Background
Non-small cell lung cancer (NSCLC), including adenocarcinoma and squamous cell carcinoma, is the predominant form of lung cancer, and accounts for the majority of cancer deaths worldwide [
1]. Despite recent advances in clinical and experimental oncology, the prognosis of lung cancer is still unfavorable, with a 5 year overall survival rate of only around 11% [
2]. Thus, a detailed understanding of the mechanisms underlying NSCLC development and progression are essential for improving the diagnosis, prevention and treatment of this disease. Recently, accumulating evidence has shown that non-coding RNAs (ncRNAs) may be involved in NSCLC pathogenesis, providing new insights into the biology of the disease.
Recent improvements in genome-wide surveys and high throughput transcriptome analysis have revealed that human genome contains only ~20,000 protein-coding genes, representing <2% of the total genome while a substantial fraction of the human genome can be transcripted into many short or long noncoding RNAs with limited or no protein-coding capacity [
3,
4]. Among the different classes of ncRNAs, microRNAs have received the most attention and have been shown to play many important roles in cancer via post-transcriptional silencing of specific target mRNAs [
5,
6]. However, one of the emerging areas in ncRNA research is the newly discovered class of long non-coding RNAs (lncRNAs). The participation of lncRNAs in a wide repertoire of biological processes has been intensely researched, because virtually every step of mRNA biology, from transcription to mRNA splicing, RNA decay and translation can be influenced by these molecules [
7‐
9]. Multiple lines of evidence link dysregulation of lncRNAs to diverse human diseases, especially cancers. Several studies suggest that many lncRNAs can be associated with chromatin-modifying complexes to affect epigenetic information and to confer multiple properties that are required for tumor progression and a metastatic phenotype [
10‐
12]. Therefore, identification of cancer-associated lncRNAs and the interplay between lncRNAs and protein-coding genes are important topics in the field of cancer biology, in which lncRNAs may provide a missing piece of the oncogene and tumor suppressor network puzzle.
HOTAIR (
Hox transcript antisense intergenic RNA ) is one of the few well-documented lncRNAs, with a length of 2158 bp and a functional role in trans-silencing [
13]. Recently, HOTAIR has been determined to be a negative prognostic indicator in breast, colon, liver, and pancreatic cancer patient survival, and increased HOTAIR expression in patients has been correlated with enhanced breast and colon cancer metastasis. Meanwhile, HOTAIR knockdown can inhibit cell invasion and proliferation, alter cell cycle progression and induce apoptosis, indicating that HOTAIR can play a direct role in the modulation of cancer progression [
14‐
17]. Nevertheless, little is known about the impact of HOTAIR on NSCLC carcinogenesis or metastasis.
To better understand the role of HOTAIR in NSCLC development and progression, we investigated the expression pattern of HOTAIR in NSCLC tissues and analyzed its relationship to clinical pathological features. We also explored HOTAIR function during NSCLC progression using in vitro and in vivo assays and investigated the molecular mechanisms by which HOTAIR contributes to the phenotypes of NSCLC cells.
Methods
Tissue collection
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.
Cell lines and culture conditions
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.
RNA extraction and quantitative real-time PCR
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.
Plasmid construction
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).
Cell transfection
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 assays
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.
Flow-cytometric analysis of apoptosis
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.
Cell migration and invasion assays
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.
Western blot analysis and antibodies
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).
Tail vein injection of cells for metastasis in athymic mice
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 × 10
7 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].
Statistical analysis
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.
Discussion
NSCLC ranks among the most common and lethal malignant diseases. Poor prognosis of early stage NSCLC is crucially linked to the onset of tumor metastasis [
19]. The processes inducing and stimulating metastasis are complex and still not well understood. Schmidt
et al. have established a role for lncRNA in metastasis formation in NSCLC. They identified the lncRNA, MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1), as a prognostic marker for metastasis and patient survival in NSCLC [
20]. However, the roles of lncRNAs in the carcinogenesis of NSCLC are far from being fully elucidated.
In this paper, we have investigated the involvement of the lncRNA, HOTAIR, in NSCLC carcinogenesis and metastasis. HOTAIR was initially identified as one of 231 lncRNAs associated with the human HOX loci, which, however, can repress transcription
in trans across 40 kilobases of the HOXD locus in foreskin fibroblasts [
21]. Notably, HOTAIR over-expression targets polycomb repressive complex 2 (PRC2), a complex comprised of EZH2, SUZ12 and EED. This has a genome-wide effect, serving to alter H3K27 methylation and gene expression patterns, thus increasing cancer invasiveness and metastasis. Conversely, knockdown of HOTAIR or PRC2 component expression can inhibit cancer invasiveness [
15,
17]. HOTAIR can also interact with a second histone modification complex, the LSD1/CoREST/REST complex, which coordinates the targeting of PRC2 and LSD1 to chromatin for coupled histone H3K27 methylation and K4 demethylation [
22]. Given its important role in the epigenetic regulation of gene expression, it is not surprising that HOTAIR is deregulated in different types of cancer [
14‐
17,
23,
24]. It remains unclear, however, whether HOTAIR plays an oncogenic role in NSCLC.
The current study indicated that the expression of HOTAIR was dramatically upregulated in NSCLC tissues compared with normal tissues. Specifically, HOTAIR expression was found to be significantly higher at later stages of tumor development and in tumors that had undergone extensive metastasis. Moreover, the overall survival time of patients with lower HOTAIR expression levels was significantly longer than that of patients with higher HOTAIR expression levels. These findings indicate that HOTAIR plays a direct role in the modulation of cancer progression, and may be useful as a novel prognostic or progression marker for NSCLC.
To further assess the role of HOTAIR in NSCLC, we investigated the effects of gain or loss of function of HOTAIR on various aspects of NSCLC biology. First, we demonstrated that RNAi-mediated suppression of HOTAIR in SPC-A1 cells led to a significant inhibition of migration and invasion, and to the promotion of apoptosis. Conversely, introducing HOTAIR into A549 cells, which express relatively low levels of endogenous HOTAIR, induced malignant tumor cell behaviors. To further quantify metastatic potential in vivo, we performed tail vein xenografts and compared the rates of lung colonization. The inhibition of HOTAIR expression resulted in a significant reduction in the number of lung metastatic nodules. In conclusion, HOTAIR knockdown can inhibit the invasion and metastasis of NSCLC in vitro and in vivo; thus, HOTAIR represents a new prognosis marker and a promising target for NSCLC treatment.
To explore the molecular mechanism by which HOTAIR contributes to the invasion and metastasis of NSCLC, we investigated potential target proteins involved in cell motility and matrix invasion. Firstly, a hallmark of EMT is the loss of E-cadherin expression and aberrant expression of N-cadherin and Vimentin [
25‐
28]. Therefore, the protein levels of these EMT-induced markers were investigated after HOTAIR depletion. However, our results indicated that the inhibitory effects on cell migration and invasion were not associated with the epithelial-mesenchymal transition. Metalloproteases (MMPs) are important in many aspects of biology, ranging from cell proliferation, differentiation and remodeling of the extracellular matrix (ECM) to vascularization and cell migration [
29]. Here, loss of HOTAIR in NSCLC cells led to a significant decrease in MMP2 and MMP9 protein levels, and the relationship between HOTAIR and MMPs is currently under further investigation in our laboratory.
Of note, HOTAIR can epigenetically regulate HOXD expression, such as HOXD10, by targeting PRC2, leading to H3K27me3 [
14,
30]. Here, we found that HOTAIR can suppress HOXA5 protein levels, another member of the HOX family. HOXA5 is involved in the developmental regulation of the lung. Mandeville
et al. observed impaired postnatal lung development in
HoxA5
-/-
mice, indicating that HOXA5 has a critical role in lung ontogeny, and implying an involvement in lung maturation and function [
31]. Similarly, Packer
et al. reported that HOXA5 is likely to be involved in the development and patterning of the mouse lung [
32]. Moreover, dysregulation of HOXA5 expression has been associated with lung tumorigenesis and other diseases in humans [
33‐
35]. In our previously study, we found that HOXA5 was significantly downregulated in NSCLC tissues and inhibition of
HOXA5 expression in A549 cells significantly promotes cell migration and invasion [
36]. Consistent with the above findings, ectopic HOTAIR expression in A549 cells also induced corresponding malignant tumor cell behaviors. Taken together, these results indicate that the oncogenic functions of HOTAIR may be partially exerted through its affect on the expression of HOXA5; however, further experiments are needed to elucidate the precise molecular mechanisms by which HOTAIR regulates HOXA5.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LXH, DW and WZX were involved in the conception and design of the study. LJ was involved in the provision of study material and patients. LXH, LZL and SM performed the data analysis and interpretation. LXH wrote the manuscript. WZX approved the final version. All authors read and approved the final manuscript.