Contributed equally
HOX transcript antisense RNA (HOTAIR), a long intergenic non-coding RNA (lncRNA), functions as a molecular scaffold to link and target the histone modification complexes PRC2 and LSD1, then reprograms chromatin states by coupling histone H3K27 methylation and H3K4 demethylation for epigenetic gene silencing to promote cancer metastasis. It is associated with poor survival in several solid cancers. In this study, we show that HOTAIR expression increased in oral squamous cell carcinoma (OSCC) compared with non-tumor tissue and is associated with metastasis, the stage and histological differentiation. In addition, overexpression of HOTAIR indicated poor overall survival (OS) and disease-free survival (DFS) in OSCC patients. Knockdown of HOTAIR by siRNA in OSCC cells decreased cell proliferation and colony formation, increased cell invasion and migration, and induced apoptosis
Oral squamous cell carcinoma (OSCC) is one of the most common cancer affecting the head and neck region, causing ~42,440 new cases and 8,390 deaths annually in the United States. In spite of the many advances in surgical treatment, radiotherapy and chemotherapy used in OSCC, the 5-year survival rate after diagnosis for OSCC remains at ~50% (
Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nts, and without a functional open reading frame (ORF) in most cases, but they might play widespread roles in gene regulation and other cellular processes, including their involvement in epigenetic regulation, gene transcription, and post-transcription regulation (
OSCC samples and adjacent histological normal tissues were obtained from 76 patients who were admitted to the Department of Maxillofacial Surgery and and Otorhinolaryngology Head and Neck Surgery of Tianjin Medical University between January 2001 and March 2009. None of the patients received treatment prior to radical surgical treatment. Tumor tissues and non-malignant tissues that were ≥1.5 cm distal to the tumor margins were snap-frozen in liquid nitrogen and then stored at −80°C until use. The clinicopathological characteristics of patients are summarized in
Two OSCC cell lines (TSCCA, Tca8223) were purchased from the Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China). CAL-27 cell line was purchased from Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China), and Tb3.1 cell line was obtained from Shanghai Ninth People’s Hospital (Shanghai, China). Cells were cultured in RPMI-1640, DMEM or MEM (Invitrogen, CA, USA) medium supplemented with 10% fetal bovine serum (FBS), (Hyclone, UT, USA) in humidified air with 5% CO2 at 37°C.
Total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen, NY, USA) 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 F: 5′-AAATATGGCGGCGTCTACACGGA-3′, R: 5′-TCCAGAACCCTCTGACATTTGCCT-3′; E-cadherin F: 5′-TGCCCAGAAAATGAAAAAGG-3′, R: 5′-GTGTATGTGGCAATGCGTTC-3′; GAPDH F: 5′-GAAGGTGAAGGTCGGAGTC-3′, R: 5′-GAAGATGGTGATGGGATTTC-3′; EZH2 F: 5′-AATCAGAGTACATGCGACTGAGA-3′, R: 5′-GCTGTATCCTTCGCTGTTTCC-3′. qRT-PCR and data collection were performed on an ABI 7500 (Applied Biosystems, CA, USA). qRT-PCR results were analyzed and expressed relative to CT (threshold cycle) values, and then converted to fold changes.
Three individual FAM-siRNAs (siHOTAIR1, siHOTAIR2 and siHOTAIR3) siSUZ12 and siEZH2, negative control FAM-siRNA (silencer negative control siRNA) were purchased from GenePharma (Shanghai, China). siRNA oligonucleotides (10 nmol/l) in Opti-MEM (Invitrogen, NY, USA) were transfected into cells using Lipofectamine 2000 (Invitrogen, NY, USA) following the manufacturer’s protocol. Transfection efficiencies were detected by fluorescence microscope 6 and 48 h post-transfection, HOTAIR expression levels were measured. Target sequences for HOTAIR siRNAs were as follows: siHOTAIR1 sense, 5′-GCACAGAGCAACUCUAUAATT-3′; antisense, 5′-UUAUAGAGUUGCUCUGUGCTT-3′. siHOTAIR2 sense, 5′-GCCUUUGGAAGCUCUUGAATT-3′; antisense, 5′-UUCAAGAGCUUCCAAGGCTT-3′. siCT sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. siEZH2 sense, 5′-TTCATGCAACACCCAACACT-3′; siEZH2 antisense, 5′-GAGAGCAGCAGCAAACTCCT-3′.
Cells were seeded in 6-well plates in normal cell growth media and incubated to confluence. A 20-μl tip was used to make a straight scratch, simulating a wound. The medium was changed to MEM or 1640 containing 2% FBS. After 48-h incubation, the area occupied by migrated cells in the scratch was evaluated.
The Matrigel invasion assay was done using the BD Biocoat Matrigel Invasion Chamber (pore size, 8 mm, 24-well; BD Biosciences, CA, USA) following the manufacturer’s protocol. Cells were plated in the upper chamber in serum-free medium. The bottom chamber contained medium with 10% FBS. After 48 h, the bottom of the chamber insert was stained with methanol and 0.1% crystal violet, imaged, and counted using an IX70 inverted microscope (Olympus, Tokyo, Japan). Each Matrigel invasion assay was conducted in at least 3 replicates.
TSCCA and Tca8113 cells, transiently transfected with siHOTAIR or siCT, were harvested 48 h after transfection by trypsinization. After double staining with FITC-Annexin V and propidium iodide, cells were analyzed by flow cytometry 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 apoptotic cells was compared to the control from each experiment. All samples were assayed in triplicate.
A total of 500 HOTAIR siRNA-transfected Tscca and Tca8113 cells were placed in a fresh 6-well plate and maintained in media containing 10% FBS, replacing the medium every 3 days. After 14 days, cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were manually counted. For each treatment group wells were measured in triplicate.
Cells were lysed using RIPA protein extraction reagent (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, Beijing, China). Approximately 30 μg of protein extract was separated by 10% SDS polyacrylamide gel electrophoresis, then transferred to nitrocellulose membrane (Sigma, MO, USA) and incubated with mouse anti-human E-cadherin, EZH2, H3K27me3 (Abcam, MA, USA). ECL chromogenic substrate was used to visualize the bands and the density of the band on the western blotting was measured using the ChemiDoc XRS imaging system and QuantityOne software (Bio-Rad). β-actin was used as a control.
ChIP assay was performed with the EZ-Magna ChIP kit (Millipore) according to the manufacturer’s instructions. For each ChIP assay, 2 μg of antibodies were used: EZH2 (Cell Signaling Technology), H3K27me3 (Abcam) and H3 (Cell Signaling Technology). The percentage of the bound DNA was quantified against the original DNA input by qRT-PCR analysis. Primer sequences used are as follows: E-cadherin-a sense, 5′-TGTCCGCCCCGACTTGTCTCTC-3′; antisense, 5′-GTCCTCTGGCCCCAGCCTCTCT-3′. E-cadherin-b sense, 5′-AGACCCCATCTCCAAAACGAACAAA-3′;antisense,5′-GCATAGACGCGGTGACCCTCTAGCC-3′. E-cadherin-c sense, 5′-TGTCCGCCCCGACTTGTCTCTC-3′; antisense, CGGTCCTCTGGCCCCAGCCTCT-3′.
Statistical tests for data analysis included the Wilcoxon test, χ2 test, Fisher’s exact test and Student’s two-tailed t-test. Kaplan-Meier analysis and log-rank test were applied to evaluate the prognostic significance of HOTAIR expression level in terms of patient survival. The statistical analyses were performed using SPSS 19.0 statistical software package (SPSS, Chicago, IL, USA). The data represent the mean ± SD. P-values of P<0.05 were considered to be statistically significant.
Expression level of HOTAIR was detected in 76 OSCC samples and adjacent normal tissues by qRT-PCR and normalized to GAPDH. HOTAIR was more highly expressed in OSCC tumor compared with non-tumor tissues (P<0.001) (
Next, we performed qRT-PCR analysis to examine the expression level of HOTAIR in human OSCC cell lines. Of these 4 OSCC cell lines, TSCCA and Tca8113 expressed significantly higher levels of HOTAIR (
Given that overexpression of HOTAIR was significantly associated with progression in patients with OSCC we examined whether HOTAIR regulated the proliferation of OSCC cells. MTT assay was used to examine the effect of HOTAIR silence on the proliferation of TSCCA and Tca8113 cells
To further assess the role of HOTAIR in apoptosis and cell death of OSCC cells, we analyzed the HOTAIR knocked down cells using flow cytometry. In addition, the results indicated that knockdown of HOTAIR promoted both early apoptosis and later apoptosis of OSCC cells Tca8113and TSCCA (
We use wound healing scratch assay and Matrigel invasion assay to evaluate whether HOTAIR alters invasion and migration of OSCC cells. As shown, silence of HOTAIR decreased their migration and invasion ability. Conversely, the 2 OSCC cell lines Tca8113 and TSCCA treated with siCT did not affect the cell invasion and migration. Based on our results, expression of HOTAIR promoted migration and invasion of OSCC cells
To define functional correlation between HOTAIR and epithelial-mesenchymal transition (EMT), we examined the effects of HOTAIR knockdown on E-cadherin expression. We first assayed the expression level of HOTAIR and E-cadherin in OSCC tissues. A significant negative correlation is observed between the E-cadherin mRNA levels and the HOTAIR expression levels in OSCC tissues (r2=−0.327, P=0.004) (
HOTAIR is thought to regulate transcription by directing the action of PRC2 complexes to control the epigenetic state of the cell and subsequently regulate gene expression. We performed western blotting to test the enrichment of EZH2 and H3K27me3, and expression of E-cadherin in OSCC cell lines treated with HOTAIR siRNA. We observed enrichment of EZH2 and H3K27me3 significantly decreased and with the expression of E-cadherin significantly increased in the HOTAIR knockdown OSCC cells (
To check whether expression of the E-cadherin was controlled by EZH2, we analyzed the E-cadherin expression after EZH2 and/or HOTAIR knockdown. E-cadherin mRNA and protein levels increased in Tca8113 and TSCCA cells treated with siEZH2 and siEZH2+siHOTAIR groups (
To further address how HOTAIR regulated E-cadherin through enrichment of EZH2, we performed ChIP-arrays in OSCC cell lines with HOTAIR knockdown. In addition, results showed that HOTAIR silence decreased the binding of EZH2 and H3K27me3 with the E-cadherin promoter in OSCC cells, whereas, we did not detect the binding of IgG with E-cadherin promoter after HOTAIR inhibition (
Evidence has shown that HOTAIR can act as a key regulation factor in the molecular mechanisms underlying the development and progression of cancer (
Several reports have suggested that HOTAIR is upregulated in breast cancer, colorectal cancer, esophageal squamous cell carcinoma (ESCC) and promotes cancer invasion and metastasis and poor survival (
Emerging evidence suggests that HOTAIR regulates proliferation, invasion and apoptosis of a variety of tumor cells
Determination of the targeted proteins associated with HOTAIR and the genes regulated by HOTAIR would reveal the molecular mechanisms underlying cancer development and progression by HOTAIR. HOTAIR functions in the recruitment and binding of the PRC2 and LSD1 complexes to the HOXD locus on chromosome 2 where genes involved in metastasis are regulated through H3K27 methylation and H3K4 demethylation (
In conclusion, this study has shown that HOTAIR, defined as an oncogene is overexpressed in OSCC patients and indicates poor prognosis, and poor outcome of patients with high HOTAIR expression due to the ability of HOTAIR to promote cancer development and progression by inducing cancer cell proliferation and invasion and reducing cancer cell apoptosis. Moreover, HOTAIR regulates EMT related marker E-cadherin through recruitment of EZH2 and H3K27me3. Therefore, these findings indicate that HOTAIR plays a vital role in the development and progression of OSCC and downregulation of such lncRNAs could be a valuable predictive marker as well as novel therapeutic target in future OSCC treatment.
This study was supported by National Basic Research Program of China (973 Program), no. 2010CB529301 (to Xishan Hao) and supported in part by the China National Natural Scientific Fund (81172573) (to Lun Zhang).
HOTAIR expression and prognostic significance in OSCC cancer. (A) HOTAIR expression in non-tumor and OSCC tissues from patients. (B) Relative expression of HOTAIR in OSCC patients with tumor localized in the oral cavity (N0) or those with tumors spread to cervical lymph nodes (N1). (C and D) Kaplan-Meier plot for disease-free survival (C) and overall survival (D) of OSCC patients expressing high and low levels of HOTAIR. *P<0.05.
HOTAIR knockdown inhibits the malignant properties of OSCC cells. The expression of HOTAIR in OSCC cell lines as determined by real-time PCR (A) and HOTAIR is efficiently knocked down by siRNA (B) in OSCC cell lines TSCCA and Tca8113. Silencing of HOTAIR suppressed the proliferation of TSCCA and Tca8113 cells as determined by an MTT assay (C and D). The effect of HOTAIR on tumorigenesis of OSCC cells was examined by a colony formation assay, HOTAIR knockdown significantly reduced the anchorage-independent cell growth of TSCCA and Tca8113 cells compared with the negative control (E and F). Annexin V-fluorescein isothiocyanate (FITC)/PI staining of cells transfected with siCT and siHOTAIR for 48 h. The percentage of apoptotic cells were counted and the results are summarized in a bar graph (G and H). *P<0.05.
HOTAIR promotes OSCC cell invasion and migration. Wounds were introduced by scratching confluent monolayers of TSCCA and Tca8113 cells transfected with siCT, siHOTAIR. Migration was monitored by light microscopy at 0 and 48 h (A and B). Matrigel-based invasion assay was performed using modified Boyden chambers with 10% FBS as a chemoattractant. Representative images are presented. The cell numbers per field were counted and the results are summarized in a bar graph (C and D). *P<0.05.
HOTAIR negatively correlates to E-cadherin expression in OSCC tissues and cell lines. Negative correlation between E-cadherin mRNA levels and the HOTAIR levels in 76 OSCC samples (A). Relative expression level of HOTAIR and E-cadherin in Tca813 and TACCA cells treated with siRNA for 48 h (B and C). Western blot analysis of E-cadherin after siHOTAIR treatment for 48 h in Tca813 and TACCA cells (D). *P<0.05.
HOTAIR is associated with EZH2 and regulates E-cadherin. Enrichment of EZH2 and H3K27me3 significantly decreased and the expression of E-cadherin significantly increased in the HOTAIR knockdown OSCC cells (A). Relative E-cadherin mRNA and protein level after siHOTAIR and/or siEZH2 treatment in OSCC cells (B and C). *P<0.05.
HOTAIR represses E-cadherin expression by associating with EZH2. ChIP analysis of Tca8113 and TSCCA cells treated with siHOTAIR were conducted on E-cadherin promoter (A) using the anti-EZH2 and anti-H3K27me3 antibodies and IgG. Enrichment was determined relative to input controls (B–G). *P<0.05.
HOTAIR expression and clinicopathological factors in OSCC.
HOTAIR expression | ||||
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Clinicopathological factors | n (%) | High | Low | P-value |
Gender | 0.479 | |||
Male | 47 (62) | 22 | 25 | |
Famale | 29 (38) | 16 | 13 | |
Age (years) | 0.243 | |||
≤60 | 45 (59) | 20 | 25 | |
>60 | 31 (41) | 18 | 13 | |
Smoking and/or alcohol | 0.238 | |||
Yes | 47 (62) | 26 | 21 | |
No | 29 (38) | 12 | 17 | |
Location | 0.065 | |||
OTSCC |
42 (55) | 25 | 17 | |
OSCC | 34 (45) | 13 | 21 | |
OTSCC (excepted) | ||||
Tumor size (cm) | 0.758 | |||
≤2 | 21 (28) | 11 | 10 | |
2–4 | 41 (54) | 19 | 22 | |
>4 | 14 (18) | 8 | 6 | |
Histological differentiation | 0.019 | |||
High | 29 (38) | 9 | 20 | |
Median | 35 (46) | 20 | 15 | |
Poor | 12 (16) | 9 | 3 | |
Stage | 0.001 | |||
I–II | 32 (42) | 9 | 23 | |
III–IV | 44 (58) | 29 | 15 | |
Lymph node metastasis | 0.000 | |||
N1 | 34 (45) | 26 | 8 | |
N0 | 42 (55) | 12 | 30 |
OTSCC, oral tongue squamous cell carcinoma.