . Interestingly, our previous work demonstrated that ZNF217, a transcriptional activator of TGF-β , is a target of miR-200c in breast cancer cells 8 and promotes epithelial-to-mesenchymal transition (EMT) in breast cancer 23 . Given that TGF-β signaling is elevated in keloids, we hypothesized that lncRNA-ATB might suppress the autocrine secretion of TGF-β 2 by targeting ZNF217 via miR-200c in keloid fibroblasts.
To test this hypothesis, in the present study, we detected the expression of lncRNA-ATB, miR-200c, and ZNF217 in keloid tissue and keloid fibroblasts. Through gain-and loss-of-function studies, we examined the effect of lncRNA-ATB on autocrine secretion of TGF-β 2 in keloid fibroblasts as well as the interactions among TGF-β 2, lncRNA-ATB, miR-200c, and ZNF217. The findings of the present study may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

Downregulation of lncRNA-ATB suppresses autocrine secretion of TGF-β2 in keloid fibroblasts.
Because lncRNA-ATB has been demonstrated to play an important role in TGF-β signaling in hepatocellular carcinoma 7 , we investigated whether lncRNA-ATB also acts similarly in keloids and keloid fibroblasts, which play a critical role in the progression of keloids. As anticipated, qRT-PCR analysis showed that lncRNA-ATB expression was significantly increased in both keloid tissue and keloid fibroblasts compared to normal skin tissue and normal fibroblasts, respectively (Fig. 1A,B).
To identify lncRNAs that are regulated by TGF-β , we treated keloid fibroblasts continuously with TGF-β 1 and -β 2. The results of qRT-PCR showed that the lncRNA-ATB expression was increased (Figs S1A,B), which is consistent with the data obtained from hepatocellular carcinoma cell lines 7 . Because TGF-β had a profound impact on lncRNA-ATB expression, we wonder whether lncRNA-ATB has a biological effect on TGF-β signaling. We thus used a lentivirus-mediated delivery system to stably knock down the expression of lncRNA-ATB in keloid fibroblasts. As shown in Fig. 1C,D, qRT-PCR assays and enzyme-linked immunosorbent assays (ELISAs) revealed that the level of lncRNA-ATB and TGF-β 2 were significantly decreased in keloid fibroblasts. To quantify the autocrine secretion of TGF-β 2 into the supernatant of keloid fibroblasts, an ELISA was performed 48 and 72 h after downregulation of lncRNA-ATB. Abrogation of lncRNA-ATB showed that the TGF-β 2 downregulation was more pronounced at 72 h after transfection (Fig. 1D). This result implies that downregulation of lncRNA-ATB suppresses the autocrine secretion of TGF-β 2 in keloid fibroblasts.
ZNF217 is overexpressed in keloid tissue. Given that ZNF217, a transcriptional activator of TGF-β , promotes EMT in breast cancer 8 and that TGF-β signaling is elevated in keloids, we surmised that ZNF217 might modulate the autocrine secretion of TGF-β 2 in keloids. We first determined the expression of ZNF217 protein by immunohistochemistry in keloid tissue samples from our retrospective cohort of 57 patients with keloids, whose clinicopathological data are presented in Supplemental Table 1. By signal intensity analysis, we found that ZNF217 expression was significantly increased in keloid tissue compared with normal skin tissue (P = 0.013; Fig. 2A,B). To confirm this finding, we next evaluated ZNF217 protein and mRNA expression in keloid tissue and normal skin tissue by Western blotting and qRT-PCR, respectively. The results showed that ZNF217 was overexpressed in keloid fibroblasts at both the protein and mRNA levels, compared to levels in normal skin tissue (Fig. 2C,D). Altogether, these data suggest that ZNF217 is overexpressed in keloid tissue and fibroblasts.

Stable downregulation of ZNF217 expression decreases autocrine secretion of TGF-β2 in vitro.
To determine if TGF-β 2 is regulated by ZNF217, we carried out chromatin immunoprecipitation (ChIP) experiments based on several ZNF217-binding sites 23 found in the + 3 kb to − 1 kb promoter region of the TGF-β 2 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-β 2 ( Fig. 3B).
To further investigate whether the autocrine secretion of TGF-β 2 is regulated by ZNF217, we used a ZNF217 siRNA delivery system to stably transfect keloid fibroblasts. The transfection of ZNF217 siRNA decreased the expression of ZNF217 by 1.8-fold change compared with control lentivirus-infected keloid fibroblasts (Fig. 3C). ZNF217 knockdown significantly decreased TGF-β 2 mRNA and protein expression (Fig. 3D,E). Collectively, these findings suggest that downregulation of ZNF217 may decrease the autocrine section of TGF-β 2 in keloid fibroblasts.
lncRNA-ATB regulates ZNF217 expression in keloid fibroblasts. Because lncRNA-ATB and TGF-β 2 reciprocally regulate each other in keloid fibroblasts, we wondered whether lncRNA-ATB could modulate ZNF217 expression. We stably knocked down lncRNA-ATB and found that the depletion of lncRNA-ATB decreased ZNF217 expression at both the mRNA and protein levels (Fig. 4A,B). These results indicate that downregulation of lncRNA-ATB inhibits ZNF217 expression in keloid fibroblasts.

lncRNA-ATB is physically associated with miR-200c.
Because previous studies suggested that miR-200c may have a role in the pathogenesis of keloids 7,19-22 , 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.
To further validate the endogenous direct binding between miR-200c and lncRNA-ATB, we constructed luciferase reporters containing the 500-nt 3′ lncRNA-ATB, which includes wild-type (wt) or mutated miR-200c binding sites (Fig. 5G). We found that overexpression of miR-200c reduced the luciferase activities of the WT reporter vector but not empty vector or mutant reporter vector in keloid fibroblasts (Fig. 5H). Taken together, these data suggest that miR-200c may function as a downstream effector of lncRNA-ATB in keloid fibroblasts.

Upregulation of miR-200c accounts for ZNF217 downregulation by lncRNA-ATB knockdown.
Consistent with our previous finding that ZNF217 is a target of miR-200c in breast cancer cells 8 , here we found that the mRNA levels of ZNF217 were inversely correlated with miR-200c levels in keloid fibroblasts (Fig. 6A).
To further validate the silencing of ZNF217 by miR-200c, we performed a dual luciferase reporter gene assay. The 3′ -untranslated region (UTR) fragments of ZNF217, containing the potential miR-200c binding site and ChIP assays using anti-ZNF217 antibody or human IgG as a control on the TGF-β 2 promoter region, followed by PCR using ZNF217 binding site-specific primers for measurement of TGF-β 2 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-β 2 mRNA and protein expression levels in KF treated with ZNF217specific siRNAs, respectively.   mutations in their seed sequences, were cloned into a vector with the firefly luciferase reporter gene (Fig. 6B). Then, these constructs were co-transfected with an miR-200c inhibitor or a control miRNA into keloid fibroblasts with lncRNA-ATB knockdown. As expected, the miR-200c inhibitor dramatically increased the expression of luciferase flanked by the 3′ -UTR of ZNF217, whereas the mutations in the predicted miR-200c binding sites of 3′ -UTR of ZNF217 abrogated the effect of miR-200c suppression (Fig. 6C). These results show that ZNF217 is a direct target of miR-200c in keloid fibroblasts with lncRNA-ATB knockdown.

Discussion
In this study, 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 fibroblasts. We discovered that knockdown of lncRNA-ATB inhibited the autocrine production of TGF-β 2 in keloid fibroblasts. We also found that miR-200c is endogenously associated with lncRNA-ATB and identified ZNF217, as a bona fide functional target of miR-200c that could be upregulated by lncRNA-ATB knockdown. Collectively, our study provides a possible novel signaling axis involving lncRNA-ATB/miR-200c/ZNF217/TGF-β 2 in keloid fibroblasts.
TGF-β 1 and -β 2 act as fibrosis activators and mitogens for fibroblasts and can stimulate ECM deposition 24 . They regulate ECM deposition at different levels, including the promotion of ECM protein expression, the inhibition of the expression of proteases capable of degrading the ECM, the stimulation of the expression of protease inhibitors, and the regulation of integrin expression and of molecules that act as receptors for several ECM components 25 . Excessive scarring in keloid formation secondary to excessive production of ECM is the characteristic of fibrotic disease. Intriguingly, recent studies have shown that TGF-β signaling can collaborate with other molecules to regulate target genes and lncRNAs 7,26 . In a mouse breast cancer cell line, lncRNA-Smad7 induced by TGF-β regulates the anti-apoptotic and tumor-progressive phenotypes 26 . In multiple myeloma cells, lncRNA MALAT1 transcript activates LTBP3-regulated molecules in tumor suppressor and oncogenic pathways 27 . However, the molecular events by which TGF-β regulates or is regulated by lncRNAs in the pathogenesis of keloids, which is known to be effected by TGF-β signaling, are largely unknown.
Compared to small non-coding RNAs, lncRNAs are less well understood. Nonetheless, the few lncRNAs that have been functionally characterized are linked to diseases such as cancer, signifying that lncRNAs may not be merely transcriptional "noise" 28 . Yuan et al. reported that lncRNA-ATB acts not only as an endogenous miRNA sponge but also as an mRNA binder as part of its competing endogenous RNA (ceRNA) activity 7 . While we were preparing this manuscript, Iguchi et al. reported that the levels of lncRNA-ATB expression were significantly higher in patients with hematogenous metastases, suggesting that lncRNA-ATB may be involved in the progression of colorectal cancer and be a novel indicator of poor prognosis in patients with this malignancy 18 . Here, in our study, the finding that lncRNA-ATB is overexpressed in keloids suggests its potential utility as a therapeutic target. Soluble antisense oligonucleotides against lncRNA-ATB or other agents that block the interactions of lncRNA-ATB with target miRNAs and mRNAs may be developed to specifically block the prometastatic branch of TGF-β signaling 29 . Due to its sequence homology with mRNAs, lncRNA-ATB can sequester miR-200c like a molecular "sponge" or as a ceRNA and thereby annul miRNA function. Consistent with this, we found that knockdown of lncRNA-ATB remarkably increased endogenous miR-200c levels in keloid fibroblasts. Identifying the functional role of lncRNAs and relative miRNAs aberrantly expressed in tumor tissues as well as the underlying mechanism would provide a novel avenue for the exploration of miRNA-based diagnostics and therapeutics.
The elucidation of gene-regulatory networks that control gene expression programs represents a major challenge in cell biology 30 . Subsequent to the discovery of miRNAs, multiple lines of evidence demonstrated that miRNA-associated networks play a critical role in disease initiation and progression 30,31 . In addition, recent studies demonstrated that TGF-β signaling can be targeted by different miRNAs 32,33 , strongly suggesting that expression and function of TGF-β family members are under rigorous surveillance of tumor-suppressive miRNAs in normal fibroblasts. miR-200c, belonging to the miRNA-200 family, might play an oncogenic role in non-small cell lung cancer 34 , but might be a tumor suppressive miRNA in breast cancer cell lines 35 . The role of miR-200c in the skin has not been firmly established 36 . Confirming the role of miRNA-200c in keloids may improve the understanding of the pathogenesis of cutaneous fibrotic diseases 36 . In this study, we found that miR-200c, by targeting ZNF217, regulates the autocrine secretion of TGF-β , thus creating a signaling axis involving lncRNA-ATB, miR-200c, ZNF217, and TGF-β 2. During the process of keloid progression, the oncogenic lncRNA-ATB is activated and contributes to the imbalance between miR-200c and ZNF217, resulting in abnormal TGF-β expression. As an important tumor-suppressive miRNA, miR-200c regulates a large cohort of oncogenes and functions as the master regulator of multiple pathways. Thus, the process of keloid progression would be, at least in part, accelerated upon the loss of miR-200c.

Conclusions
Our results provide the first evidence that in keloids, lncRNA-ATB governs autocrine secretion of TGF-β 2, at least in part, by downregulating the expression level of ZNF217 via miR-200c. Our findings, together with those of previous studies, indicate that miR-200c is part of a regulatory pathway that inhibits autocrine production of TGF-β 2. Thus, both lncRNA-ATB and miR-200c might be potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

Materials and Methods
Tissue samples. Keloid tissueand paired normal skin tissue samples were surgically obtained from 57 patients who were diagnosed pathologically with keloids at the Department of Burns and Cutaneous Surgery of Xijing Hospital between April 2009 and July 2014. All the protocols were approved by the Ethics Committee of Xijing Hospital affiliated to Fourth Military Medical University (China). Written informed consent was obtained for each participant according to the guidelines. The collection of skin samples was carried out in accordance with the approved guidelines. The collected skin samples were divided into three portions; one was fixed in 4% paraformaldehyde for histopathological examination, the second was soaked in liquid nitrogen for the preparation of total RNA and total protein lysates, and the third was used for the isolation and culture of fibroblasts.
Cell culture. Cultures of keloid fibroblasts and normal skin fibroblasts, which were isolated from 17 pairs of keloid tissue samples and normal skin samples, respectively, were established as described previously 1,3 . All cells were maintained in a humidified incubator at 37 °C in an atmosphere containing 5% CO 2 . Fibroblasts at the third to fifth passages were used in all experiments unless otherwise indicated.
Quantitative real-time PCR analysis. RNA extraction and real-time PCR were performed as previously ELISA. Cell culture supernatants were collected in sterile tubes and stored at − 20 °C until use. The concentration of TGF-β 2 was determined using an ELISA kit (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions 37 . All analyses and calibrations were carried out in duplicate. All concentrations were recorded as ng/mL. To quantitate the autocrine secretion of TGF-β 2, an ELISA was performed 48 and 72 h after lncRNA-ATB knockdown. Immunohistochemical staining, microscopy, and image analysis. Immunohistochemical staining, microscopy, and image analysis were performed as previously described [38][39][40] . The primary antibody used was anti-ZNF217 rabbit polyclonal antibody (1:200; ab181741, Abcam, Cambridge, UK), and goat anti-rabbit IgG (Alexa Fluor 594, Invitrogen) was used as a secondary antibody. The signal intensity of tissue areas from five randomly selected views was blindly determined and statistically analyzed.
ChIP. ChIP experiments were performed as previously described 7 . Briefly, keloid fibroblasts were cultured, washed twice with phosphate-buffered saline (PBS), and cross-linked using 1% formaldehyde for 10 min. Following lysis of the cells and sonication, DNA-protein complexes in the lysates were subjected to immunoprecipitation using anti-ZNF217 (ab117798, Abcam) or control normal IgG. After precipitation of the immunocomplex with protein G-agarose, isolated DNA was used as template in PCR with specific primers spanning the target region of the TGF-β 2 promoter. Primers used for amplification of the human TGF-β 2 promoter were as follows: forward, 5′ -CGG GAG ACT TGA TTG TCC TT-3′ and reverse, 5′-CGT TGA GGG AGT GTG GAA AT-3′ .
Vector construction, lentivirus production, and construction of stable cell lines. The cDNA encoding lncRNA-ATB or lncRNA-508851 (another lncRNA that is also induced by TGF-β but does not have a predicted miR-200c targeting site) was PCR-amplified and subcloned into pcDNA3.1 vector (Invitrogen), named PC3-ATB and PC3-508851, respectively. The PC3-ATB with point mutations in the miR-200c response elements was synthesized using a QuikChange Site-Directed Mutagenesis kit (Stratagene) and named PC3-ATB-mut (miR-200c) 7 . The 525-nt region at the 3′ end of either lncRNA-ATB or lncRNA-ATB-mut (miR-200c) was amplified using PCR and subcloned into the pmirGLO vector (Promega, Madison, WI) for a luciferase reporter assay using the one-step directed cloning kit (Novoprotein, Shanghai, China) 7 . The human miR-200c precursor sequence was amplified by PCR using the primers 5′ -GAA TTC GGG GAT CTG GGC GCA GGG GCC-3′ and 5′ -CTC GAG CCG ACC TGG AGC GCG GAG C-3′ and cloned into a pcDNA3.1 vector, and the resulting vector was named PC3-200c. Lentivirus production and generation of cell lines stably expressing lncRNA-ATB, lncRNA-ATB-mut (miR-200c), or lncRNA-508851 were performed as previously described 7 . For construction of lentiviral vector expressing the human lncRNA-ATB gene, lncRNA-ATB cDNA was PCR-amplified and subcloned to the lentiviral vector, named Lv-ATB, using Lv-Luc as control 7 . We used a scrambled shRNA as the negative control for shRNA-ATB-1, shRNA-ATB-2, and shRNA-508851 and designated this as shRNA-control 7 .
Luciferase assay. The 3′ -UTR fragments of ZNF217 containing the putative binding site for miR-200c and 3′ UTR-mutation were modified as previously described 8 . The fragments were subcloned into pmirGLO vector, a dual-luciferase miRNA target expression vector (Promega). pmirGLO, pmirGLO-ATB, or pmirGLO-ATB-mut (miR-200c) was cotransfected with miR-200c mimic or miR NC (a negative control), mirGLO (as a control), or pmirGLO-ZNF217 into keloid fibroblasts by Lipofectamine-mediated gene transfer 7 . Luciferase assays were performed 48 h after transfection using the dual-luciferase reporter assay system. Firefly luciferase activity was normalized to renilla luciferase expression for each sample.