Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma

Metastatic melanoma, the primary cause of skin cancer-related death, warrants new therapeutic approaches that target the regulatory machinery at molecular level. While long noncoding RNAs (lncRNAs) are dysregulated in a number of cancer types, limited data are available on the expression and function of lncRNAs in melanoma metastasis. The primary objective of this study was to investigate the role of 6 metastasis-related lncRNAs in pairs of primary melanoma and matched lymph node metastatic tissues. Among the tested lncRNAs, HOTAIR was the most highly expressed in lymph node metastasis. The role of HOTAIR in melanoma cell motility and invasion was further evaluated by knocking down HOTAIR with siRNAs. Knockdown of HOTAIR resulted in the reduction of motility and invasion of human melanoma cell line A375, as assessed by wound healing assay and Matrigel-based invasion assay. siHOTAIR also suppressed the degradation of gelatin matrix, suggesting that HOTAIR promotes gelatinase activity. Together, our study shows that HOTAIR is overexpressed in metastatic tissue, which is associated with the ability of HOTAIR to promote melanoma cell motility and invasion. These data indicate that lncRNAs may be involved in the metastasis of melanoma and provide support for further evaluation of lncRNAs in melanoma.


Introduction
Although the incidence of malignant melanoma accounts for only approximately 1% of cutaneous tumors, it is among the few cancers with a remarkably high mortality rate. This is mainly due to its high potential to metastasize to the vital organs such as lungs, liver, and brain. Approximately 80% of skin cancer-related mortality is due to melanoma (http://www.chinaswzl.com/cancer/hsslby/2493.html).
The worldwide incidence of melanoma is increasing [1]. Statistical analysis of the melanoma cases collected in recent years in our department shows that the incidence of melanoma is much higher than the Chinese national average and is still increasing, especially in the younger population in southern industrial cities in China. This phenomenon may be caused by complex environmental factors and long sun exposure (Yu B, unpublished data).
Noncoding RNAs (ncRNAs) are emerging as new regulators in the cancer paradigm. They have demonstrated potential roles in both oncogenic and tumor suppressive pathways [2,3]. ncRNAs are largely grouped into two major classes based on transcript sizes: small ncRNAs (<200 kb) and long ncRNAs (lncRNAs) (>200 kb) [4,5]. Small ncRNAs include a broad range of well-known and newly discovered RNA species, with many being associated with 5 or 3 regions of genes. This class includes the well-documented miRNAs. It has been widely reported that cancer-specific miRNAs can be 2 BioMed Research International detected in the blood, sputum, and urine of cancer patients and serve as diagnostic and prognostic markers [6][7][8].
LncRNAs, ranging from 200 nucleotides to over 10 kb, are abundantly transcribed by the mammalian genome [9,10]. LncRNAs have been found to be dysregulated in a wide range of human diseases and disorders, including various cancers. For example, PCGEM [11] and DD3 [12] are overexpressed in prostate cancer as compared to adjacent normal prostate tissue, implicating a role for these lncRNAs in prostate tumorigenesis [13]. BC200 RNA overexpression is correlated with the progression of breast cancer and has been proposed to be a new molecular marker for breast cancer [14]. Increased expression of MALAT-1 RNA indicates a worse clinical outcome in lung cancer patients [15]. Gupta et al. recently revealed an important role for HOTAIR in breast cancer metastasis. HOTAIR is highly induced (up to 2,000fold) in breast cancer metastatic tissues [16]. In addition, overexpression of HOTAIR in primary breast tumors is a powerful predictor of eventual metastasis and death [16]. These studies provide evidence and support that lncRNAs may be involved in tumorigenesis and tumor progression.
However, research on the expression and function of lncRNAs in melanoma is still limited. In this study, we analyzed the expression profiles of 6 well documented metastasis-related lncRNAs in 3 pairs of primary melanoma and matched lymph node metastatic tissues using real-time quantitative RT-PCR. Further, we investigated the role of HOTAIR in melanoma cell motility and invasion. Our study provides novel insights into the role of lncRNAs in the metastatic progression of melanoma and identifies a potential new target for the treatment of metastatic melanoma.

Tissue Samples.
Three pairs of primary melanoma and matched lymph node metastatic tissues were obtained from the Department of Gastric Cancer and Soft Tissue Sarcoma Surgery at the Fudan University Shanghai Cancer Center. The protocol was approved by the Institutional Review Board of Fudan University. Patients enrolled in the study were provided with written informed consent. Fresh tissue samples were collected and cut into fragments <0.5 cm in any single dimension. The tissues were then immersed into 2 mL RNAlater (Ambion, Foster City, CA). All the tissue samples were frozen within 30 minutes after surgery and stored in liquid nitrogen until use. Tissue sections from each sample were reviewed and classified by a pathologist.

Cell
Culture. Human metastatic melanoma cell line A375 was obtained from the Typical Cell Culture Collection Committee of the Chinese Academy of Sciences. A375 cells were maintained in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovine serum (FBS).

Quantitative Reverse Transcriptase PCR (qRT-PCR) of Six lncRNAs.
Total RNA was isolated from melanoma and lymph node metastatic tissues using the RNeasy kit (Qiagen, Grand Island, NY) according to the manufacturer's instructions.

IncRNA
Sequence Reverse transcription (RT) reactions were performed with 1 g total RNA using a PrimeScript RT reagent kit (TaKaRa BIO, Shiga, Japan). Random hexamer primers were used in the RT reactions. Real-time qPCR was performed on a Bio-Rad CFX-96 real-time PCR system (Bio-Rad, Hercules, CA) using SYBR Premix DimerEraser kit (TaKaRa, Shiga, Japan). GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan). All assays were performed in triplicates.   the upper chambers with cotton swabs. Chamber membranes were fixed and stained using Diff-Quik Stain Set (Dade Behring Inc., Newark, DE) and examined under a bright-field microscope. Invasion was assessed by counting 6 fields per membrane (×20 objective).

In Situ Zymography.
Glass coverslips were coated with 0.2 mg/mL Oregon green 488-conjugated gelatin (Invitrogen), cross-linked in 0.5% glutaraldehyde for 15 minutes at 4 ∘ C, and incubated with 5 mg/mL NaBH 4 for 3 minutes. The coverslips were then sterilized with 70% ETOH for 15 minutes and incubated in serum-free media for 1 hour at 37 ∘ C. A375 cells transfected with siRNA-NS or siHOTAIR (I and II) were plated on gelatin-coated coverslips, incubated at 37 ∘ C for 24 hours, and processed by fluorescence microscopy procedures. Cell morphology was photographed under a light microscope.

Statistical Analysis.
Statistical significances between groups were determined by two-tailed Student's t-test. For paired melanoma tissues, the difference of lncRNA expression was evaluated with Wilcoxon matched pairs signed ranks test. < 0.05 was considered statistically significant.

LncRNA Expression in Melanoma and Matched Lymph
Node Metastatic Tissues. Expression profiles of 6 lncRNAs that have been found to be associated with cancer or metastasis [17] were examined by qRT-PCR in 3 pairs of primary melanoma and matched lymph node metastatic tissues. We observed that most of the lncRNAs (5 out of 6) were differentially regulated in melanomas versus matched metastatic tissues (Figure 1(a)). Among them, HOTAIR was significantly overexpressed in metastatic lymph nodes compared to matched primary melanoma ( < 0.01) (Figure 1(a)).
Further, data mining of publically available gene profiling database Gene Expression Omnibus (GEO) showed that HOTAIR expression is markedly higher in melanomas compared with nontumor tissues, and the highest expression was observed in tumors spread to regional lymph nodes (Figure 1(b)), suggesting that HOTAIR expression is linked to the progression and metastasis of melanoma.

Knockdown of HOTAIR Suppressed Metastatic Melanoma A375 Cell Motility.
Cell migration is an essential step in metastasis. Therefore, we next examined the ability of HOTAIR to affect cell motility using the scratch "wound" healing assays in human metastatic melanoma cell line A375. HOTAIR expression in A375 cells was knocked down by siRNAs and confirmed by real-time qPCR (Figure 2(a)). The wound healing assay showed that siControl-transfected A375 cells covered almost the entire damaged area by 24 hours (Figure 2(b)). In contrast, the wounded area was only partially covered by siHOTAIR-transfected cells after 24 hours of incubation (Figure 2(b)), suggesting that HOTAIR promotes melanoma cell motility. Cells/field * * siControl siHOTAIR I siHOTAIR II ×20 ×20 ×20 Figure 3: Knockdown of HOTAIR inhibits the invasion of melanoma A375 cells. Matrigel-based invasion assay was performed using modified Boyden chambers with 10% FBS as a chemoattractant. Representative images were presented. The cell numbers per field were counted and the results are summarized in a bar graph. * P < 0.05.

Knockdown of HOTAIR
∼3-fold reduction of the invasiveness of A375 cells ( < 0.01; Figure 3). Taken together, these data indicate that knockdown of HOTAIR inhibits in vitro parameters associated with metastasis including motility and invasion.

Degradation of Matrix In Situ Was Suppressed by
Knockdown of HOTAIR. Tumor cell invasion associated with metastasis requires both specialized cell migration and the ability to degrade basement membrane by secreted or membrane-bound proteases [18]. We sought to determine whether matrix metalloproteinase (MMPs), known to be upregulated in many metastatic tumors, might be responsible for HOTAIR-potentiated invasion. Gelatinase activity, indicating the activity of MMP-2 and MMP-9, was assessed by in situ zymography. The ability of A375 cells to degrade matrix in situ was markedly suppressed by siRNA-HOTAIR (I and II), as indicated by the reduced black holes representing matrix degradation (Figure 4(b)). This result suggests that HOTAIR promotes gelatinase activity in melanoma cells.

Discussion
Increasing studies provide evidence and support that ncRNAs are key factors in gene regulation and influence normal and cancer cell phenotypes [19][20][21][22]. LncRNAs belong to a novel class of ncRNA. They contain longer than 200 nucleotides and have no protein-coding capacity [23]. Several lncRNAs have been reported to control transcriptional alterations, implying that the difference in lncRNA profiling between normal and cancer cells is not just the secondary effect of cancer transformation [24]. On the contrary, lncRNAs are strongly associated with cancer progression [24]. However, little information regarding the expression profiles of lncRNAs in melanoma is available. Hence, in this study, we selected 6 well-documented lncR-NAs associated with metastasis including MALAT1 [15,16], HOTAIR [16], NEAT-1 [25], HULC [26], MEG-3 [27], and UCA1 [28] to evaluate their expression in primary melanoma and matched lymph node metastasis tissues. We observed that HOTAIR is the only consistently overexpressed lncRNA among the 6 lncRNAs in melanoma metastasis compared to matched primary tumors. Four other lncRNAs were differentially expressed in melanoma versus lymph node metastasis. HOTAIR was initially identified as one of the 231 ncRNAs associated with human HOX loci [16]. HOTAIR promoted metastasis in breast cancer [16]. There is growing evidence that HOTAIR may have prometastasis activity in several cancer types, including breast [16], pancreatic [29], and hepatocellular carcinoma (HCC) [30]. Whether HOTAIR performs the same function in the progression of melanoma remains unknown. To understand the role of HOTAIR in melanoma progression, a series of in vitro assays were performed. Studies using wound healing assay demonstrate that knockdown of HOTAIR inhibits the migration of melanoma cells. The invasiveness of melanoma cells is also markedly suppressed by knocking down HOTAIR, as demonstrated by the Matrigel-based Boyden chamber assay. Recently, Geng et al. found that HOTAIR expression is elevated in HCC tumors compared with adjacent nontumor tissues [31]. HOTAIR expression is correlated with lymph node metastasis in HCC [31]. siRNA-mediated knockdown of HOTAIR in HCC cells was accompanied by a deduction in MMP-9, suggesting that MMP-9 may be involved in HOTAIRmediated regulation of HCC progression [31]. As MMP-9 plays an important role in tumor metastasis, gelatinase activity (indicating the activity of MMP-2 and MMP-9) was assessed by in situ zymography, which is a unique technique for revealing proteolytic activity at specific sites in tissues or cell cultures. Results indicate that the degradation of matrix was suppressed by knockdown of HOTAIR, supporting the hypothesis that HOTAIR promotes the activity of MMP-9 and/or MMP-2. BRAF-activated noncoding RNA (BANCR) shows increased expression in melanoma [32]. Knockdown of BANCR reduced melanoma cell migration [32]. These data suggest that lncRNAs may play an important role in the progression of melanoma. Further research on lncRNA expression profiles is needed to define the impact of lncRNAs on the progress of melanoma.
Interestingly, in our study, the expression levels of lncRNA MALAT1 show no significant difference between primary melanoma and matched metastatic tissues. On the contrary, MALAT1 was documented by several studies to associate with metastasis in other cancer types [15]. Increased expression of MALAT1 was first observed in metastatic nonsmall cell lung cancer [15], followed by endometrial stromal sarcoma of the uterus [33], and more recently in six other types of cancer, including HCC, breast, pancreas, lung, colon, and prostate cancers [34]. Overall, these results suggest that the effect of lncRNAs on cancer progression may be cancertype specific.
In conclusion, we demonstrate that HOTAIR lncRNA is predominantly upregulated in lymph node metastasis tissues compared with primary melanoma. Knockdown of HOTAIR inhibits the motility and invasiveness of melanoma cells, and the latter is associated with decreased degradation of extracellular matrix. Although further mechanistic investigation into the regulation of metastasis by HOTAIR is necessary, the observed prometastatic activity of HOTAIR in multiple preclinical model systems supports HOTAIR to be a potential target for melanoma metastasis therapy.