Cervical cancer samples were obtained from 111 female patients who underwent surgery at Yonsei Severance Hospital, Yonsei University, between 2007 and 2012. Specimens from patients with newly diagnosed invasive [FIGO (International Federation of Gynecology and Obstetrics) stage IA-IVB] cervical cancer who had not received prior treatment were included in the study. Forty samples of normal cervix from patients undergoing simple hysterectomy because of uterine leiomyomata were obtained as controls. Specimens from patients with concomitant gynecological cancer were excluded from the study. All specimens were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. The study was conducted according to the principles in the Declaration of Helsinki and was approved by the ethical committee of Yonsei Severance Hospital. Informed consent was obtained from all patients. The clinical information is summarized in Table I.

SiHa (squamous cervical carcinoma), HeLa (epitheloid cervical carcinoma) and Caski (epidermoid cervical carcinoma established from a metastasis in the small bowel mesentery) human cervical cancer cell lines obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). SiHa and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium, and Caski cells were cultured in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD, USA). The human keratinocyte cell line HaCaT was cultured in RPMI-1640 medium. The culture media were supplemented with 10% (vol/vol) fetal bovine serum and penicillin/streptomycin. The cell lines were maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Cells with a passage number <20 were used in all experiments.

Total RNA was extracted from cancerous/non-cancerous specimens or cell lines using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA), and 2 μg of total RNA was reverse transcribed into first-strand cDNA by using a reverse transcription reagent kit (Invitrogen) according to the manufacturer’s protocol. qRT-PCR was performed using the SYBR^® Green real-time PCR kit (Toyobo, Co., Ltd., Osaka, Japan) in a 20-μl reaction volume, which contained 10 μl of SYBR-Green Master PCR Mix, 5 pmole each of forward and reverse primers, 1 μl of diluted cDNA template, and appropriate amounts of sterile distilled water. Conditions for the amplification of genes were as follows: initial denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 60 sec, and elongation at 72°C for 60 sec; and final elongation at 72°C for 5 min. qRT-PCR was performed on the ABI StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). All quantifications were performed with U6 as the internal standard. The PCR primer sequences were as follows: HOTAIR, 5′-GGTAGAAAAAGCAACCACGAAGC-3′ (sense) and 5′-ACATAAACCTCTGTCTGTGAG TGCC-3′ (antisense); E-cadherin, 5′-ATTCTGATTCTGC TGCTCTTG-3′ (sense) and 5′-AGTAGTCATAGTCCTGGTCCT-3′ (antisense); β-catenin, 5′-TGCAGTTCGCCTTCACTATG-3′ (sense) and 5′-ACTAGTCGTGGAATGGCACC-3′ (antisense); vimentin, 5′-TGGATTCACTCCCTCTGGTT-3′ (sense) and 5′-GGTCATCGTGATGCTGAGAA-3′ (antisense); snail, 5′-GAGGCGGTGGCAGACTAG-3′ (sense) and 5′-GACACATCGGTCAGACCAG-3′ (antisense); twist, 5′-CGGGAGTCCGCAGTCTTA-3′ (sense) and 5′-TGAATCTTGCTCAGCTTGTC-3′ (antisense); and U6, 5′-CTCGCTTCGGCAGCACA-3′ (sense) and 5′-AACGCTTCAGGAATTTGCGT-3′ (antisense). Relative gene expression was analyzed using the 2^−ΔΔCT method, and the results were expressed as extent of change with respect to control values. qRT-PCR experiments were replicated at least 3 times.

HOTAIR siRNA (siHOTAIR-1 and siHOTAIR-2) and negative control siRNA (siNC) were purchased from Bioneer (Daejeon, Korea). Cells (5×10⁴ cells/well) were seeded into 6-well plates and were transfected with 10 nM siRNA in phosphate-buffered saline (PBS) using the G-Fectin kit (Genolution Pharmaceuticals Inc., Seoul, Korea) according to the manufacturer’s protocol. These siRNA-transfected cells were used in the in vitro assays 48 h post-transfection. The target sequences for HOTAIR siRNAs were as follows: siRNA-1, 5′-UUUUCUACCAGGUCGGUAC-3′ and siRNA-2, 5′-AAUUCUUAAAUUGGGCUGG-3′.

The human HOTAIR transcript variant 3 cDNA was amplified by PCR and was inserted into the pLenti6/V5-D-TOPO vector according to ViraPower™ Lentiviral Expression systems (Invitrogen). Briefly, plasmid was transfected into the 293FT cell line and then lentivirus was infected in desired cell line. Selection of HOTAIR stable transfected cells was performed in medium containing blasticidin (Invitrogen).

Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan). Cells (2×10³ cells/well) were seeded into 96-well flat-bottomed plates in 100 μl of complete medium. The cells were incubated overnight to allow for cell attachment and recovery and were then transfected with siNC or siHOTAIR for 24, 48, 72 and 96 h. CCK-8 solution (10 μl) was added to each well, and the cells were incubated for an additional 2 h. Absorbance was measured at 450 nm using a microplate reader. Three independent experiments were performed in triplicate.

The Matrigel invasion assay was performed using the BD BioCoat Matrigel Invasion Chamber (pore size: 8 mm, 24-well; BD Biosciences, Bedford, MA, USA) according to the manufacturer’s protocol. siHOTAIR-transfected cells and siNC-transfected cells (5×10⁴ cell/plate) were plated in the upper chamber in serum-free medium, and complete medium was added to the bottom chamber. The Matrigel invasion chamber was incubated for 48 h at 37°C under 5% CO₂. Non-invading cells were removed from the upper chamber using cotton-tipped swabs. Cells that had invaded through the pores onto the lower side of the filter were stained (Diff-Quik; Sysmex, Kobe, Japan), and these were counted using a hemocytometer. The number of invaded siHOTAIR-transfected cells was expressed as fold-change relative to siNC-transfected cells, which was set at 1. The assay was replicated at least 3 times.

Cells transfected with siNC or siHOTAIR (5×10⁵ cells/well) were seeded into 6-well culture plates with serum-containing medium and were cultured until the cell density reached ~90% confluence. The serum-containing medium was removed, and cells were serum starved for 24 h. When the cell density reached ~100% confluence, an artificial homogeneous wound was created by scratching the monolayer with a sterile 200-μl pipette tip. After scratching, the cells were washed with serum-free medium. Images of cells migrating into the wound were captured at 0, 24 and 48 h using a microscope. The assay was performed in triplicate.

Cells were transfected with siNC or siHOTAIR for 48 h, washed with ice-cold 0.01 M PBS (pH 7.2), and lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM saline, 1% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS)] supplemented with protease inhibitors. Protein concentrations were determined using Bio-Rad protein assay reagent according to the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Samples were boiled for 5 min, subjected to 10% SDS-PAGE, and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat dried milk in 1X Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.6) at room temperature for 1 h and were then incubated with primary antibody at 4°C overnight under constant agitation. The primary antibodies used included: rabbit anti-human VEGF (1:500 dilution; Abcam, Cambridge, MA, USA), rabbit anti-human MMP-9 (1:1,000 dilution; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-human E-cadherin (1:1,000 dilution; Cell Signaling Technology), rabbit anti-human β-catenin (1:1,000 dilution; Cell Signaling Technology), mouse anti-human Vimentin (1:1,000 dilution; Sigma, St. Louis, MO, USA), mouse anti-human Snail (1:1,000 dilution; Cell Signaling Technology), rabbit anti-human Twist (1:1,000 dilution; Abcam), and mouse anti-human β-actin antibody (1:5,000 dilution; Sigma). Membranes were washed 3 times with 1X TBST, incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000 dilution; Abcam) or anti-mouse secondary antibody (1:2,000 dilution; Abcam) for 1 h at room temperature under constant agitation, and then washed 3 times with 1X TBST. Proteins were visualized using an enhanced chemiluminescence system (ECL™; Amersham, Little Chalfont, UK), and band intensities were quantified using the Luminescent image analyzer (LAS 4000 mini; Fujifilm, Uppsala, Sweden).

SPSS software (standard version 20.0; IBM) was used for all statistical analyses. Data are expressed as the mean ± standard deviation (SD). The association between HOTAIR expression and clinicopathological characteristics was assessed using the Pearson’s χ² test, Student’s t-test, and Fisher’s exact test. Overall survival was analyzed by the Kaplan-Meier method, and the differences between groups were estimated by the log-rank test. Multivariate survival analysis was performed for the significant parameters in the univariate analysis using the stepwise Cox regression model analysis. All statistical tests were two-sided, and P<0.05 was considered to indicate a statistically significant result.

The expression of HOTAIR lncRNA was determined in cervical cancer tissues (n=111) and corresponding normal tissues (n=40) using qRT-PCR. HOTAIR expression in cervical cancer tissues was >30-fold that in non-cancerous tissues (Fig. 1A), suggesting that the expression of HOTAIR is upregulated in cervical cancer. To evaluate the prognostic value of HOTAIR for predicting clinical outcome in cervical cancer, HOTAIR expression levels were determined in an independent panel consisting of 111 cervical cancer patients with extensive clinical follow-up (Table I). The patients were divided into low (n=22) and high (n=89) HOTAIR expression groups, and clinicopathologic features were compared between the two groups. Age, stage, cell type and lymphatic invasion were not significantly different between the low and high HOTAIR expression groups. In contrast, HOTAIR expression was correlated with lymph node metastasis (P=0.0437). Multivariate Cox regression model analysis was performed to further evaluate the prognostic significance of HOTAIR expression and clinicopathologic characteristics on recurrence (Table II). HOTAIR expression was a significant prognostic indicator for recurrence in cervical cancer patients (relative risk=5.281; P=0.0493). As shown in Fig. 1B, HOTAIR expression levels were correlated with overall survival HOTAIR (log-rank test; P=0.035). These data suggest that HOTAIR expression represent an independent prognostic factor for survival and that the overexpression of HOTAIR might play an important role in the program of cervical cancer.

To determine the functional role of HOTAIR in cervical cancer, siRNA was used to downregulate HOTAIR expression. For this, HOTAIR expression in SiHa, Caski and HeLa cervical cancer cell lines was first determined using qRT-PCR. As shown in Fig. 2A, HOTAIR expression levels were higher in HeLa cells than in SiHa and Caski cells. Therefore, HeLa cells were used for siRNA-mediated knockdown of HOTAIR expression. The knockdown efficiency of the 2 HOTAIR-specific siRNAs (siHOTAIR-1 and siHOTAIR-2) was evaluated, and siHOTAIR-2 was found to have higher silencing efficiency than siHOTAIR-1 did (Fig. 2B). Therefore, siHOTAIR-2 was selected for use in the subsequent in vitro biological assays. To determine the role of HOTAIR in cervical cancer cell growth, siHOTAIR-transfected cells were used in the CCK-8 assay. siRNA-mediated knockdown of HOTAIR decreased cell proliferation by 30% at 96 h post-transfection in HeLa cells (Fig. 2C). Also, HOTAIR siRNA inhibited cell proliferation in SiHa and Caski cells. This finding indicates that HOTAIR is involved in the proliferation of cervical cancer cells.

To investigate the effect of HOTAIR on migration and invasion, siHOTAIR-transfected cells were used in wound healing and Matrigel invasion assays, respectively. The width of the wound closure was larger in siHOTAIR-transfected cells than in siNC-transfected of HeLa, SiHa and Caski cells (Fig. 3A). Therefore, downregulation of HOTAIR decreased the migration of cervical cancer cells. We also tested whether HOTAIR knockdown has an inhibitory effect on HeLa cell invasion. Knockdown of HOTAIR inhibited HeLa cell invasion >80% (Fig. 3B). To further assess the role of HOTAIR in the pathogenesis of cervical cancer, SiHa cell lines stably expressing ectopic HOTAIR were established (Fig. 3C). Consistent with the previous results, stable HOTAIR overexpression in SiHa cells resulted in a significantly increase the invasion ability of SiHa cells (Fig. 3D). Collectively, these results indicate that HOTAIR has an important role in the migratory and invasive phenotype of cervical cancer cells.

VEGF and MMP-9 play an important role in tumor progression by promoting migration and invasion (28,29). Therefore, the effect of HOTAIR on the expression levels of these proteins was determined in HeLa cells. VEGF and MMP-9 protein expressions were significantly lower in siHOTAIR-transfected cells than in siNC-transfected cells (Fig. 4A and B). In contrast, HOTAIR overexpression in SiHa cells promoted VEGF and MMP-9 protein expression (Fig. 4C). In addition, the high expression level of HOTAIR in cervical cancer tissues associated with upregulation of VEGF and MMP-9 expression levels compared with the low expression groups (Fig. 4D). Taken together, our findings suggest that HOTAIR may promote cervical cancer cell migration and invasion through the upregulation of VEGF and MMP-9 expression.

Because the EMT is important in cell migration and invasion, we also investigated whether direct inhibition of HOTAIR could reverse EMT-related markers in HeLa cells using real-time RT-PCR and western blot assays following HOTAIR knockdown. As anticipated, the siHOTAIR resulted in an increase in the expression of E-cadherin and a decrease in the expression of β-catenin and vimentin (Fig. 5). Next, we assessed the effect of HOTAIR knockdown on the expression of following transcription factors known to promote EMT: Snail and Twist. siHOTAIR-transfected cells expressed lower level of snail and twist compared with the siNC-transfected cells (Fig. 5). Collectively, the dysregulation of the expression of EMT-related genes partially explains the involvement of HOTAIR in cervical cancer cell migration and invasion.