Samples from 120 patients undergoing colectomy between October 2007 and December 2010 by the same surgical team at the Shanghai Jiao Tong University Affiliated First People’s Hospital were collected and archived for the study. Fresh colon cancer samples and matched adjacent non-tumor tissues were obtained from 52 males and 68 females with ages ranging from 35 to 88 years. The samples were stored at −80°C immediately following removal. None of the patients had received any preoperative therapy, and patients with stage II, III and IV disease underwent standard adjuvant chemotherapy according to The National Comprehensive Cancer Network Practice Guidelines for Colon Cancer (1). Follow-up was carried out completely (median follow-up time, 55.5 months; range, 10–72 months). The study was approved by the Ethics Committee of Shanghai Jiaotong University Affiliated First People’s Hospital. Informed written consent was obtained from each patient enrolled in our study according to the Declaration of Helsinki. Colon cancer cell lines were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO₂ at 37°C.

Total RNAs from all samples were isolated with AllPrep DNA/RNA Mini kit (Qiagen, Hilden, Germany). After the RNA integrity and purity were checked, the first single-stranded cDNAs were synthesized from 3 μg of RNA, according to the manufacturer’s instructions (Fermentas, Shenzhen, China). One microliter of the cDNAs from each sample was used as a template for qRT-PCR analysis with the ABsolute qPCR SYBR-Green Mix (Fermentas), employing the ABI Prism 7900 system (Applied Biosystems Inc., Foster City, CA, USA).

The following primers were used for qRT-PCR: HOTAIR sense 5′-GGTAGAAAAAGCAACCACGAAGC-3′ and antisense 5′-ACATAAACCTCTGTCTGTGAGTGCC-3′; GAPDH sense 5′-GGAGCGAGATCCCTCCAAAAT-3′ and antisense 5′-GGCTGTTGTCATACTTCTCATGG-3′.

The expression level of HOTAIR was normalized to the transcription level of GAPDH. The quantitative PCR reaction for each sample was repeated in triplicate. The relative HOTAIR expression was calculated using the 2^−ΔΔCt comparative method.

Two colon cancer cell lines were transfected with 50 nM positive siRNAs (si-HOTAIR 5′-GAACGGGAG UACAGAGAGAUU-3′) or negative control siRNA (Mock 5′-CUACAACAGCCACAACGUCdTdT-3′) employing Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. Total RNA isolation was performed 72 h later for real-time PCR analysis, while other functional assays were carried out 48 h post-transfection. All RNA interference assays were carried out in triplicates independently.

For cell proliferation assay, cells were seeded in 96-well plates (2×10³ cells/well) in triplicate. At the appropriate time (24, 48, 72, 96 and 120 h), the cells were incubated with MTT (5 mg/ml; Sigma, St. Louis, MO, USA) for 4 h at 37°C. MTT solution was then carefully removed and replaced with dimethyl sulfoxide (DMSO; Sigma). Absorbance was measured at a wavelength of 570 nm.

Cell migration and invasion assays were carried out using Transwell chambers (micropore size, 8 μm, 24-well; BD Biosciences, Franklin Lakes, NJ, USA) without Matrigel (for migration assay) or with Matrigel (for invasion assay). Both were performed according to the manufacturer’s protocol. Briefly, the treated cells were plated in the upper chamber at a concentration of 5×10⁴ in 500 μl FBS-free media. The bottom chambers maintained suitable media with 10% FBS for 48 h. Then, the bottom of the chamber insert was fixed and stained with Giemsa solution. The stained cells were counted under a microscope at ×200 magnification; meanwhile, images were captured and saved. The experiments were carried out in triplicate.

The total protein was extracted from cells using Thermo Scientific Pierce RIPA buffer combined with Thermo Scientific Halt proteinase inhibitor cocktail and then quantified using Thermo Scientific BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The equivalent amounts of protein (20 μg) for each sample were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 5% non-fat milk solution for 1 h, and then probed with primary polyclonal antibodies at 4°C overnight, followed by secondary antibodies (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. β-actin was used as a loading control. After washing with TBST (Tris-buffered saline with 0.01% Tween-20), the proteins were detected by enhanced electrochemiluminescence (Millipore) and radiography. The primary antibodies used above were anti-E-cadherin (1:1,000; Abcam, Cambridge, UK) and anti-Vimentin (1:1,000; Abcam), anti-MMP9 (1:500; Cell Signaling Technology, Boston, MA, USA) and anti-β-actin antibody (1:1,000; Cell Signaling Technology).

All statistical analyses were set with a significance level of P≤0.05 and carried out using SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA). The differences between 2 groups were analyzed with the Student’s t-test and χ² test, as appropriate. The relationship between HOTAIR expression and clinicopathological characteristics was estimated with the Mann-Whitney U test and the Kruskal-Wallis test appropriately. Survival curves were calculated by Kaplan-Meier method, with the log-rank test employed for the comparison of differences. Variables estimated with significant means by univariate analysis were subjected to the multivariate Cox proportional hazards regression model.

qPCR was applied to examine the expression level of HOTAIR in 120 pairs of cancer and matched normal tissues, and 32 metastasized lymph node tissues. As shown in Fig. 1, HOTAIR was overexpressed from 2- to nearly 1,600-fold in the cancer tissues. According to a HOTAIR expression level of >5-fold compared to normal tissues, 120 patients were divided into a high expression group (n=40) and a low expression group (n=80). In addition, 27 of 32 (84.4%) lymph node metastasis tissues showed high expression of HOTAIR, with significant means between primary cancer tissues and metastatic lymph node tissues (Table I).

The correlation between HOTAIR expression and clinicopathological characteristics is summarized in Tables II and III. Increased HOTAIR expression was significantly associated with AJCC stage (P<0.001), lymph node metastasis (pN stage, P=0.002), organ metastasis (pM stage, P<0.001), vascular invasion (P=0.019) and histologic differentiation (P<0.001). No significant correlations were found between HOTAIR expression and age, gender or tumor location. The Kaplan-Meier curves (Fig. 2) revealed that patients in the high expression group had a significant poorer clinical prognosis than those in the low expression group, both for metastasis-free survival and overall survival (log-rank test P<0.001). With regard to Cox univariate proportional hazards regression model, the level of HOTAIR expression, lymph node metastasis, organ metastasis, AJCC stage, degree of differentiation and vascular invasion were prognostic factors. Additionally, multivariate analysis, including the variables above with P<0.05, indicated that HOTAIR expression was an independent clinical risk indicator for both metastasis-free survival (HR, 3.878; 95% CI, 1.369–10.981; P=0.011) and overall survival (HR, 3.915; 95% CI, 1.226–12.499; P=0.021).

As reported by other research groups, HOTAIR may play a pro-oncogenic role in certain types of cancers such as breast (5), hepatocellular (6), gastrointestinal (8), pancreatic (9) and lung cancer (11). To examine the effect of HOTAIR on colon cancer, a series of functional experiments concerning cell proliferation and cell metastasis was performed. We quantified the expression levels of HOTAIR in the colon cancer cell lines by qRT-PCR, and compared the levels to the level in MCF-7, a breast cancer cell line that overexpresses endogenous HOTAIR (Fig. 3). HOTAIR expression levels in colon cancer cell lines SW480 and HT29 were higher than that in the other 4 cell lines. HOTAIR knockdown was carried out by using small interfering RNA transfection in the cell lines. The efficiency of siRNA-mediated knockdown in the SW480 or HT29 cells was >60% (data not shown). As illustrated by Fig. 4, results from the MTT assay showed that the proliferation rate of cells depleted of HOTAIR was slightly slower than the rate of cells treated with mock siRNA throughout 5 continuous days.

We next ascertained whether manipulation of the HOTAIR level affects cell metastasis in vitro. Migration and invasiveness of tumor cells are key aspects of metastasis. In the Transwell migration assay, fewer SW480 and HT29 colon cancer cells treated by si-HOTAIR migrated to the 8-μm pores of the Transwell chambers when compared to the cells treated with Mock (Fig. 5A and B). Furthermore, similar results were obtained in the invasion assay using a Matrigel environment study (Fig. 5C and D). All of these studies were repeated three times and the differences were statistically significant (P<0.05). Based on these outcomes, it was suggested that HOTAIR is involved in the progression of colon cancer mainly by promoting cancer cell migration and invasiveness.

Epithelial-mesenchymal transition (EMT) with alterations in gene expression profiles of cells initiates the invasion-metastasis cascade. Therefore, we ascertained whether HOTAIR promotes EMT to regulate tumor progression. Several key genes involved in EMT of colon cancer were selected for further western blot analysis (Fig. 6). When SW480 and HT29 cells were treated by HOTAIR knockdown, expression of E-cadherin, a hallmark of epithelial cell was increased, while expression of vimentin, a mesenchymal cell marker, was decreased. Additionally, in the si-HOTAIR-treated cells, expression of matrix metalloproteinase 9 (MMP9), which functions as an important proteinase to degrade the extracellular matrix for facilitating cell motility, was inhibited as well. These data indicate that the regulation of EMT is partly involved in the mechanism of HOTAIR-induced metastasis.