The long non-coding RNA HOTAIR indicates a poor prognosis and promotes metastasis in non-small cell lung cancer

Forty-two paired NSCLC and adjacent non-tumor lung tissues were obtained from patients who underwent surgery at Jiangsu Province Hospital between 2006 and 2007 and were diagnosed with NSCLC (stage II, III, and IV) based on histopathological evaluation. Clinicopathological characteristics including tumor-node-metastasis (TNM) stage were collected. No local or systemic treatment was conducted in these patients before surgery. All collected tissue samples were immediately snap-frozen in liquid nitrogen and stored at -80°C until use. The study was approved by the Research Ethics Committee of Nanjing Medical University, China. Written informed consent was obtained from all patients.

Three NSCLC adenocarcinoma cell lines (A549, SPC-A1, NCI-H1975), a NSCLC squamous carcinomas cell line (SK-MES-1), and a normal human bronchial epithelial cell line (16HBE) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 or DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) in humidified air with 5% CO₂ at 37°C.

Total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. qRT-PCR assays were performed to detect HOTAIR expression using the PrimeScript RT reagent Kit and SYBR Premix Ex Taq (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used were as follows: HOTAIR sense, 5’-CAGTGGGGAACTCTGACTCG-3′ and antisense, 5′-GTGCCTGGTGCTCTCTTACC-3′; GAPDH sense, 5′-GGGA GCCAAAAGGGTCAT-3′ and antisense, 5′-GAGTCCTTCCACGATACCAA-3′. qRT-PCR and data collection were performed on an ABI 7500. qRT-PCR results were analyzed and expressed relative to CT (threshold cycle) values, and then converted to fold changes.

To generate a HOTAIR expression vector, we amplified a full-length HOTAIR fragment by PCR from SPC-A1 cDNA. Oligonucleotides for amplification of HOTAIR (sense, 5′-CATGGATCCACATTCTGCCCTGA TTTCCGGAACC-3′ and antisense, 5′-ACTCTCGAGCCACCACACACACACA ACCTACAC-3′) were designed to incorporate external HindIII and XhoI sites, respectively. The PCR product was verified and subcloned into the mammalian expression vector pCDNA3.1 (Invitrogen).

Plasmid vectors (pCDNA3.1-HOTAIR and pCDNA3.1-NC) for transfection were prepared using DNA Midiprep or Midiprep kits (Qiagen, Hilden, Germany). Three individual small interfering RNA (siRNAs) and scrambled negative control siRNA (si-NC) were purchased from Invitrogen (Invitrogen). The target sequences for HOTAIR siRNAs were as follows: (si-HOTAIR1, 5′-AAAUCCAGAACCCUCUGACAUUUGC-3′, si-HOTAIR2, 5′-UUAAGUCUA GGAAUCAGCACGAAGC-3′ and si-HOTAIR3, 5′-CAUAUUAUAGAGUUGCU CUGUGCUG-3′. pCDNA3.1-HOTAIR or pCDNA3.1-NC was transfected into cultured A549 cells, and HOTAIR siRNAs or si-NC were transfected into cultured SPC-A1 cells. A549 and SPC-A1 cells were grown on six-well plates to confluency and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were harvested for qRT-PCR or western blot analyses.

Cell proliferation was monitored using Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science). Si-HOTAIR-transfected SPC-A1 cells (3000/well), and pCDNA3.1-HOTAIR-transfected A549 cells (2000/well) were allowed to grow in 96-well plates. Cell proliferation was documented every 24 h following the manufacturer’s protocol. All experiments were performed in quadruplicate. For the colony formation assay, a total of 500 HOTAIR siRNA-transfected SPC-A1, or pCDNA3.1-HOTAIR-transfected A549 cells were placed in a fresh six-well plate and maintained in media containing 10% FBS, replacing the medium every 4 days. After 14 days, cells were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich). Visible colonies were manually counted. For each treatment group wells were assessed in triplicate.

SPC-A1 cells, transiently transfected with si-HOTAIR or si-NC, were harvested 48 h after transfection by trypsinization. After double staining with FITC-Annexin V and propidium iodide, cells were analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells, and then the relative ratio of early apoptotic cells was compared to the control from each experiment. All samples were assayed in triplicate.

For the migration assays, 48 h after transfection, 5 × 10⁴ cells in serum-free media were placed into the upper chamber of an insert (8 μm pore size, Millipore). For the invasion assays, 1 × 10⁵ cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (Sigma-Aldrich). Media containing 10% FBS were added to the lower chamber. After incubation for 24 hours, the cells remaining on the upper membrane were removed with cotton wool, whereas the cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet, imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan). Experiments were independently repeated three times.

Cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, CA, USA) and phenylmethylsulfonyl fluoride (Roche). Protein concentration was measured using the Bio-Rad protein assay kit. Approximately 50 μg of protein extract was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to nitrocellulose membrane (Sigma) and incubated with specific antibodies. ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad, CA, USA). GAPDH was used as a control. Antibodies (1:1,000) for HOXA5, E-cadherin, N-cadherin, vimentin, MMP-2, and MMP-9 were purchased from Cell Signaling Technology (MA, USA).

Male athymic mice (5 weeks old) were purchased from the Animal Center of the Chinese Academy of Science (Shanghai, China) and maintained in laminar flow cabinets under specific pathogen-free conditions. SPC-A1 cells transfected with si-HOTAIR or si-NC were harvested from 6-well cell culture plates, washed with PBS, and resuspended at a concentration of 2 × 10⁷ cells/mL. A volume of 0.1 mL of suspended cells was injected into the tail veins of 10 mice. The mice were sacrificed 6 weeks after injection and the lungs were dissected out, photographed and visible tumors on the lung surface were counted. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University (Permit Number: 200933). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering [18].

Student’s t-test (2-tailed), one-way ANOVA, and the Mann–Whitney U test were conducted to analyze data using SPSS 16.0 software (IBM, IL, USA). P-values of less than 0.05 were considered significant.

The level of HOTAIR was detected in 42 NSCLC samples and adjacent, histologically normal tissues by qRT-PCR, and normalized to GAPDH. HOTAIR expression was significantly up-regulated in cancerous tissues compared with corresponding normal tissues (Figure 1A). Furthermore, correlation of HOTAIR expression with pathological features of NSCLC patients, revealed a significant association between HOTAIR up-regulation and advanced pathological stage (I/II, n = 25) vs. (III/IV, n = 17) and NSCLC lymph-node metastasis (Figure 1B and C). However, HOTAIR expression was not associated with patients’ gender or tumor position (Table 1).

Kaplan-Meier survival analysis and log-rank tests using patient post-operative survival were performed to further evaluate the correlation between HOTAIR expression and NSCLC patient prognosis. According to the median ratio of relative HOTAIR expression (8.57) in tumor tissues, the 42 NSCLC patients were classified into two groups: High-HOTAIR group (n = 21): HOTAIR expression ratio ≥ median ratio; and Low-HOTAIR group (n = 21): HOTAIR expression ratio ≤ median ratio. The Kaplan-Meier survival curve showed that patients with high HOTAIR expression levels had significantly shorter survival times compared to those with low HOTAIR expression levels (P <0.001; log-rank test) (Figure 1D). These findings support the hypothesis that over-expression of HOTAIR may play a key role in NSCLC progression and development.

We next performed qRT-PCR analysis to examine the expression of HOTAIR in four human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes. Of these, SPC-A1 and NCI-H1975 expressed significantly higher levels of HOTAIR compared with the normal bronchial epithelial cell line (16HBE), while A549 cells expressed relatively low levels of HOTAIR (Figure 2A).

To investigate the functional effects of HOTAIR in NSCLC cells, we modulated its expression through RNA interference and over-expression experiments. pCDNA3.1-HOTAIR was transfected into A549 cells and three individual HOTAIR siRNAs were transfected into SPC-A1 cells. qRT-PCR analysis of HOTAIR levels was performed 48 h post-transfection. HOTAIR expression was increased 97-fold in A549 cells, compared with control cells. In SPC-A1 cells, when compared with control cells (si-NC), HOTAIR expression was knocked down by 75% by si-HOTAIR3, the most effective siRNA. Thus, si-HOTAIR3 was used in subsequent experiments (Figure 2B and C).

To assess the role of HOTAIR in NSCLC, we investigated the effect of targeted knockdown or over-expression of HOTAIR on cell proliferation and apoptosis. MTT assays revealed that cell growth was not influenced in SPC-A1 cells transfected with si-HOTAIR compared with controls (Figure 3A). Similarly, colony-formation assays revealed that clonogenic survival was not changed following inhibition of HOTAIR in SPC-A1 cells (Figure 3B). Consistent with the above results, alteration of HOTAIR expression in A549 cells also had no significant effect on cell proliferation compared with control cells (data not shown).

We then tested the effect of HOTAIR on apoptosis. SPC-A1 cells were seeded in six-well plates and transfected with si-HOTAIR or si-NC. We conducted apoptosis assays using an annexin V-propidium iodide apoptosis detection kit to determine whether knockdown of HOTAIR induces NSCLC cell apoptosis. The results shown in Figure 3C demonstrated a significantly higher percentage of apoptotic cells for si-HOTAIR-treated cells compared to those transfected with the untargeted-control (si-NC). Taken together, these results indicate that inhibition of HOTAIR induces NSCLC cells apoptosis, but has no effect on cell vitality.

Cell invasion is a significant aspect of cancer progression, and involves the migration of tumor cells into contiguous tissues and the dissolution of extracellular matrix proteins. To investigate whether HOTAIR has a direct functional role in facilitating NSCLC cell migration and invasion, we evaluated cancer cell invasion through Matrigel and migration through a transwell. As shown in Figure 4A, inhibition of HOTAIR impeded the migration of SPC-A1 cells by approximately 68% compared with control. Similarly, invasion of SPC-A1 cells was reduced by 65% following inhibition of HOTAIR. Conversely, transfection of A549 cells with pCDNA-HOTAIR promoted cell migration and invasion ability ~1.9-fold (Figure 4B). These data indicate that HOTAIR can promote the migratory and invasive phenotype of NSCLC cells.

To validate the effect of HOTAIR on metastasis of NSCLC cells in vivo, SPC-A1 cells transfected with si-HOTAIR were injected into an athymic mouse xenograft model via the tail vein. The metastatic nodules on the surface of lungs were counted after 6 weeks. Inhibition of HOTAIR expression resulted in a significant reduction in the number of metastatic nodules compared with the control group (Figure 4C). This in vivo data complemented the functional in vitro studies of HOTAIR, and demonstrated that HOTAIR was capable of promoting NSCLC cell metastasis in vivo.

To explore the molecular mechanisms by which HOTAIR contributes to the phenotypes of NSCLC cells, we investigated potential targets involved in tumor invasion and metastasis. Often, cancer cells undergo morphological alterations and change their cell-cell or cell-matrix interactions. The epithelial- mesenchymal transition (EMT) is an important event in the progression of tumor invasion and metastasis. In this study, western blot assays were performed to detect the expression of EMT-induced markers (E-cadherin, N-cadherin and Vimentin) between HOTAIR knockdown SPC-A1 cells and control cells. These results revealed no significant difference between HOTAIR knockdown cells and control cells (Figure 5A). Matrix metalloproteinases (MMPs) have a pivotal role in the degradation of extracellular matrix (ECM) proteins, and thereby enhance the invasive, proliferative and metastatic potential of cancer. Therefore, we next sought to determine whether there was any interaction between MMPs and HOTAIR. As expected, clear reductions in the levels of MMP2 and MMP9 proteins were observed that correlated with the inhibition of HOTAIR in SPC-A1 cells. In addition, suppression of HOTAIR in SPC-A1 cells induced the up-regulation of HOXA5 protein, which is involved in NSCLC cell migration and invasion. Conversely, over-expression of HOTAIR in A549 cells resulted in down-regulation of HOXA5 protein (Figure 5A). These data indicate that HOTAIR may influence the invasive and metastatic potential of NSCLC cells by altering MMPs and HOXA5 expression, but not by deregulating the expression of EMT-induced markers.