Long non-coding RNA HOTAIR, a driver of malignancy, predicts negative prognosis and exhibits oncogenic activity in oesophageal squamous cell carcinoma

Abstract

Background: HOX transcript antisense RNA (HOTAIR), which is expressed from the homebox C gene (HOXC) locus, is capable of reprogramming chromatin organisation and promoting cancer cell metastasis and can simultaneously bind the polycomb repressive complex 2, which enhances H3K27 trimethylation, and the LSD1-CoREST-REST complex, which is critical for H3K4 demethylation. Clinically, the overexpression of HOTAIR is a powerful predictor of the tumour progression and overall survival in patients with diverse cancers. The relationship between HOTAIR and oesophageal squamous cell carcinoma (ESCC), however, remains unclear. We investigated the role of HOTAIR in the pathogenesis of ESCC.

Methods: We used quantitative real-time PCR to determine the level of HOTAIR in ESCC cell lines and 100 ESCC samples from patients; 56 adjacent non-neoplastic tissues were used as controls. We measured the effect of HOTAIR knockdown and overexpression in ESCC cell lines using colony formation assays, anchorage-independent growth assays, the CCK-8 assay, transwell migration and invasion assays, and Annexin V-binding assays. We analysed the growth of ESCC xenograft tumours in nude mice. Changes in the gene expression and methylation levels in ESCC cell lines were analysed using gene expression microarrays and the Infinium HumanMethylation450K BeadChip assay, respectively. results: The levels of HOTAIR were increased in ESCC cell lines and patient samples compared with the controls; the expression levels correlated with the disease stage and survival time. The knockdown of HOTAIR in the KYSE510 and KYSE180 ESCC cell lines using small hairpin RNAs (shRNAs) reduced the ability of the cells to form foci, migrate, and invade the extracellular matrix in culture, altered cell cycle progression, and increased the sensitivity of the cells to apoptosis. The HOTAIR knockdown reduced cancer cell metastasis in vivo, and the tumours formed by HOTAIR-silenced ESCC cells were smaller, both in size and weight, than the tumours and metastases formed by the shRNA vector control cells in a mouse xenograft model. The results of the gene microarray study showed that HOTAIR reprogrammed the gene expression profile of ESCC cells, and the gene ontology analysis revealed an enrichment in genes that are important for tumorigenesis, such as genes involved in cell migration and the regulation of the cell cycle. Comparing the gene expression profiles and DNA methylation analysis between the KYSE180 and KYSE180_HOTAIR cells revealed that only a small proportion of the methylation changes were correlated with gene expression changes.

Conclusion: HOX transcript antisense RNA is upregulated in ESCC cell lines and patient samples, and promotes ESCC cell proliferation and tumour metastasis in mice. The knockdown of HOTAIR resulted in significant changes in gene expression, and data analysis suggested that HOTAIR-mediated gene regulation has a critical role in ESCC progression and is a novel epigenetic molecular target for treating ESCC patients.

Figure 1

High levels of HOTAIR are correlated with progression and prognostic significance in ESCC. (A) HOX transcript antisense RNA expression in normal oesophageal epithelial tissues and ESCC tissues from patients. The majority of cases exhibited elevated levels of HOTAIR in tumours compared with levels observed in noncarcinoma tissues (P<0.001). Relative gene expression determinations were made with the comparative delta-delta CT method (2^−ΔΔCt). (B) The relative expression levels of HOTAIR were confirmed via real-time PCR in 11 EC cell lines. (C) HOTAIR levels categorised according to tumour-nodule-metastasis stage. (D) Oesophageal SCC patients with lymph node metastasis displayed significantly higher HOTAIR expression levels (P<0.05). (E) The total of 100 ESCC patients included in this study were divided into an elevated HOTAIR expression group (n=30) and a low HOTAIR expression group (n=70) the value of relative HOTAIR expression (125-fold). (F) The Kaplan–Meier overall survival curves by HOTAIR levels. Patients with elevated HOTAIR expression (n=30) showed reduced survival times compared with patients with low levels of HOTAIR expression (n=70; log-rank test; P=0.0334).

Figure 2

Silencing HOTAIR inhibits the malignant properties of ESCC cells. (A) Silencing HOTAIR in two specific short hairpin RNA-transduced stable ESCC cell lines. Relative gene expression determinations were made with the comparative delta-delta CT method (2^−ΔΔCt). (B) The representative pictures (left panel) and quantification (right panel) of colony numbers of indicated cells as determined by an anchorage-independent growth assay. Colonies larger than 100 μm in diameter were scored. (C) The representative pictures (left panel) and quantification (right panel) of Giemsa-stained cells. (D) The ESCC KYSE510 or KYSE180 cells were transfected with control siRNA or two individual siRNAs against HOTAIR as indicated. Cell viability was detected at the indicated time points after transfection using CCK-8 assays. (E) The representative pictures (top panel) and quantification (bottom panel) of invaded cells were analysed using the transwell matrix penetration assay. (F) KYSE180 cells stably transfected with the indicated siRNAs were treated with Nocodazole (Noc) for 0 h (top panel), 8 h (bottom panel). Cell cycle distribution was measured by propidium iodide (PI) staining followed by flow cytometry. The percentage of cells in G1, S, or G2 phase transfected with control siRNA is defined as control. Data are represented as mean±s.d. from three independent experiments. (G) Annexin V-fluorescein isothiocyanate (FITC)/PI staining of cells transfected with indicated siRNA for 48 h. Each bar represents the mean±s.d. of three independent experiments. *P<0.05; **P<0.01.

Figure 3

HOX transcript antisense RNA promotes the aggressiveness of ESCC cells in vitro. (A) Overexpression of HOTAIR in KYSE510 and KYSE180 cell lines analysed by qRT–PCR. Relative gene expression determinations were made with the comparative delta-delta CT method (2^−ΔΔCt). (B) The representative pictures (left panel) and quantification (right panel) of colony numbers of indicated cells as determined by an anchorage-independent growth assay. Colonies larger than 100 μm in diameter were scored. (C) Cell viability was detected at the indicated time points after transfection using CCK-8 assays. (D) The representative pictures (left panel) and quantification (right panel) of Giemsa-stained cells. (E) Scratch wound assay results. Overexpression of HOTAIR in KYSE510 and KYSE180 cells produced a higher scratch closure rate than observed in controls infected with empty vector. (F) The representative pictures (top panel) and quantification (bottom panel) of migrated and invaded cells were analysed using transwell matrix penetration assay. (G) Comparison of DNA content between empty vector and HOTAIR overexpressed in both KYSE510 and KYSE180 cell lines by flow cytometry. Summary of cell proportions in different phases of cell cycle is shown. The results are expressed as means±s.d. of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 4

HOX transcript antisense RNA contributes to ESCC progression in vivo. (A) Lung metastasis assay using an intravenous cancer mouse model and histological analysis from nude mice injected via the lateral tail vein with KYSE180 cells with control siRNA or siRNAs against HOTAIR (a). Representative Haematoxylin-eosin (H&E) images in lung metastases are shown (b). (B) Representative images of tumour-bearing mice (left panel; a) and images of the tumours from all mice in each group (right panel; a). Tumour volumes were measured on the indicated days (left panel; b), and tumour weights were determined (right panel; b). (C) H&E and immunohistochemical staining demonstrated that suppression of HOTAIR inhibited the aggressive phenotype of ESCC cells in vivo, as indicated by the expression of Ki67-, TUNEL-, and CD31/PECAM1-positive cells. *P<0.05; **P<0.01.

Figure 5

Gene expression profiling data and overall relation between differential methylation and expression. (A) Heat map of expression profiles for differentially expressed genes overlapped with cancer-associated genes set in the Molecular Signatures Database. The colour of the heat map represents the relative expression of each sample (red: overexpressed; green: underexpressed). Clustering is performed using an average clustering algorithm and a Euclidean distance dissimilarity metric of the normalised expression. (B–D) A selection of the top enriched GO biological process. Gene ontology molecular function, and Kyoto Encyclopedia of Genes and Genomes pathway for genes with a P-value⩽0.05. The numbers for each pathway indicate the fold enrichment based on the Fisher exact test. (E) Total number of hypermethylated CpG sties in KYSE180 and KYSE180_HOTAIR cells over a basal β-value of 0.6. (F) Scatter plots of β-values show differentially methylated loci between KYSE180 cells vs KYSE180_HOTAIR cells as measured by Infinium HumanMethylation450K BeadArrays. The differential methylation cutoff (pink and blue dotted line) was estimated by controlling false discovery rate <0.05. The colour scale indicates the genes with corresponding levels of methylation (increases from blue to red). (G) Graphic showing the percentage of differentially methylated CpG sites in KYSE180_HOTAIR cells with respect to KYSE180 cells, percentage of hypermethylation and hypomethylation. (H) CpG hypermethylation and hypomethylation events observed in KYSE180_HOTAIR cells in comparison with control KYSE180 cells according to functional genomic distribution (promoter, intergenic, 5′-UTR, 3′-UTR, gene body, and 1st exon). (I) CpG hypermethylation and hypomethylation events observed in KYSE180_HOTAIR cells in comparison with control KYSE180 cells according to chromosome location. (J) Overall relation between differential methylation and expression. Shown is a scatter plot of the relative levels (according to DiffScro in Infinium HumanMethylation450K BeadArrays) of methylation and fold change (according to log2 transformed data) in gene expression from microarray. Pink-coloured dots represent that a set of genes decrease methylation and increased expression, blue-coloured dots represent that a set of genes increased methylation and decreased expression in KYSE180_HOTAIR cells. (K) A summary of coordinate and independent regulation of genes by methylation changes in KYSE180_HOTAIR cells compared with KYSE180 cells. (L) Gene expression was determined by qRT–PCR in HOTAIR knockdown and HOTAIR overexpression cells, and relative gene expression determinations were made with the comparative delta-delta CT method (2^−ΔΔCt). The results are expressed as means±s.d. of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.